Portada Libro Resumen Nitrogen 2013

Second International Symposium on the
Nitrogen Nutrition of Plants
18 - 22 November 2013. Puerto Varas, Chile.
Sponsors
INDEX
Sponsors
Introduction
Organizing Committee
General Information
Location Information
General Services Information
Program
Lectures
Sessions
Session 1
Session 2
Session 3
Session 4
Session 5
Session 6
Session 7
Poster Sessions
2
5
7
8
9
10
11
20
27
28
36
41
50
56
63
70
76
Session 1
Session 2
Session 3
Session 4
Session 5
Session 6
Session 7
77
85
90
96
102
107
118
Tours Information
List of Participants
131
134
Welcome to the Second International Symposium on
the Nitrogen Nutrition of Plants - Nitrogen2013
We welcome you to Puerto Varas, Chile and to the Second International
Symposium on the Nitrogen Nutrition of Plants. This symposium was planned
during the first International Symposium held at Inuyama-city, Japan in 2010. The
international nitrogen meetings reflect the natural growth of the nitrogen community
over the years, building on efforts initiated by ENAAG (European Nitrate and
Ammonium Assimilation Group) and NAMGA (Nitrate Assimilation: Molecular and
Genetic Aspects Group) groups. ENAAG and NAMGA had jointly and independently
organized Nitrogen meetings for many years, with the last joint meeting held in
Lancaster, UK in 2007. ENAAG group met in Jarandilla de la Vera (Spain, 1986),
Naples (Italy, 1989), Tiberias (Israel, 1992), Darmstadt (Germany, 1995), Luso
(Portugal, 1998), Reims (France, 2001), and Wageningen (The Netherlands, 2004),
while NAMGA group held meetings in Gatersleben (DDR, 1982), St. Andrews (UK,
1987), Bombannes (France, 1990), Tampa (USA, 1997) and Cordoba (Spain, 2002).
Historically, the ENAAG groups have focused on the more physiological and
agronomic aspects while the NAMGA groups have emphasized molecular and
genetic aspects. The aim of the international meetings is to integrate these aspects
as well as highlight new developments relevant to the international nitrogen
community.
Chile’s history has long be associated to the use of
nitrogen minerals for agriculture. Sodium nitrate (16% N)
is one of the first known mineral fertilizers, also known as
Chilean nitrate. It is considered one of the first natural
minerals containing fixed N and a natural source of
nitrate. Although its deposits were found in several
countries, Chile possessed commercially viable deposits
of sodium nitrate, hence the name Chilean nitrate. The
first commercial mining of this material was done in the
early 19th century by the Spaniards. Commercial
development of Chilean nitrate deposits first occurred on
extremely arid islands off the coast of Chile and Peru around 1840. These island
deposits – derived from whole cliffs of seabird excrement or guano, deposited over
thousands of years – were quickly depleted and by 1870 markets turned to the
so-called caliche deposits located in the Atacama Desert in northern Chile. Unlike
the guano deposits, this mined substance is a crude mineral conglomerate of salts
possibly formed from nitrogen fixation by microorganisms approximately 10 – 15
million years ago.
In Chile, the economic period that ensued is called “el ciclo del salitre,” or the nitrate
cycle. Nitrate exports grew rapidly between 1880 and 1913, accounting for over two
thirds of total exports and 15% of GDP over the period. Chile imposed an export tax
that remained unchanged until 1930. This policy yielded revenues that accounted
for about half of total government revenues, and underwrote a great deal of public
investment. The performance of the nitrate export industry after World War I
declined due to development of the synthetic nitrogen industry, primarily in
Germany. Currently, it is a fertilizer of small, localized and special applications but it
is still used as a standard against which the salt index of various fertilizers is
measured. Currently, Chilean nitrate constitutes 0.14% of the total US fertilizer
application, and is used primarily by niche markets.
We are honored to have the opportunity to organize the second International
Symposium on the Nitrogen Nutrition of Plants here in Puerto Varas, Chile. The aims
of this meeting are to review progress in N nutrition, genomics, systems biology,
signaling and use efficiency in plants among other topics. The conference features a
mix of invited speakers drawn from a wide variety of backgrounds and experiences
as well as speakers selected from submitted abstracts. The format of the meeting
includes seven oral sessions and two poster sessions. Each oral session includes
invited speakers and speakers selected from submitted abstracts covering the
following topics (1) Nitrogen signaling, (2) Ammonium transport and assimilation, (3)
Nitrate transport and allocation, (4) Genomics and Systems Biology, (5) Nitrogen
interactions with other nutrients/signals, (6) Nitrogen-use efficiency and
ecophysiological aspects of nitrogen nutrition and (7) Nitrogen nutrition in plant and
bacterial systems.
The conference takes place in Hotel Patagónico in Puerto Varas, located in Región
de los Lagos, Chile. Puerto Varas is approximately a one hour flight away from
Santiago. It is a beautiful town in the southern lake district of Chile with many great
places to visit in the area such as national parks, glaciers and hot springs. Puerto
Varas is also known as the gateway to the Chilean Patagonia.
A Special Issue of the Journal of Experimental Botany will be produced to
accompany the Symposium. This will contain 15 reviews written by contributors to
the Symposium and covering a broad range of topics related to the N nutrition of
plants that you will find a useful supplement to the Symposium.
We hope that all of you have an exciting and stimulating five days and that the
weather holds so that we may enjoy the science as well as the views. We are sure
that this conference will provide opportunities to interact with old friends and
colleagues, as well as to meet new people for future scientific exchanges.
Finally, we thank our many sponsors for their financial support.
On behalf of the Organizing Committee,
Rodrigo A. Gutiérrez.
ORGANIZERS
International Nitrogen Steering Committee
Rodrigo A. Gutiérrez (Chair)
Department of Molecular Genetics and Microbiology. P. Universidad Católica de
Chile, Chile.
Gloria M. Coruzzi
Department of Biology. Center for Genomics and Systems Biology, New York
University, USA.
Alain Gojon
CNRS/INRA- Biochemistry & Plant Molecular Physiology – Montpellier, France.
Hitoshi Sakakibara
Plant Science Center. Riken, Japan.
Brian Forde
Lancaster Environment Centre, Department of Biological Sciences, Lancaster
University, United Kingdom.
Tomoyuki Yamaya
Graduate School of Agricultural Science. Tohoku University, Japan.
Executive Committee
Carolina Córdova – Conference Coordinator (ccordova@bio.puc.cl)
Karem Tamayo – Administrative and Financial Assistance (kptamayo@uc.cl)
7
General Information
Venue
All Scientific sessions will be held in the Volcán Osorno Conference Room (A+B)
located in the first basement floor (level -1) of Hotel Patagónico.
Registration Desk
The registration desk will be open at the following times:
Monday, 18 November
Tuesday, 19 November
12:00 – 19:00 (Hotel Reception)
9:00 – 13:00/14:00 – 18:00 (Río Puelo Room)
Conference Administrative Office hours.
Wednesday, 20 November 11:00 – 13:00/15:00 – 17:00 (Río Puelo Room)
Thursday, 21 November 11:00 – 13:00/15:00 – 17:00 (Río Puelo Room)
Friday, 22 November 11:00 – 13:00/15:00 – 17:00 (Río Puelo Room)
Name Badges
Participants are requested to wear name badges all times. Name badges will be
used as identification for hotel staff to grant access to conference rooms, lunch and
dinner as well as social activities organized by the conference.
Wireless access
Hotel Patagónico provides wireless access to all conference attendees.
Wi-Fi : Hotel Patagonico (no password required)
Mobile Phone
Please ensure that all mobile phones are switched off or in silent mode during all
scientific sessions.
Lunch and Dinner
Lunch and Dinner will be served for all registered participants in the Alerce
Restaurant located on the first floor.
8
Location Information
Hotel Patagónico
Contact:
Klenner 349
Puerto Varas, Chile.
reservas@hotelpatagonico.cl
(56 65) 201000
(56 65) 201014
(56 2) 4142013
Check In: 15:00 hrs
Check Out: 12:00 hrs
Floor plan
9
General Services Information
Health:
Hospital “Clínica Alemana”
Address: Calle Dr. Otto Bader 810, Puerto Varas
Phone: 56 65 582100
Police:
Primera Comisaría de Puerto Varas
Address: Calle San Francisco 241, Puerto Varas
Phone: 56 65 765100
10
Program Outline
11
Program
Monday, November 18
12:00 - 19:00
Registration + Poster setup
19:00 - 20:00
Welcome and Keynote Lecture
Mark Stitt (Max Planck Institute, Potsdam-Golm, Germany)
Using nitrogen: protein synthesis and plant growth.
20:00 - 22:00
Reception (cocktail party with drinks and food)
Tuesday, November 19
9:00 - 9:45
Lecture
Yi-Fang Tsay (Institute of Molecular Biology, Academia
Sinica, Taipei, Taiwan.)
Roles of NRT1 transporters in nitrate sensing and allocation.
Session 1
Nitrogen signaling (Rodrigo Gutiérrez)
9:45 – 10:15
Alain Gojon, (CNRS/INRA Montpellier, France)
How can NRT1.1 control so many different responses of
the plant to nitrate?
10:15 – 10:45
Hitoshi Sakakibara (RIKEN Plant Science Center, Japan)
Dual regulation of de novo cytokinin biosynthesis in response
to nitrogen nutrition: the role of glutamine metabolism as an
additional signal.
10:45 – 11:30
Coffee Break
12
Program
11:30 – 12:00
Brian Forde (Lancaster University, UK)
Glutamate signalling in root development: molecular and
chemical genetic approaches to uncovering the pathway.
12:00 – 12:30
Shuichi Yanagisawa (The University of Tokyo, Japan)
Members of the NIN-like protein family are transcription
factors governing nitrate-inducible gene expression.
12:30 – 12:45
José Miguel Alvarez (PUC, Chile)
TGA1 and TGA4 transcription factors modulate nitrate
responses in Arabidopsis thaliana roots.
12:45 – 13:00
Tatsuo Omata (Nagoya University, Japan)
Effects of PII deficiency and the toxicity of PIPX on
growth of the Cyanobacterium synechococcus elongatus.
13:00 – 14:30
Lunch
14:30 – 15:00
Anne Krapp (INRA Centre de Versailles-Grignon, France)
NIN-like proteins: key regulators of plant responses to
nitrogen availability.
Session 2
Ammonium transport and assimilation (Hitoshi
Sakakibara)
15:00 – 15:30
Nicolaus von Wirén (Leibniz Institute of Plant Genetics
and Crop Plant Research, Gatersleben, Germany)
Regulation of ammonium transport and sensing in plant roots.
15:30 – 16:00
Coffee Break
16:00 – 16:30
Antonio Márquez (Universidad de Sevilla, Spain)
Reassimilation of ammonium in Lotus japonicus.
13
Program
16:30 – 16:45
Mitsue Miyao-Tokutomi (National Institute of Agrobiological
Sciences, Japan)
Ammonium assimilation in the root of rice plants:
comparison between water and soil cultures.
16:45 – 17:00
Katrin Fischer-Schrader (University of Cologne, Germany)
Dual binding motifs required in 14-3-3 mediated inhibition
of nitrate reductase.
17:00 – 19:00
Poster session - Even numbers
19:00 – 21:30
Dinner
Wednesday, November 20
9:00 – 9:45
Lecture
Gloria Coruzzi (New York University, USA)
The systems biology approach to NUE: from predictive
network modeling to trait evolution.
Session 3
Nitrate transport and allocation (Tomoyuki Yamaya)
9:45 – 10:15
Françoise Daniel-Vedele (INRA Centre de VersaillesGrignon, France)
Nitrate and nitrite fluxes in plants.
10:15 – 10:45
Francisco Cánovas (Universidad de Málaga, Spain)
Nitrogen metabolism in forest trees.
10:45 – 11:00
Peter Buchner (Rothamstead, UK)
Low affinity nitrate transporters in wheat (a 20:20
wheat® project)
11:00 – 11:30
Coffee Break
14
Program
11:30 – 12:00
Tomoyuki Yamaya (Tohoku University, Japan)
Distinct functions of GS1 and NADH-GOGAT1
isoenzymes in rice.
12:00 – 12:30
Aurora Galván (Universidad de Córdoba, Spain)
Nitrate signaling in Chlamydomonas. The role of
NIT2, NZF1, NRT2 AND NRT1.
12:30 – 12:45
Po-Kai Hsu (Academia Sinica, Taiwan)
NRT1.11 and NRT1.12 are responsible for redistributing
nitrate from mature leaves to young leaves.
12:45 – 13:00
Laurence Lejay (INRA, France)
Post-transcriptional regulation of the root nitrate uptake
transporter NRT2.1 in Arabidopsis thaliana.
13:00 – 14:30
Lunch
Session 4
Genomics and Systems Biology (Gloria Coruzzi)
14:30 – 15:00
Rodrigo A. Gutiérrez (P. Universidad Católica de Chile, Chile)
Nitrogen regulatory networks controlling plant root growth.
15:00 – 15:30
Miyako Kusano (RIKEN Plant Science Center, Japan)
The study of two cytosolic glutamine synthetase isoforms
of rice using reverse genetic, metabolite and transcript
profiling approaches, and microscopic analysis.
15:30 – 16:00
Coffee Break
16:00 – 16:15
Darren Plett (Australian Centre for Plant Functional
Genomics, Australia)
Nitrate responsive transcription in maize is highly dynamic
across the lifecycle.
15
Program
16:15 – 16:30
Virginie Lauvergeat (INRA, France)
Understanding scion development in grapevine: how do
rootstocks contribute to nitrogen uptake and signaling?
16:30 – 19:00
Poster session - Odd numbers
19:00 – 21:30
Dinner
Thursday, November 21
9:00 – 9:45
Lecture
Jen Sheen (Harvard Medical School, USA)
Probing nitrate signaling network.
Session 5
Nitrogen interactions with other nutrients/signals
(Brian Forde)
9:45 – 10:15
Anna Amtmann (University of Glasgow, UK)
A combinatorial input of N, P, K, S and light shapes the
root architecture of Arabidopsis and produces a
quantitative readout of the underlying signaling network
10:15 – 10:45
Toru Fujiwara (The University of Tokyo, Japan)
Molybdenum transport in Arabidopsis thaliana.
10:45 – 11:00
Anna Medici (INRA, France)
Nitrate early regulated (NER) transcription factors link
nitrate and phosphate signaling in the control of root
meristem activity.
11:00 – 11:30
Coffee Break
16
Program
11:30 – 12:00
Gabriel Krouk (CNRS Montepellier, France)
A systems view of nitrogen signalling interactions.
12:00 – 12:30
Guillaume Pilot (Virginia Tech, USA)
Amino acid export and its control by the GDU-LOG2
proteins.
12:30 – 12:45
Aline Matsumura (Sao Paulo University, Brazil)
Nitrate reductase activity control in pineapple plants
subject to low temperatures in different phases of
light/dark cycle.
12:45 – 14:30
Lunch
Session 6
Nitrogen-use efficiency and ecophysiological
aspects of nitrogen nutrition (Alain Gojon).
14:30 – 15:00
Steven Rothstein (University of Guelph, Canada)
Understanding plant response to growth under nitrogen
limitation conditions to improve crop nitrogen.
15:00 – 15:30
Céline Masclaux-Daubresse (INRA Centre de
Versailles-Grignon, France)
Autophagy machinery controls nitrogen remobilization
to the seeds in Arabidopsis thaliana.
15:30 – 15:45
Vanessa Melino (Australian Centre for Plant Functional
Genomics, Australia)
Genotypic diversity for root plasticity and N uptake.
15:45
Coffee Break
– 16:15
17
Program
16:15 – 16:45
Bertrand Hirel (INRA Centre de
Versailles-Grignon, France)
Improving nitrogen use efficiency in crops for
sustainable agriculture.
16:45 – 17:00
Mamoru Okamoto (Australian Centre for Plant Functional
Genomics, Australia)
High-throuput phenotyping of nitrogen response and use
in wheat with lemnatec scanalyzer 3D.
17:00 – 17:15
Maaike de Jong (The Sainsbury Laboratory, UK)
Plasticity in the shoot branching regulatory network.
17:15 – 17:45
Conference Photo (Hotel Terrace)
20:00 – 01:00
Banquet and party
Friday, November 22
9:00 – 9:45
Lecture
Giles Oldroyd (John Innes Centre, UK)
The first step towards engineered nitrogen fixation
in cereals.
Session 7
Nitrogen nutrition in plant and bacterial systems
(Bertrand Hirel).
9:45 – 10:15
Jean-Michel Ané (University of Wisconsin-Madison, USA)
Regulation of the mevalonate pathway by symbiotic
receptor-like kinases and its role in early symbiotic
signaling.
18
Program
10:15 – 10:45
Adriana Hemerly (Universidad Federal do Rio de
Janeiro, Brazil)
Plant signaling during sugarcane colonization with
endophytic nitrogen-fixing bacteria.
10:45 – 11:00
Brent Kaiser (University of Adelaide, Australia)
A membrane localised bHLH transcription factor
involved in legume nodule development and
ammonium transport.
11:00 – 11:30
Coffee Break
11:30 – 11:45
Rejane Pratelli (Virginia Tech, USA)
Screening for amino acid exporters and their
regulators in arabidopsis and soybean.
11:45 – 12:00
Tatiana Kraiser (PUC, Chile)
Molecular mechanisms control functional association
between Arabidopsis thaliana and Sinorhizobium
meliloti bacterium.
12:00 – 13:00
David Fischoff (Monsanto)
Crop Improvement for Nitrogen Use Efficiency:
Challenges and Opportunities.
13:00 -
Closing remarks and departure
19
LECTURES
Lectures
USING NITROGEN: PROTEIN SYNTHESIS AND PLANT GROWTH
Mark Stitt
Max Planck Institute of Molecular Plant Physiology, Golm Germany
Email: mstitt@mpimp-golm.mpg.de
Plants pace their metabolism and growth in the face of a fluctuating environment.
The daily alternation between light and darkness has an especially marked impact
on plants, because photosynthesis is only possible in the light. Metabolism and
growth in the night depend entirely on reserves, like starch, that are accumulated in
the light and remobilised in the dark. Starch turnover is exquisitely regulated by the
clock and metabolic signals, pacing starch breakdown such that the reserves are
almost but not entirely consumed at dawn. This must be accompanied by
coordinated by changes in the growth rate. Growth is usually studied by monitoring
physical size, e.g., leaf expansion. As mature plants cells are highly vacuolated,
expansion is mainly due to water uptake. I will discuss approaches that give
information about the synthesis of cellular components, with a focus on protein
synthesis and protein turnover. I will first show that proet4in synthesis closely tracks
sucrose levels. I will then present a modelling strategy, in which we use quantitative
data on ribosome abundance, polysome loading and transcript abundance to
model the rates of protein synthesis and, additionally, provide insights into protein
turnover and the associated costs. This approach also highlights the importance of
amino acid turnover for the energy budget at night. I will then describe methods to
experimentally validate the model, by supplying 13C-CO2 to intact plants and
analysing the labelling kinetics of metabolic pools and protein to measure the rate of
protein synthesis and protein turnover in intact plants. As examples, I will discuss
how growth is distributed in a highly flexible manner between the day and the night
to optimise ribosome use to the prevailing conditions, and how changes in
ribosome abundance, ribosome usage and protein turnover contribute to
differences in growth rates between Arabidopsis accessions.
21
Lectures
ROLES OF NRT1 TRANSPORTERS IN NITRATE SENSING AND ALLOCATION
Yi-Fang Tsay, Cheng-Hsun Ho, Shan-Hua Lin, Po-Kai Hsu, Hui-Yu Chen
Instiute of Molecular Biology, Academia Sinica, Taipei, Taiwan.
Email:
yftsay@gate.sinica.edu.tw
Nitrate is one of the major nitrogen sources for higher plants. CHL1 (NRT1.1) is a
dual affinity nitrate transporter involved in nitrate uptake. CHL1 also function as a
nitrate sensor to monitor external nitrate changes and then modulate the expression
of nitrate related genes. As a sensor, using dual-affinity binding and
phosphorylation switch, CHL1 can detect a wide range of nitrate concentration
changes and lead to different levels of transcriptional responses. Further study
showed that CHL1 could not only detect concentration changes of nitrate, but also
monitor temporal changes of nitrate. The dynamic interaction between CHL1 and
other signaling components including a protein phosphatase could elicit temporal
changes of nitrate responses.
Nitrate taken into the plants can be assimilated immediately in the root, or
transported to the shoot to be assimilated there. In addition, excess nitrate can be
stored in vacuole for future use. Proper nitrate allocation among different tissues is
important for efficient nitrogen utilization. In Arabidopsis, there are 53 NRT1
transporters. One of the NRT1 transporters NRT1.7 is expressed in the phloem of
older leaves. Phenotype of nrt1.7 mutant indicated that NRT1.7 is involved in
remobilizing stored nitrate from older leaves into younger leaves. Growth retard of
nrt1.7 mutant during N starvation indicated that this remobilization process is
important for plant to sustain vigorous growth at N deficiency. Nevertheless,
another two transporters NRT1.11/1.12, also expressed in phloem, are important for
growth enhanced by high nitrate. NRT1.11/1.12 are responsible for transferring
xylem born nitrate to phloem in the larger leaves to feed younger leaves with high
nutrient demand but low transpiration rate. These studies indicated that under
nitrate sufficient or nitrate deficient condition, plants use different strategies of
nitrate allocation to maximize their growth.
22
Lectures
THE SYSTEMS BIOLOGY APPROACH TO NUE: FROM PREDICTIVE NETWORK
MODELING TO TRAIT EVOLUTION
Gloria M. Coruzzi1, Gabriel Krouk2, Sandrine Ruffel2, Ulises Rosas1, Angelica
Cibrian-Jaramillo3, Daniela Ristova1, Ying Li1, Kranthi Varala1, Amy MarshallColon1, Alessia Para1, Tara Moran1, Nancy Francoeur1, Manpreet Katari1, Miriam
Gifford4, Kenneth D. Birnbaum1, Michael Purugganan1, Dennis E. Shasha5
1New York University , 2BPMP, CNRS/INRA/SupAgro- Montpellier, France,
3LANGEBIO, Mexico, 4School of Life Sciences, Warwick, UK , 5Courant Institute
of Mathematical Sciences, New York University
Email: gc2@nyu.edu
A systems biology approach enables researchers to predict how changes in gene
network states can effect trait improvements such as NUE [1]. A first step towards
this goal is the Arabidopsis multinetwork whose edges connect gene nodes
according to metabolic, protein, and regulatory interactions [2], as embodied in the
software platform “VirtualPlant” and extended to crops (www.virtualplant.org ) [3].
The derived N-regulatory subnetworks uncovered new hypotheses such as the
master clock gene CCA1 as a hub of an organic-N regulated network [4] and
miR-TFs that mediate N-regulation of lateral root growth [5]. Next, using time-series
data, we “learned” N-networks that could accurately predict gene expression states
at future time points, the ultimate goal of systems biology [6]. To enable rapid
validation of TF hubs, we developed a cell-based system TARGET (Transient Assay
Reporting Genome-wide Effects of Transcription factors) [7]. To next explore how
plants respond to nitrogen as an integrated root/shoot system, we exploited a
split-root set-up at the genome-wide level uncovering genes controlling the
“economics” of N-supply and demand [8], and also using whole root responses in
Arabidopsis natural variants [9,10]. To further exploit evolutionary approaches, we
constructed a phylogenomic tree BIGPLANTv1.0 using 22,833 orthologs spanning
150 plant genomes [11]. Queries of BIGPLANTv1.0 using PhyloBrowse
(http://nypg.bio.nyu.edu/bp/ ), reveals genes supporting species divergence,
enabling a novel approach to “trait-to-gene” discovery.
Refs: [1] Krouk (2013) Genome Biol.14:123; [2] Gutierrez (2007) Genome Biol,
8:R7; [3] Katari (2010) Plant Physiol. 152:500; [4] Gutiérrez (2008) PNAS 105:4939;
[5] Gifford (2008) PNAS, 105:803; [6] Krouk (2010) Genome Biol. 11:R123; [7]
Bargmann (2013) Mol Plant PMID: 23335732; [8] Ruffel et al (2011) PNAS
108:18524; [9] Rosas et al (2013) PNAS PMID: 23980140; [10] Gifford et al 2013,
PLoS Gen; [11] Lee et al (2011) PLoS Gen (12):e1002411.
23
Lectures
PROBING NITRATE SIGNALING NETWORK
Kun-hsiang Liu, Matthew McCormack, Jen Sheen
Department of Molecular Biology and Center for Computational and Integrative
Biology Massachusetts General Hospital
Email:
sheen@molbio.mgh.harvard.edu
Nitrate is a central nutrient regulator of gene expression, metabolism, proliferation
and root and shoot growth in plants. It remains challenging to elucidate the molecular mechanisms of nitrate signaling and dissect the genetic basis of myriad nitrateassociated traits in plant growth and development. To broadly explore the molecular
and genetic basis of nitrate-associated plant traits and transcriptional network, we
have initiated integrated genetic and functional genomic screens based on distinct
nitrate-dependent traits and nitrate-inducible reporter and marker genes in Arabidopsis roots and leaves. We will present our characterization of novel mutants with
distinct nitrate insensitive phenotypes in the regulation of root architecture,
transcription and signaling processes.
24
Lectures
THE FIRST STEP TOWARDS ENGINEERED NITROGEN FIXATION IN CEREALS
Giles E D Oldroyd
1Department of Cell and Developmental Biology, John Innes Centre, Norwich
Research Park, Norwich, UK
Email: giles.oldroyd@jic.ac.uk
Sustained crop yields are dependent on fertiliser application, but it comes at a
high price, both in the cost of the fertiliser and the environmental damage that
results from its use. A number of plant species have evolved beneficial interactions
with micro-organisms that facilitate the uptake of nutrients. Legumes form symbiotic
interactions with mycorrhizal fungi that facilitate phosphate uptake and with rhizobial
bacteria that provide the plant with a source of nitrogen. The establishment of these
symbioses involves a molecular communication between the plant and the
symbiotic micro-organisms in the soil. Mycorrhizal fungi and rhizobial bacteria
release signals that are recognised by the host plant and lead to developmental
changes associated with the accommodation of the symbionts. Genetic dissection
in the legume Medicago truncatula has defined the signalling pathways involved in
these symbioses. A number of the genes required for the mycorrhizal interaction
are also necessary for the rhizobial interaction, indicating a conserved symbiosis
signalling pathway. This implies that the evolution of nodulation involved the
recruitment of a signalling pathway already functioning in mycorrhizal signalling.
This signalling pathway is present in most plant species, including cereals
suggesting that engineering the perception of rhizobial bacteria in cereals is
simplified and requires an understanding of the legume specific components that
activate and are activated by the common symbiosis signalling pathway. We are in
the process of engineering this signalling pathway in cereals to promote the
recognition of rhizobial bacteria as the first step in engineering biological nitrogen
fixation into cereal crops.
25
Lectures
CROP IMPROVEMENT FOR NITROGEN USE EFFICIENCY: CHALLENGES
AND OPPORTUNITIES
David Fischhoff
Monsanto Company, St. Louis, Missouri
Email: david.a.fischhoff@monsanto.com
The world’s population is now projected to increase to 9.6 billion by 2050. This
increased population accompanied by dietary shifts based on increasing incomes in
the developing world will double the demand for grain for food and for animal feed;
but, arable land per capita is projected to decrease by 50%. At the same time crop
production in some parts of the world will be challenged by global climate change.
In sum, these pressures dictate that crop productivity will need to see significant
increases in the coming decades. Since nitrogen can be a limiting factor in crop
production, and nitrogen use itself may come under increasing environmental
pressure, in order for agriculture to achieve the needed gains, the improvement of
nitrogen use efficiency in crop plants is a major goal for crop improvement
programs. This target is being approached using the tools of biotechnology and
plant breeding, and also through the development of improved agronomic
practices. Substantial progress has been made in developing biotechnology
products for some traits such as insect resistance and herbicide tolerance in crop
plant such as maize, cotton and soybean. In addition, great progress has been
made in the basic understanding of more complex traits effecting plant growth and
development, including nitrogen metabolism, in model systems such as Arabidopsis
and to some degree in crop plants themselves. However, translating this basic
knowledge into commercially useful biotechnology traits, such as improved nitrogen
use efficiency in maize, has proven more difficult. The processes used for
developing complex biotechnology traits in crop plants, some of the challenges in
this type of genetic engineering, and possibilities for the future will be illustrated. In
addition, prospects for improving nitrogen use efficiency in crops through plant
breeding will be discussed. Improved agronomic practices, such as recent
developments in precision agriculture, also hold promise for improving nitrogen
utilization in crops, and developments in this area will be addressed. Finally,
possibilities from new emerging areas of research such as the plant microbiome will
be described.
26
SESSIONS
SESSION 1
NITROGEN SIGNALING
Chair: Rodrigo Gutiérrez
Session 1
HOW CAN NRT1.1 CONTROL SO MANY DIFFERENT RESPONSES OF
THE PLANT TO NITRATE?
Alain Gojon, Eleonore Bouguyon, Philippe Nacry, François Brun, Gabriel Krouk,
Marjorie Pervent, Benoît Lacombe, Sophie Léran
B&PMP, CNRS/INRA/SupAgro/UM2, Montpellier France
Email: alain.gojon@supagro.inra.fr
The nitrate transporter NRT1.1 has been proposed to have a dual
transport/sensing function, acting as a transceptor protein. An increasing list of plant
responses to nitrate has been reported to be under the control of NRT1.1. These
include: short-term (within minutes) or long-term (within days) regulation of nitrogen
transport and metabolism, modulation of germination, adaptation of root
development and growth, control of shoot growth, etc?The mechanisms involved
are largely unknown, with the exception of the NRT1.1-dependent regulation of
lateral root development, which relies on nitrate-regulated auxin transport facilitation
by NRT1.1 in lateral root primordia. Structure/function analysis indicates that several
of the responses to nitrate triggered by NRT1.1 can be uncoupled by point
mutations in the protein, suggesting the occurrence of independent signal
transduction mechanisms for these responses. The picture emerges that NRT1.1
plays a highly versatile role in nitrate signaling, with different forms of the protein in
charge of specific signaling responses, depending on the experimental conditions
or the tissues. Furthermore, new regulatory mechanisms were found that control the
expression of the NRT1.1 protein in specific tissues, in accordance with its
particular signaling function in these tissues.
29
Session 1
DUAL REGULATION OF DE NOVO CYTOKININ BIOSYNTHESIS IN
RESPONSE TO NITROGEN NUTRITION: THE ROLE OF GLUTAMINE
METABOLISM AS AN ADDITIONAL SIGNAL
Hitoshi Sakakibara, Tomoe Kamada-Nobusada, Takatoshi Kiba
RIKEN Center for Sustainable Resource Science
Email: sakaki@riken.jp
Cytokinin, a phytohormone, plays an important role in plant growth and
development, and its activity is finely controlled by environmental factors in
morphological and metabolic optimization. Recent studies have revealed that
cytokinin is a signaling molecule involved in the propagation of nitrogen signals
throughout the whole plant body for an integrated network of intracellular and
inter-organ signaling of nitrogen availability. We previously identified that an
Arabidopsis gene for adenosine phosphate-isopentenyltransferase (IPT) AtIPT3 is
regulated by nitrogen source in nitrate specific manner. In addition to the system,
we have recently identified existence of another regulation system of cytokinin de
novo biosynthesis in response to nitrogen status. In rice, OsIPT4, OsIPT5, OsIPT7,
and OsIPT8 are up-regulated by exogenously applied nitrate and ammonium.
L-methionine sulfoximine, a potent inhibitor of glutamine synthetase, abolished the
nitrate- and ammonium-dependent induction of OsIPT4 and OsIPT5, while
glutamine application induced their expression. Thus, neither nitrate nor ammonium
but glutamine or a related metabolite is essential for the induction of these IPTs in
rice. On the other hand, glutamine-dependent induction of IPT3 occurs in
Arabidopsis, at least to some extent. In transgenic lines repressing the expression of
OsIPT4, which is the dominant IPT in rice roots, the nitrogen-dependent increase of
cytokinin in the xylem sap was significantly reduced, and seedling shoot growth
was retarded despite sufficient nitrogen. We conclude that plants possess multiple
regulation systems for nitrogen-dependent cytokinin biosynthesis to modulate
growth in response to nitrogen availability. We will show our recent data and
discuss the physiological significance of the dual regulation system.
30
Session 1
ARABIDOPSIS PLASTID AMOS1/EGY1 INTEGRATES ABA
SIGNALING TO REGULATE GLOBAL GENE EXPRESSION RESPONSE
TO AMMONIUM STRESS
Baohai B Li1, Qing Q Li1, Liming Li Xiong2, Herbert J Kronzucker3, Ute U Krämer4,
Weiming W Shi5.
1State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science,
Chinese Academy of Sciences, 2Plant Stress Genomics Research Center, King
Abdullah University of Science and Technology , 3Department of Biological
Sciences, University of Toronto, 4Department of Plant Physiology, Ruhr University
Bochum, 5State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil
Science, Chinese Academy of Sciences
Email:
wmshi@issas.ac.cn
Ammonium (NH4+) is a key intermediate of nitrogen (N) metabolism in most living
organisms. However, sensitivity to NH4+ widely occurs in animals, plants, and
microorganisms. Extensive studies of the underlying mechanisms of NH4+ toxicity
have been reported in plants, however, the expression and regulation of
NH4+-responsive genes are only marginally recognized. In this study, we identified
a novel ammonium-overly-sensitive 1 (amos1) mutant in Arabidopsis thaliana that
displays severe chlorosis but normal root development under moderate NH4+
stress. Map-based cloning shows amos1 to carry a mutation in a plastid
metalloprotease EGY1. We establish a previously unrecognized molecular
connection between the function of plastid AMOS1/EGY1 and the activation of
NH4+-responsive genes in the nucleus, which is required for NH4+ tolerance in
Arabidopsis.
Furthermore, we show ABA signaling acts as an important downstream
component of the AMOS1/EGY1-dependent plastid retrograde signaling pathway to
regulate the expression of NH4+-stress responsive genes, and to enhance
chloroplast functionality under NH4+ stress.
Additionally, H2O2 in chloroplasts is proposed to constitute an
AMOS1/EGY1-dependent plastid retrograde signal. The findings provide novel
insight into the transcriptional regulation of plant acclimation to NH4+ stress, which
integrates plastid retrograde signaling and the ABA signaling pathway.
31
Session 1
GLUTAMATE SIGNALLING IN PLANTS: TOWARDS A BETTER
UNDERSTANDING THROUGH MOLECULAR AND CHEMICAL GENETICS
Brian G Forde
Lancaster University
Email:
b.g.forde@lancaster.ac.uk
Previous studies showed that external glutamate (L-Glu), perceived at the primary
root tip, is able to trigger major changes in Arabidopsis root architecture. Although
plants possess a family of glutamate receptor-like (GLR) genes related to ionotropic
glutamate receptors in animals, up to now it has been unclear how an external
L-Glu signal is transduced to a downstream physiological or developmental
response in plants. A systematic screen of T-DNA insertion mutants affecting the 20
Arabidopsis GLR genes and has identified only one whose disruption affects the
sensitivity of root growth to L-Glu. In a chemical genetics approach to dissecting the
L-Glu signalling pathway, we screened >1500 small molecules bioactive in yeast
and identified two structurally unrelated groups of molecules able to antagonise
L-Glu’s effect on root growth and branching. Evidence that one of these small
molecules targets the evolutionarily conserved Ste11 MAP3K in yeast led to the
identification of the Arabidopsis MEKK1 gene as a key component of the L-Glu
signalling pathway that modulates root architecture. These results provide the first
genetic evidence for the existence in plants of an L-Glu signalling pathway
analogous to that found in animals.
32
Session 1
MEMBERS OF THE NIN-LIKE PROTEIN FAMILY ARE TRANSCRIPTION
FACTORS GOVERNING NITRATE-INDUCIBLE GENE EXPRESSION
Shuichi Yanagisawa
Biotechnology Research Center, The University of Tokyo, Tokyo 113-8657, Japan
Email: asyanagi@mail.ecc.u-tokyo.ac.jp
In land plants, nitrate, a major nitrogen source, functions as a signaling molecule
that modulates the expression of a wide range of genes and that regulates growth
and development. Although such a critical role of nitrate has been established for
decades, the molecular mechanism for nitrate-regulated gene expression has
remained elusive. To reveal this mechanism, we identified key transcription factors
meditating nitrate signals by yeast one-hybrid screening using the
nitrate-responsive cis-element (NRE) identified in the nitrite reductase gene (NIR1)
promoter. Consequently, members of the NIN-LIKE PROTEIN (NLP) family were
found to be DNA-binding proteins that interact with the NRE in the NIR1 promoter
and an NRE-like sequence at the locus for a nitrate reductase gene (NIA1). We also
found that the proteins activate NRE-dependent transcription and that the region
N-terminal to the RWP-RK DNA domain contains transcriptional activation and
nitrate-responsive domains. Furthermore, the suppression of NLP function impaired
the nitrate-inducible expression of a number of genes, including genes involved in
nitrate assimilation and putative transcription factor genes. Thus, we propose that
NLPs are transcription factors with a central role in nitrate-responsive gene
expression. Recently, we also found that NODULE INCEPTION (NIN), a key
regulator of symbiotic nitrogen in legumes, could bind to the NRE and activate
transcription from an NRE-containing promoter and that the N-terminal region of
NIN, which is homologous to those of NLPs, did not respond to nitrate signaling.
Based on this finding, we speculate that NIN is a variant of NLPs which lost
nitrate-responsiveness by mutations in its N-terminal region and has evolved into a
transcription factor that is specifically involved in nodulation.
33
Session 1
TGA1 AND TGA4 TRANSCRIPTION FACTORS MODULATE NITRATE
RESPONSES IN ARABIDOPSIS THALIANA ROOTS
Jose M Alvarez1, Eleodoro J Riveras1, Diana E Gras1, Elena A Vidal1, Orlando L
Contreras-Lopez1, Karem P Tamayo1, Maria I Gomez1, Sandrine Ruffel2, Laurence
Lejay2, Xavier Jordana1, Rodrigo A Gutiérrez1.
1Pontificia Universidad Catolica de Chile , 2Institut de Biologie Intégrative des
Plantes
Email: jmalvarh@gmail.com
Nitrogen (N) nutrient and metabolites regulate plant growth and development and
act as potent signals to control gene expression in Arabidopsis. Using an integrative
bioinformatics approach we identified TGA1 and TGA4 as putative regulatory
factors that mediate N responses in Arabidopsis thaliana roots. We showed that
both TGA1 and TGA4 mRNAs accumulate strongly after nitrate treatments in root
organs. Phenotypic analysis of tga1 and tga4 double mutant plants indicated that
TGA1 and TGA4 are necessary for both primary and lateral root growth in a nitrate
dependent manner. Global gene expression analyses revealed that 97% of the
genes with altered expression in the tga1/tga4 double mutants are regulated by
nitrate treatments indicating these transcription factors have a specific role in nitrate
responses in Arabidopsis roots. Among the nitrate-responsive genes that depend
on TGA1/TGA4 for normal regulation of gene expression, we found the nitrate
transporters NRT2.1 and NRT2.2. Specific binding of TGA1 to its cognate DNA
sequence on the target gene promoters was confirmed by chromatin
immunoprecipitation assays. These results identify TGA1 and TGA4 as important
regulatory factors of the nitrate response in Arabidopsis roots.
34
Session 1
EFFECTS OF PII DEFICIENCY AND THE TOXICITY OF PIPX ON GROWTH
OF THE CYANOBACTERIUM SYNECHOCOCCUS ELONGATUS
Tatsuo Omata1, Yajun Chang1, Nobuyuki Takatani1, Kazuma Uesaka1, Ihara
Kunio1, Shin-ichi Maeda1, Makiko Aichi2
1Nagoya University and JST-CREST, 2Chubu University and JST-CREST
Email:
omata@agr.nagoya-u.ac.jp
The PII protein is conserved in bacteria, archaea and plants, playing key roles in
regulation of nitrogen assimilation. In cyanobacteria, PII is required for
ammonium-promoted inactivation of ABC-type NRT and NR and activation of
N-acetyl-L-glutamate kinase. It has also been shown to regulate PipX, a
transcriptional coactivator of the NtcA regulon encoding a variety of proteins related
to nitrogen assimilation. Because all the PII-less mutants thus far constructed from
the unicellular cyanobcterium Synechococcus elongatus carrry spontaneous
mutations in pipX, regulation of PipX is supposed to be an essential function of PII,
attentuating "toxic effects" of PipX on cell growth. The toxicity of PipX, however, has
not been clearly defined because of the lack of PII-deficient mutants carrying
wild-type pipX. In this study, we developed a reliable method to construct a PII-less
mutant of S. elongatus without a pipX mutation and determined the contribution of
PipX to the detrimental effects of PII deficiency. Growth defects of the mutant were
severe under the nitrogen-replete conditions (i.e., in the presence of ammonium),
but were apparent also under the nitrogen-limited conditions. The growth
impairment observed under the nitrogen-limited conditions was ascribed to the
toxicity of PipX. The prominent phenotypes observed under the nitrogen-replete
conditions (e.g. reduced pigmentation and death of most of the cells after transfer to
ammonium-containing media) were also ascribed to PipX. However, inactivation of
pipX only partially rescued the growth defect in the presence of ammonium,
indicating the presence of a yet unknown PII function(s) required for normal growth.
Effects of ammonium addition on the nitrite uptake activity of the PII-less mutant
revealed a new function for PII in regulation of the activity of the bispecific ABC-type
cyanate and nitrite transporter. Refefence: Chang Y. et al.[2013] Plant Cell Physiol.
published on line. doi:10.1093/pcp/pct092?
35
SESSION 2
AMMONIUM TRANSPORT
AND ASSIMILATION
Chair: Tomoyuki Yamaya
Session 2
DISTINCT FUNCTIONS OF GS1 AND NADH-GOGAT1 ISOENZYMES IN RICE
Tomoyuki Yamaya
Graduate School of Agricultural Science, Tohoku University
Email: tyamaya@biochem.tohoku.ac.jp
Under anaerobic conditions in paddy field where rice plants are cultivated,
ammonium is a major form of available inorganic nitrogen. Most of the ammonium
taken up by the roots is assimilated within the roots by glutamine synthetase (GS)
and glutamate synthase (GOGAT). In the top part of rice, approximately 80% of the
total nitrogen in the panicle arise from remobilization through the phloem from
senescing organs. The major forms of nitrogen in the phloem sap are Gln and Asn.
Thus, GS/GOGAT is also important in the remobilization processes. Ammonium is
also generatd in plants during photorespiration, secondary metabolism, and
catabolic reactions. In rice, there are three cytosolic GS (GS1) isoenzymes (GS1;1,
GS1;2, and GS1;3) and two NADH-GOGAT isoenzymes (NADH-GOGAT1 and
NADH-GOGAT2), but physiological functions of those enzymes have not clearly
understood. In this talk, evidences supporting discinct functions of those
isoenzymes are described, using reverse genetics approaches. The results indicate
that GS1;2 and NADH-GOGAT1 are important in the primary assimilation of
ammonium in rice roots. GS1;1 and NADH-GOGAT2 are responsible for the
remobilization of leaf nitrogen during senescence. NADH-GOGAT1 is also important
in the reutilization of Gln in sink organs. OsGS1;3 expressed specifically in spikelet,
but function of GS1;3 is not clear yet. None of isoenzyme was able to compensate
for the function of each molecule.
37
Session 2
REGULATION OF AMMONIUM TRANSPORT AND SENSING IN PLANT ROOTS
Nicolaus R.W. von Wirén
IPK Gatersleben
Email: vonwiren@ipk-gatersleben.de
Together with nitrate ammonium represents the most important nitrogen source
for the nutrition of plants. High-affinity uptake of ammonium by plant roots is
mediated by a family of AMMONIUM TRANSPORTER (AMT)-type transport
proteins, in which individual members differ in biochemical properties and cell
type-specific localization to generate an overall transport capacity that is tightly
regulated by nitrogen at different regulatory levels.
Besides acting as a nutrient, external ammonium is perceived by plant roots as a
signal. Ammonium shuts off AMTs by C-terminal phosphorylation leading to
trans-inactivation of neighbouring subunits in a trimeric AMT protein complex. In
addition, ammonium sensing displays at the morphological level. This is based on
the observation that localized ammonium supply enhances lateral root branching, to
which individual AMTs contribute to a different extent. These AMT-dependent
changes in root morphology are reminiscent of ammonium-induced morphological
changes in hyphal structures of yeast and fungi, emphasizing common features in
ammonium signaling.
38
Session 2
REASSIMILATION OF AMMONIUM IN LOTUS JAPONICUS
Antonio J Marquez, Marco Betti, Carmen M Perez-Delgado, Margarita GarciaCalderon, Alfredo Credali, José M Vega
Departamento de Bioquimica Vegetal y Biologia Molecular, Facultad de Quimica,
Calle Profesor Garcia Gonzalez, 1 ; 41012-Sevilla (Spain)
Email:
cabeza@us.es
In this talk we will summarize some of the recent results concerning the analysis
of two metabolic pathways involved in the release of internal sources of ammonium,
extremely important for nitrogen remobilization in plants: on the one hand,
photorespiratory metabolism, and, on the other hand, asparagine breakdown
mediated by aparaginase, both being studied in the model legume Lotus japonicus.
The use of photorespiratory mutants deficient in plastidic glutamine synthetase
(GS2), enabled to investigate the transcriptomics and metabolomic changes
associated to photorespiratory ammonium accumulation in this plant. The results
obtained indicate the existence of a coordinate regulation of genes involved in
photorespiratory metabolism (1). Other types of results will be shown that
emphasize how photorespiratory metabolism affects nodule function in this plant,
particularly in the above mentioned GS-deficient mutants (2). Finally, we describe
the structural and functional basis for the involvement of K+ as a crucial cofactor of
NSE1, the most abundant K+-dependent asparaginase isoform of L. japonicus
plants (3) and how TILLING mutants were used to demonstrate by reverse genetics
the importance of this particular isoform in plant growth and seed production (4).
(1) Pérez-Delgado CM, García-Calderón M, Sánchez DH, Udvardi MK, Kopka J,
Márquez AJ, Betti M (2013) Plant Physiol. doi:10.1104/pp.113.217216.
(2) García-Calderón M, Chiurazzi M, Espuny MR, Márquez AJ (2012) Mol. Plant
Microbe Interact. 25, 211-219.
(3) Credali A, Díaz-Quintana A, García-Calderón M, de la Rosa MA, Márquez AJ,
Vega JM (2011) Planta 234, 109-122.
(4) Credali A, García-Calderón M, Dam S, Perry J, Díaz-Quintana A, Parniske M,
Wang TL, Stougaard J, Vega JM, Márquez AJ (2013) Plant Cell Physiol. 54,
107-118.
Acknowledgements: Project P10-CVI-6368 and BIO-163 (Junta de Andalucía,
Spain) and PIF fellowship to CMP.
39
Session 2
AMMONIUM ASSIMILATION IN THE ROOT OF RICE PLANTS:
COMPARISON BETWEEN WATER AND SOIL CULTURES
Shin-Ichi Miyazawa, Masae Konno, Masayuki Muramatsu, Mitsue Miyao
National Institute of Agrobiological Sciences (NIAS)
Email:
mmiyao@affrc.go.jp
Rice plants preferentially use ammonium as the nitrogen source, and thrive
better with ammonium than nitrate. It is considered that ammonium is assimilated
in the root and that the resultant assimilation products are transported to the shoot
in forms of Gln and Asn. Amino acids transported to the shoot were reexamined by
analyzing the content and the composition of free amino acids of the root, the shoot
xylem sap and the leaf blade of rice plants (cv. Nipponbare) grown hydroponically
and in soil. When hydroponically grown plants were transferred to a fresh culture
solution containing 2 mM ammonium after the onset of the day period (8:30), Gln
was most abundant in both the root and the xylem sap, accounting for around 90%
of the total amino acids. Its concentration in the xylem sap reached about 3 mM
before the end of the day period (17:00). The Gln levels showed similar changes in
the root, the xylem sap and the leaf blade, increasing during the day period and
decreasing during the subsequent night period, an indication that Gln was the major
nitrogen assimilate transported to the shoot. The levels of Asn were much below
those of Gln, 4% and 7% of the total in the root and the xylem sap, respectively, at
17:00. When plants were grown in soil, which had been supplemented with
compound fertilizer with ammonium sulfate as the nitrogen source (0.3 g N per
plant), Ala and Ser as well as Gln and Asn were abundant in the xylem sap, and they
accounted for 43, 12, 19, and 9% of the total, respectively, at noon. These results
indicated that the nitrogen assimilates transported to the shoot largely differed
between water and soil cultures, and that Ala is the major assimilate in the rice root
of soil culture.
40
Session 2
DUAL BINDING MOTIFS REQUIRED IN 14-3-3 MEDIATED INHIBITION
OF NITRATE REDUCTASE
Jen-Chih Chi, Guenter Schwarz, Katrin Fischer-Schrader.
Institute for Biochemistry, University of Cologne, Germany
Email:
k.schrader@uni-koeln.de
Regulation of nitrate reductase (NR) activity is fundamental for the survival of
plants, as nitrate reduction is interlaced with the complex network of C- and
N-metabolism. Upon environmental changes, fast inhibition of NR is required in
order to suppress the accumulation of nitrite, which is toxic to the plant upon
persistent exposure. The fastest mode of NR inhibition was identified almost 20
years ago and involves binding of a 14-3-3 protein to a conserved phospho-serine
motif (Ser534 in Arabidopsis NIA2) located in hinge 1 between the N-terminal
molybdenum and the central heme domain [1]. Despite elucidation and detailed
characterization of this well-established binding mode, 14-3-3 mediated inhibition of
plant NR remained incomplete, with approximately 20 % residual activity [2,3]. Here
we report an additional 14-3-3 binding site located within the N-terminus of NR, that
has a novel and atypical motif for 14-3-3 binding. Activity, inhibition and binding
studies of the N-terminal NR fragment [4] and its variants confirmed that (i) this novel
site binds to 14-3-3 independently from the already known phospho-Ser534 site in
hinge 1, (ii) the binding of this N-terminal site to 14-3-3 has a lower affinity than the
well-known phospho-site, (iii) the N-terminal motif interacts with 14-3-3 outside the
typical binding groove for phosphorylated targets, and (iv) the novel 14-3-3 binding
site contributes to 50 % of total NR inhibition at saturating 14-3-3 concentrations
leading to full and complete inhibition of NR activity. The involvement of both
binding sites in full NR inhibition allows higher plants a better fine-tuning of nitrate
reduction over a wide and dynamic activity range.
[1] MacKintosh, C, Douglas P, Lillo C, Plant Physiology, 1995, 107(2), 451-457.
[2] Athwal GS, Huber SC, Plant Journal, 2002, 29, 119-129.
[3] Lambeck et al, Biochemistry, 2010, 49(37), 8177-8186.
[4] Lambeck et al, J Biol Chem, 2012, 287(7), 4562-4571.
41
SESSION 3
NITRATE TRANSPORT AND
ALLOCATION
Chair: Hitoshi Sakakibara
Session 3
NITRATE AND NITRITE FLUXES IN PLANTS
Laure David, Julie Dechorgnat, Thomas Girin, Sylvie Ferrario-Mery, Anne Krapp,
Françoise Daniel-Vedele
INRA
Email:
francoise.vedele@versailles.inra.fr
Nitrate is an essential element for plant growth, both as primary nutrient and as
signaling molecule for plant development and metabolism. Many structural genes
have been already identified playing a role in nitrate uptake, reduction and
assimilation. During the last decade, research objectives have focused on the
characterization of transport processes, from the soil into the plant, between plant
organs and between cellular compartments. All these fluxes require membrane
transporters belonging very often to multigenic families and their activities must be
tightly coordinated at the plant level but also at the cellular level as well. To
comprehensively decipher these complex interactions, we are studying each of the
seven members of the Arabidopsis NRT2 family, which codes for putative high
affinity nitrate transporters. By using expression pattern analyses in response to
environmental conditions or organ specificity, sub-cellular protein localization and
phenotypic analyses of de-regulated genotypes (mutants or overexpressors), we try
to assign a role for each of the 7 NRT2 genes in the whole plant. Surprisingly, some
of them seem to play a direct or indirect role in non-related pathways such as
responses to biotic stresses or phenolic compound metabolism. Because very little
is known on transport processes that occur after nitrate reduction, namely nitrite
transport into the chloroplast, we are focusing on NRT1 genes that are putatively
involved in this process and their interaction with the PII protein, a chloroplast
regulatory protein. Finally and because cereals are a major source of carbohydrates
and proteins for humans, we initiated a study the molecular basis of nitrate transport
in Brachypodium distachyon, a model species for studying wheat nitrogen
pathways.
43
Session 3
NITROGEN METABOLISM IN FOREST TREES
Francisco Canovas, Concepcion Avila, Fernando N. de la Torre, Rafael Cañas,
Belen Pascual.
Universidad de Malaga
Email:
canovas@uma.es
Forests are essential components of the ecosystems covering approximately
one-third of the Earth’s land area and playing a fundamental role in the regulation of
terrestrial carbon sinks. Forest trees are also of significant economic importance, as
they are used for timber and paper production worldwide. A sustainable
management of forest resources is needed to preserve natural forests and to meet
the increasing international demands in the production of wood and other
forest-derived products. New advances and developments in biotechnology will
contribute to accelerate the domestication of important traits for forest productivity.
It is critical to identify the fundamental constraints on forest productivity to
addressing these constraints with modern genomic tools. Nitrogen availability is
extremely low in forest ecosystems, and consequently, forest trees have evolved
adaptive mechanisms and biotic interactions to guarantee the strict economy of this
essential nutrient. Nitrogen assimilation and recycling play a key role in tree growth
and biomass production and we firmly believe that knowledge on nitrogen
metabolism will lead to approaches aimed at increasing forest productivity. In our
laboratory, we are interested in studying nitrogen metabolism and its regulation in
the conifer maritime pine (Pinus pinaster Aiton), a forest tree species of great
economic and ecological importance in the Mediterranean area and a relevant
model for conifer genomic research in Europe. Current research efforts are focused
on improving the understanding of the response of conifer trees to ammonium
availability and the transcriptional control of ammonium assimilation into amino
acids. An overview and update of our research programme will be presented and
discussed. Research supported by Spanish Ministry of Economy and
Competitiveness and Junta de Andalucía (Grants BIO2012-33797, PLE2009-0016
and research group BIO-114).
44
Session 3
LOW AFFINITY NITRATE TRANSPORTERS IN WHEAT
(A 20:20 WHEAT® PROJECT)
Peter Buchner, Malcolm J Hawkesford.
Plant Biology and Crop Science Department, Rothamsted Research, West
Common, Harpenden AL5 2JQ
Email:
peter.buchner@rothamsted.ac.uk
In plants, nitrate transporters are involved in nitrate uptake by the root as well as
transport and distribution of nitrate within the plant. The plant nitrate transporter
gene family may be subdivided in to low affinity NRT1 and high affinity NRT2
families. Although nitrate transporter genes are well characterized in Arabidopsis,
little is known about the gene family structure and the specific functions of nitrate
transporters in wheat. As part of the 20:20 Wheat® project, the nitrate transporter
genes in wheat are being hosphor zed to understand their function in relation to
root nitrate uptake and root structure, and their roles in the distribution of nitrate
within the plant, especially during N-remobilisation at grain N-filling. Nitrate
transporter genes belonging to the low affinity nitrate transporter gene family
homologous to well characterized Arabidopsis NRT1 genes were identified from the
wheat genome indicating much higher complexity of the low affinity NRT1 family in
wheat in comparison to the smaller genomes of Arabidopsis and Brachypodium.
Gene expression analysis indicated a much more complex pattern of wheat NRT1
gene expression as compared to the well hosphor zed Arabidopsis NRT1 genes,
suggesting different and more complex function. Gene expression is partly
regulated by nitrate availability. NRT1 expression pattern similar to genes well
known to be important for N-remobilisation implicates also involvement/importance
of NRT1 transporter during the N-remobilisation from canopy during senescence
important for wheat grain development.
45
Session 3
NIN-LIKE PROTEINS: KEY REGULATORS OF PLANT RESPONSES TO
NITROGEN AVAILABILITY
Chloé Marchive1, François Roudier2, Charlotte Renne1, Yves Texier1, Loren
Castaings1, Virginie Bréhaut1, Camille Chardin1, Eddy Blondet3, Vincent Colot2,
Françoise Daniel-Vedele1, Christian Meyer1, Anne Krapp1.
1Institut Jean-Pierre Bourgin (IJPB), INRA, 2IBENS, ENS, 3URGV, INRA
Email:
anne.krapp@versailles.inra.fr
Nitrogen is an essential macroelement for plant growth. Nitrate, beside its role as
nutrient, acts as a signal molecule for triggering many adaptive responses to
changes in N availability. How such nitrate specific mechanisms are regulated at the
molecular level is poorly understood. We identified NIN-like protein 7 (NLP7), a
member of the RWP-RK family of putative transcription factors, as an important
element involved in the adaptation to N availability. Nlp7 knockout mutants
constitutively display several traits of nitrogen starved plants and NLP7 expression
pattern is consistent with a function in the sensing of N and translational fusions
with the green fluorescent protein (GFP) show a nuclear localization for NLP7.
Indeed, immediately after nitrate exposure, NLP7 accumulates in the nucleus and
binds dozens of genes involved in nitrate signalling and assimilation leading to an
altered response of many nitrate-regulated genes in the nlp7 mutant background.
Altogether, we propose NLP7 as a master regulator of early nitrate signalling.
46
Session 3
NITRATE SIGNALING IN Chlamydomonas. THE ROLE OF NIT2,
NZF1, NRT2 AND NRT1
Aurora Galván1, Jose J Higuera1, Zaira I González1, Jose M Siverio2, Emilio
Fernández1
1Dpto de Bioquímica y Biología Molecular. Campus de Rabanales y Campus,
internacional de Excelencia Agroalimentario (CeiA3). Edif. Severo Ochoa. Universidad de Córdoba, Spain. 2Dpto de Bioquímica y Biología Molecular. Universidad de
la Laguna, Spain.
Email:
bb1gacea@uco.es
Chlamydomonas uses inorganic nitrogen (ammonium, nitrate and nitrite) as
preferred nitrogen sources. Urea, some amino acids and purine and pyrimidine
bases can also be used as alternative nitrogen sources.
Ammonium is a negative signal, which blocks the utilization of other nitrogen
sources, and nitrate is a positive signal that activates the nitrate/nitrite assimilation
route. For nitrate signaling, two factors are essential, 1) the increase of intracellular
nitrate and 2) the presence of a functional NIT2. NIT2 is a transcription factor
RWP-RK type and the major regulatory element for nitrate/nitrite assimilation. NZF1
(Nitrate Zinc Finger 1) is a protein containing three zinc finger motifs in tandem,
CCCH type, which controls NIT2 polyadenylation. NRT2 and NRT1 are nitrate
transporters that control intracellular nitrate.
The integration of these four elements NIT2, NZF1, NRT2 and NRT1 is presented,
as well as that nitrate signal affects the assimilation of alternative nitrogen sources,
such as urea and arginine.
Supported by JA-P08-CVI-042157 and MINECO-BFU2011-29338 (EU FEDER
Program)
47
Session 3
NRT1.11 AND NRT1.12 ARE RESPONSIBLE FOR REDISTRIBUTING NITRATE
FROM MATURE LEAVES TO YOUNG LEAVES
Po-Kai Hsu, Yi-Fang Tsay.
Institute of Molecular Biology, Academia Sinica. Graduate Institute of Life Sciences,
National Defense Medical Center
Email:
pkhsu@gate.sinica.edu.tw
Under many conditions, nitrate is the primary nitrogen source for most plants.
How to deliver nitrate efficiently to all the demanding tissues is important for optimal
plant growth. NRT1.11 and NRT1.12 are two of 53 CHL1 (NRT1.1) homologs in
Arabidopsis. Functional analysis in Xenopus oocytes suggested that NRT1.11 and
NRT1.12 are low-affinity nitrate transporters. The expression levels of NRT1.11 and
NRT1.12 are higher in larger expanded leaves. The transient expression of
GFP-fusion protein in mesophyll protoplasts showed that NRT1.11 and NRT1.12
are localized in plasma
membranes. Promoter-GUS studies revealed that NRT1.11 and NRT1.12 are
mainly expressed in the phloem of the major vein. Analysis of GFP-fusion protein
driven by the native promoter further confirmed the expression of the two proteins
in companion cells. Compared with wild type, more root-fed 15N-labeled nitrate
was translocated to mature leaves of nrtr1.11 nrt1.12 double mutants, but less was
translocated to younger leaves. Moreover, the enhancement of young leaf growth
following an increase in the nitrate supply was defective in nrt1.11 nrt1.12 double
mutants. These results indicate that NRT1.11 and NRT1.12 participate in
transferring nitrate from xylem-to-phloem in the major veins of mature leaves for
redistributing nitrate to low-transpiration young leaves, and this process is critical for
high-nitrate enhanced plant growth. The study of NRT1.11 and NRT1.12 suggests
that in addition to regulation of nitrate uptake and assimilation, regulation of leaf
nitrate redistribution is another strategy for plants to modulate nitrogen use
efficiency.
48
Session 3
POST-TRANSCRIPTIONAL REGULATION OF THE ROOT NITRATE UPTAKE
TRANSPORTER NRT2.1 IN ARABIDOPSIS THALIANA
Adeline Mauries1, Edith Laugier2, Eleonore Bouguyon1, Sonia Hem1, Valerie
Rofidal1, Pascal Tillard1, Veronique Santoni1, Michel Rossignol1, Alain Gojon1,
Laurence Lejay1.
1INRA , 2CNRS
Email:
lejay@supagro.inra.fr
In Arabidopsis the NRT2.1 gene encodes a main component of the root
high-affinity nitrate uptake system (HATS). Due to the strong correlation generally
found between high-affinity root NO3- influx and NRT2.1 mRNA level, it has been
postulated that transcriptional regulation of NRT2.1 is a key mechanism for
modulation of the HATS activity. However, this hypothesis has never been
demonstrated and is challenged by studies suggesting the occurrence of
post-transcriptional regulation at the NRT2.1 protein level. Over the past few years,
we combined different approach to study the regulation of NRT2.1 at the protein
level. Using an immunological approach we showed that the abundance of NRT2.1
protein in the plasma membrane is only slowly affected in response to light, sugars
and high nitrogen supply, whereas much faster changes in NRT2.1 mRNA or NO3HATS activity have been demonstrated. Furthermore, the constitutive expression of
NRT2.1 under the control of a 35S promoter did not prevent HATS activity in the
roots to be down regulated in response to repressive N or dark treatments that
strongly reduce NRT2.1 transcription and NO3- HATS activity in the wild type. In
some treatments, this was associated with a decline of NRT2.1 protein abundance,
indicating posttranscriptional regulation of NRT2.1. However, in other instances,
NRT2.1 protein level remained constant. Changes in abundance of NAR2.1, closely
followed those of NRT2.1, and thus could not explain the close-to-normal regulation
of the HATS in the 35S::NRT2.1 transformants. These results confirmed that
post-translational regulatory mechanisms are involved to control NRT2.1 activity.
More recently, we started a phosphoproteomic approach combined with
hosphor-peptides quantification using MRM. Unpublished data will be presented
concerning the characterization of the role of one phosphorylation site in the
regulation of NRT2.1 activity by NO3-.
49
SESSION 4
GENOMICS AND SYSTEMS
BIOLOGY
Chair: Gloria Coruzzi
Session 4
SHARING THE COMPONENTS OF MOLECULAR SIGNALING PATHWAYS OF
NITROGEN AND PHOSPHORUS NUTRITION IN RICE
Guohua Xu
State Key laboratory of crop genetics and germplasm enhancement, College of
Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing,
210095, China
Email: ghxu@njau.edu.cn
Nitrogen (N) ans phosphorus (P) are two most limiting nutrientes for plant
production worlwide. N- and P-starved plants share some common features in their
phenotypes from the growth of roots and shoots to the development of
reproductive organs. We observed in rice that either N- or P-deficiency could
induce exudation of strigolactones (SLs) in roots, and decrease the uptake and
translocation of both N and P from roots to shoots. In addition, supply of
ammonium in comparison to nitrate could improve Pi uptake rate and translocation
from roots to shoots. Enhancing the ability to utilize nitrate for balancing ammonium
nutrition in paddy field could increase yield N use efficiency in rice. Several genes
associated with SL synthesis and signaling, encoding the plasma membrane
H+-ATPase
51
Session 4
NITROGEN REGULATORY NETWORKS CONTROLLING PLANT ROOT GROWTH
Rodrigo A. Gutiérrez
Department of Molecular Genetics and Microbiology. Pontificia Universidad
Católica de Chile. Santiago, Chile.
Email : rgutierrez@bio.puc.cl
Nitrogen (N)–based fertilizers increase agricultural productivity but have
detrimental effects on the environment and human health. Research is generating
improved understanding of the signaling components plants use to sense N and
regulate metabolism, physiology, and growth and development. However, we still
need to integrate these regulatory factors into signal transduction pathways and
connect them to downstream response pathways. We used systems approaches
to identify gene regulatory networks involved in N responses using Arabidopsis
thaliana as a plant model system. Using next generation sequencing, microarray
technologies and integrative network bioinformatics tools we are dissecting
nitrate-regulatory networks controlling root growth. We will discuss our current
experimental efforts towards mapping gene networks leading to nitrate induced
changes in root system architecture. We will also discuss new bioinformatics tools
to identify new components of the nitrogen response in Arabidopsis thaliana.
Systems biology approaches are accelerating the identification of new components
and N-regulatory networks linked to other plant processes. A holistic view of plant N
nutrition should open avenues to translate this knowledge into effective strategies to
improve N-use efficiency and enhance crop production systems for more
sustainable agricultural practices.
52
Session 4
THE STUDY OF TWO CYTOSOLIC GLUTAMINE SYNTHETASE ISOFORMS OF
RICE USING REVERSE GENETIC, METABOLITE AND TRANSCRIPT PROFILING
APPROACHES, AND MICROSCOPIC ANALYSIS
Miyako Kusano12, Atsushi Fukushima1, Kazuhiro Funayama3, Mayumi TabuchiKobayashi3, Tomoko Nishizawa1, Makoto Kobayashi1, Mayumi Wakazaki1,
Mayuko Sato1, Kiminori Toyooka1, Kumiko Osanai-Kondo1, Yoshinori Utsumi1,
Motoaki Seki1, Soichi Kojima3, Tomoyuki Yamaya3, Kazuki Saito14
1RIKEN CSRS, 2Yokohama City University, 3Tohoku University, 4Chiba University
Email:
miyako.kusano@riken.jp
Nitrogen (N) is one of the critical limiting elements because it provides N atom to
synthesize important components such as chloroplast and amino acids for plant
growth. Cytosolic Glutamine synthetase (GS) can assimilate ammonium from
different sources for both primary nitrogen assimilation and recycling. In this study,
we aim to characterize functions of two cytosolic GS isoforms, i.e., GS1;1 and
GS1;2, in rice seedlings. Rice is an useful model plant for the internal N signal via
GS, because ice plants express only the three GS genes (GS1;1, GS1;2 and GS2) at
vegetative stages and can grow healthy in the presence of sufficient ammonium as
sole N source. We conducted metabolite and transcript profiling of single knockout
mutants of gs1;1 and gs1;2 to investigate the extent and impact toward central
metabolism by lacking these genes in rice seedlings. Metabolite profiling analysis
revealed that gs1;1 plants showed dramatic changes of the levels of amino acids,
carbohydrates and their derivatives in both roots and shoots, while there were
significant decrease in the levels of amino acids in gs1;2 roots. Transcript profiles of
gs1;1 roots displayed significant up-regulation of photosynthesis-related genes,
though the profiles of aerial parts of gs1;1 samples as well as gs1;2 samples did not
show such changes. Detailed investigation of transcript data and real-time
quantitative reverse transcription polymerase chain reaction analysis revealed the
up-regulation of two transcription factor genes encoding Golden2-like (GLK) in
gs1;1 roots, that have tight coordination for induction of chloroplast development.
Indeed, microscope and pulse amplitude modulation analyses captured
photosynthetically active chloroplast development in gs1;1 roots, though there were
no such chloroplasts in gs1;2 and WT roots. The distinct functions of GS1;1 and
GS1;2 through proper supply of internal glutamine from each GS in rice roots will be
presented and discussed.
53
Session 4
NITRATE RESPONSIVE TRANSCRIPTION IN MAIZE IS HIGHLY DYNAMIC
ACROSS THE LIFECYCLE
Darren Plett1, Ute Baumann1, Andreas Schreiber2, Luke Holtham1, Elena Kalashyan1, John
Toubia1, Antoni Rafalski3, Mary Beatty4, Kanwarpal Dhugga4, Mark Tester5, Brent N Kaiser6,
Trevor Garnett1.
1Australian Centre for Plant Functional Genomics, Waite Research Institute, University of Adelaide,
Adelaide, South Australia, 5064, AUSTRALIA, 2Centre for Cancer Biology, Frome Road, Adelaide
SA 5000, AUSTRALIA, 3 DuPont Crop Genetics, Wilmington, Delaware, 19803, USA, 4PioneerHiBred, Johnston, Iowa, 50131, USA, 5Division of Biological and Environmental Sciences and
Engineering, 4700 King Abdullah University of Science and Technology, Thuwal 23955-6900,
SAUDI ARABIA, 6School of Agriculture, Food and Wine, Waite Research Institute, University of
Adelaide, Adelaide, South Australia, 5064, AUSTRALI
Email:
darren.plett@acpfg.com.au
Nitrate (NO3-) is the predominant form of inorganic nitrogen (N) accessed by crop
plants in an agricultural system. To understand how to modify crop plants to
maximise N uptake we characterised the transcriptional response of maize to low N.
To approximate steady-state N supply and demand, which cereal crops experience
in the field, we grew dwarf maize with sufficient (2.5 mM) and limiting (0.5 mM) NO3provision for the entire lifecycle. Maize plants grown with limiting NO3- were able to
maintain the same biomass and yield as those grown with sufficient NO3- by
increasing NO3- uptake capacity, chiefly through increasing transcription of genes
encoding high affinity NO3- transporters (NRT2). We undertook a transcriptomic
analysis of leaf and root tissue at seven key time points to identify genes responding
to both NO3- provision and N demand changes due to development across the
lifecycle. We found the majority of NO3- responsive transcription occurs at 11
(D11), 18 (D18) and 29 (D29) days after emergence, and primarily in the root at D11
and D29 and in the leaf at D18 (for reference, anthesis occurs on D31). Overall,
there were surprisingly few patterns of differential gene expression over the lifecycle,
but the composition of classes of genes differentially regulated at individual time
points was unique. A second major goal of this analysis was to identify genes
which have similar NO3- responsive transcription to NRT2s, implying their
involvement in regulating NO3- uptake. We identified a cluster of 98 probe sets
which share an expression pattern with the NRT2 genes across the lifecycle. The
cluster is enriched with genes encoding lipid transport proteins and lipid metabolism
enzymes and suggests that lipid-mediated signalling or trafficking may be central to
the control of the NO3- uptake system and NRT2 regulation. Manipulation of these
genes may produce increased N uptake which is a crucial step in improving
nitrogen use efficiency in cereal crops.
54
Session 4
UNDERSTANDING SCION DEVELOPMENT IN GRAPEVINE: HOW DO
ROOTSTOCKS CONTRIBUTE TO NITROGEN UPTAKE AND SIGNALING?
Julien Lecourt, Sarah Cookson, Jean - Pascal Tandonnet, Elodie Claverie, Noé
Cochetel, Nathalie Ollat, Philippe Vivin, Virginie Lauvergeat.
Univ. Bordeaux, ISVV, EGFV, UMR1287, F-33140 Villenave d’Ornon, France.
Email: virginie.lauvergeat@bordeaux.inra.fr
In Europe, grapevine varieties (scions) are grafted on to rootstocks resistant to the
soil-dwellingaphid pest phylloxera. Scion development (vegetative growth,
phenology, berry quality and yield) is strongly dependent on the rootstock genotype.
Wepreviously showed that different scion/rootstock combinations have intrinsic
differences in shoot-toroot biomass ratio in unlimited conditions. Because N
nutrition isa key determinant of biomass allocation in plants, we explored the role of
rootshoot N signalling in grafted grapevine development. In model
herbaceousplants, there is increasing evidence that N controls plant development
through the regulation of the hormonal status and that hormonal signals interplay
withN nutrition. Nitrate, nitrogenous compounds, auxin, cytokinins and microRNA
have been shown to be involved in root-shoot signalling of N status. The
workpresented is devoted to understanding the molecular and physiological
mechanisms involved in the response of grafted plants to different levels of
Navailability. Short and long term physiological and molecular responses to N
supply have been studied in the different scion/rootstockcombinations. Approaches
of genetics, genomics and physiology have been combined and will provide tools to
analyze the geneticdiversity of rootstock N responses. Global transcriptome
analysis has been performed using microarrays. Gene expression hasbeen
correlated with biochemical responses (nitrogenous compounds, mineral and
hormonal contents, photosynthetic rate …). Today, in the context of climate change,
the challenge is to define a rootstock ideotype able toadapt to a low water, and
hence low nitrogen, environment. Understanding root-shoot N signalling in grafted
grapevine is important for the breeding andselection of new rootstock genotypes for
the future.
55
SESSION 5
NITROGEN INTERACTIONS
WITH OTHER
NUTRIENTS/SIGNALS
Chair: Brian Forde
Session 5
A COMBINATORIAL INPUT OF N, P, K, S AND LIGHT SHAPES THE ROOT
ARCHITECTURE OF ARABIDOPSIS AND PRODUCES A QUANTITATIVE
READOUT OF THE UNDERLYING SIGNALING NETWORK
Fabian Kellermeier, Patrick Armengaud, David E Salt and Anna Amtmann
1Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow
G12 8QQ, UK, 2 INRA Centre de Versailles-Grignon, RD10, 78026 Versailles,
France, 3Institute of Biological and Environmental Sciences, College of Life
Sciences and Medicine, University of Aberdeen, Aberdeen AB24 3UU, UK.
Email: anna.amtmann@glasgow.ac.uk
Plant roots translate a multi-factorial input of environmental stimuli into a
multi-factorial developmental output that manifests itself as root system architecture
(RSA). To provide a quantitative basis for unravelling the complex signalling network
that underlies RSA plasticity we have measured 13 RSA parameters of Arabidopsis
thaliana in 32 conditions consisting of binary combinations of sufficient or deficient
supply of nitrate, phosphate, potassium and sulphate and two light regimes.
Analysis of variance showed that each RSA parameter was determined by a distinct
pattern of contributions from individual environmental inputs and their interactions.
These patterns were correlated with distinct signatures of root transcript levels and
shoot nutrient concentrations. Phenotyping selected RSA features in several mutant
lines identified novel synergistic and antagonistic interactions between well-known
molecular components of nitrate and potassium signalling. The findings
demonstrate the usefulness of the generated dataset for understanding how plants
integrate multiple nutritional stimuli into complex developmental programs.
57
Session 5
MOLYBDENUM TRANSPORT IN ARABIDOPSIS THALIANA
Hanuka Fujita1, Toru Fujiwara1.
1Department of Applied Biological Chemistry, Graduate School of Agricultural and
Life Sciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, JAPAN
Email:
atorufu@mail.ecc.u-tokyo.ac.jp
Molybdenum is an essential micronutrient in plants. Mo is essential for the activity
of nitrate reductase and it is known that molybdenum deficiency causes defects in
nitrate assimilation. Proper molybdenum uptake and transport is important for
nitrogen assimilation in plants. Several years ago, we identified the first eukaryotic
molybdate transporter, MOT1, from Arabidopsis thaliana. MOT1 is an high affinity,
secondary active transporter of molybdate required for molybdate uptake into roots.
MOT1 shares similarity with sulfate transporters and it was used to be called
sultr5;2. Lack of MOT1 results in several fold reduction in Mo concentration in roots
and shoots. As Mo requirements are low, it is in our hand difficult to observe
Mo-deficiency phenotype of the wild type plants on solid media, but mot1 mutants
grew poorly on media without Mo supplementation. MOT2, the most closest
ortholog of MOT1, is also an active molybdate transporter and based on the
phenotype of the mutant plants, we concluded that MOT2 functions in root-to-shoot
translocation of Mo. It is also demonstrated that sulfate transporters also transport
molybdate. We examined possible role of sulfate transporters in molybdate
transport in A. thaliana. Among the 12 members of sultry transpoters, sultr1;2 was
found to have a significant contribution to the uptake and translocation of Mo in
plants. We have also constructed transgenic plants that overexpress MOT1 and
MOT2. Overexpression of these transporters altered Mo uptake and distribution.
58
Session 5
NITRATE EARLY REGULATED (NER) TRANSCRIPTION FACTORS LINK
NITRATE AND PHOSPHATE SIGNALING IN THE CONTROL OF ROOT
MERISTEM ACTIVITY.
Anna Medici1, Amy Marshall-Colon2, Elsa Ronzier1, Alain Gojon1, Nigel M
Crawford3, Gloria M Coruzzi2, Gabriel Krouk1
1B&PMP, 2 NYU, 3UCSD,
Email: anna.medici@supagro.inra.fr
In this work we identified two A. thaliana transcription factors, AtNER1 and
AtNER2, whose expression regulation in roots is both early induced by NO3
treatment and regulated in a NO3 dose-dependent manner. Their regulation by NO3
is also affected by NRT1.1 (nitrate sensor) mutation. In order to identify genes
directly controlled by AtNER1, we use the genome wide TARGET approach
(Bargmann et al., 2013). The analysis of the gene cluster directly up-regulated by
AtNER1 in presence of NO3 showed that meristematic and phosphate functions are
the significantly over-represented. We thus tested the root development of the TF
double mutant (ner1-1 ner2-1) on a combination of P and N concentrations. The
results show that the P starvation dependent arrest of the primary root (PR) growth
is lost in the double mutant, in N sufficient conditions. This suggests that AtNER1
and AtNER2 could act as repressors of the meristematic activity in the root apical
meristem (RAM), depending on P and N nutrient signals. The role of these
transcription factors is further supported by the localization of the GFP-fusion of
these proteins in the nucleus of elongating cells of the root meristem. In order to
identify putative cis-regulatory element of AtNER1, we carried out a MEME analysis
of the 500bp promoter regions within the 120 most induced and repressed target
genes and we selected 5 significantly over-represented motifs. The EMSA analysis,
performed first on putative MEME predicted repeated motifs and then on 30bp
fragments of target genes promoters, confirmed in vitro the interaction of AtNER1
with the two distinct Cis-Regulatory-Element. Finally our results converge in a model
where NO3 and Pi are regulators of the same N-responsive transcription factor
triggering the control of meristematic activity.
59
Session 5
A SYSTEMS VIEW OF NITROGEN SIGNALLING INTERACTIONS
Anna Medici1, Daniela Ristova2, Amy Marshal-Colon2, Elsa Ronzier1, Alain Gojon1,
Nigel M Crawford3, Kenneth Birnbaum2, Gloria M Coruzzi2, Gabriel Krouk1.
1Biochimie et physiologie moleculaire des plantes (UMR5004) Montpellier France,
2New York University (NYC, USA), 3 Division of Biological Sciences - UC San
Diego-USA
Email:
gkrouk@gmail.com
A drastic change in plant Nitrogen (N) nutrition results in systematic adaptations
ranging from metabolic to growth changes. Interestingly, experimental evidences
support the idea that it exists dedicated signalling pathways involved in the tuning of
growth in response to nutritional status of the plant. On the other hand, growth can
influence nutrition partly through hormones action. This constitutes a feed-forward
loop that entangles nutrition and growth [concept developped in (Krouk et al.,
2011)]. We aim to get deeper insights into such signalling interactions. To this
purpose, two approaches will be presented. First, genome wide investigations have
been made to understand the effect of combinatorial interactions between nitrogen
and hormone treatments in the control of i) gene expression and ii) root
development. Multi-dimensional networks have been built and functional validations
of the predicted roles for the genes belonging to these networks are currently made.
Second, by studying the genome wide effect of nitrate regulated transcription
factors [TARGET technique; (Bargmann et al., 2013)], we have found potential
connections between nitrate and phosphate signalling in the control of root
meristem activity.
These results demonstrate that N Signalling is not a monolithic pathway but
rather very adaptable to external and internal cues.
Bargmann, B.O., Marshall-Colon, A., Efroni, I., Ruffel, S., Birnbaum, K.D., Coruzzi,
G.M., and Krouk, G. (2013). TARGET: A Transient Transformation System for
Genome-Wide Transcription Factor Target Discovery. Mol Plant 6, 978-980.
Krouk, G., Ruffel, S., Gutierrez, R.A., Gojon, A., Crawford, N.M., Coruzzi, G.M.,
and Lacombe, B. (2011). A framework integrating plant growth with hormones and
nutrients. Trends Plant Sci 16, 178-182.
60
Session 5
AMINO ACID EXPORT AND ITS CONTROL BY THE GDU-LOG2 PROTEINS
Guillaume Pilot
Department of Plant Pathology, Physiology and Weed Science, Virginia Tech, USA
Email: gpilot@vt.edu
Amino acids, well known for their use in protein synthesis, are synthesized from
ammonium and nitrate. They play crucial roles in plant metabolism as storage and
transport forms of reduced nitrogen and as precursors of secondary metabolites
(e.g. alkaloids and flavonoids). Genes involved in amino acid metabolism and the
corresponding pathways are well identified, and have been used various degrees of
success to manipulate amino acid composition in storage organs, an important trait
in a landscape of increased demand for agriculture performance. Much less is
known about the membrane transporters involved in the distribution of amino acids
within the cell and in the plant. About 60 amino acid importers are identified, but the
function of only about a dozen of them is described. In spite of extensive research,
only two amino acid exporters have been characterized so far and they did not bring
much light about the regulation of the export process. The gdu1D mutant, which
over-expresses the GDU1 gene, displays reduced size, secretion of glutamine at the
hydathodes, increased amino acid content in phloem sap, xylem sap and in the
apoplasm, and most importantly increase in amino acid export from cells. GDU1 is
a plant specific one-transmembrane domain protein, unlikely to be a transporter
itself. GDU1 is localized in endosomes and at the plasma membrane in vascular
cells. It interacts with LOG2, a plasma membrane-localized RING finger ubiquitin
ligase, which is necessary for the increase in amino acid export triggered by GDU1
over-expression. GDU1-like proteins (GDU2 to GDU7) also interact with LOG2 and
lead to a Gdu1-similar phenotype when over-expressed, suggesting that they also
participate in the regulation of amino acid export in plant cells. LOG2 and the GDU
proteins likely form protein complexes involved in the regulation of amino acid
exporters, probably via ubiquitination steps. The identity and role of these elusive
exporters is currently under investigation.
61
Session 5
NITRATE REDUCTASE ACTIVITY CONTROL IN PINEAPPLE PLANTS SUBJECT
TO LOW TEMPERATURES IN DIFFERENT PHASES OF LIGHT/DARK CYCLE
Aline Matsumura1, Helenice Mercier1
1Sao Paulo University
Email:
aline.tiemi@gmail.com
Nitrate is the main nitrogen source available to plants and nitrate reductase (NR) is
the enzyme responsible for its reduction to nitrite. Previous works from our group
with pineapple plants cultivated in vitro showed that, under thermoperiod of 28ºC
day/15ºC night, NR activity increased in roots during dark phase compared to
activity in plants grown under constant temperature of 28ºC. According to these
results it was questioned what would be the influence of low temperatures in NR
daily rhythm and if leaves could also be affected. This study aimed to investigate the
effects of low temperature on NR activity of pineapple leaves and roots in different
phases of light/dark cycle. We performed 4 experiments beginning at different times
of the 24-hour cycle (beginning of light phase, middle of light phase, beginning of
dark phase, middle of dark phase). In each experiment, plants were exposed to
10ºC for 6 hours and then transferred to rewarming condition of 25°C. NR activity
was measured immediately after cold exposure period by in vivo and in vitro
methods and every 3 hours during 24 hours in rewarming condition by in vivo
method. Upon rewarming, leaves presented a delay in NR daily rhythm in all
treatments, except when low temperature was applied at the beginning of dark
phase, showing no variation throughout the cycle. In roots, NR activity presented
almost no differences in plants submitted to 10ºC or 25ºC. However, by in vitro
method, NR activity in roots increased when cold stimulus was applied at dark
phase, while in leaves, the increment occurs in both light and dark phases. This
study demonstrated that the temperature of 10ºC affected leaves and roots
differently. Roots showed an increment of NR activity by low temperature
dependent of the dark condition and only immediately after cold exposure, while the
responses of leaves varied according to the phase of the 24-hour cycle in which
they were subjected to 10ºC.
62
SESSION 6
NITROGEN-USE EFFICIENCY
AND ECOPHYSIOLOGICAL
ASPECTS OF NITROGEN
NUTRITION.
Chair: Alain Gojon
Session 6
UNDERSTANDING PLANT RESPONSE TO GROWTH UNDER NITROGEN
LIMITATION CONDITIONS TO IMPROVE CROP NITROGEN USE EFFICIENCY
Steven Rothstein1, Yong-Mei bi1, Surya Kant1, Mingsheng Peng1, Sabrina
Humbert1, Darryl Hudson1.
1Department of Molecular and Cellular Biology, University of Guelph, Canada.
Email:
rothstei@uoguelph.ca
Development of genetic varieties with improved nitrogen use efficiency (NUE) is
important for sustainable crop production. The production of high-yielding crops is
associated with application of large quantities of N fertilizers, which is a major cost
in crop production and also causes serious N pollution. To improve NUE, it is
important to understand how plants respond to growth when the available nitrogen
is the growth-limiting factor. Our approach has been to use a variety of genetic,
physiological and genomic approaches to study this problem using Arabidopsis,
rice and corn as our plant systems. Three approaches will be discussed. In the
first, a genetic approach in Arabidopsis was utilized in an attempt to identify
important genes involved in the response to growth under different nitrogen
conditions. Then this information is used to try to identify improved genetics in
important crop plants. The second apprach involved a functional genomics
assessment in rice in which a number of genes were identified via a transcriptome
analysis of plants grown under different nitrogen conditions and to test these via the
generation of rice lines in which each of these was expressed ectopically. The
resulting lines were then tested for growth under different defined nitrogen
conditions to identify those with enhanced phenotypes. The final approach involved
generating and analyzing transcriptome data from plants grown under various
stress conditions. In particular, we have been interested in combining different
types of abiotic and biotic stress conditions to identify common, unique and
synergistic responses for these different stresses. The long-term goal of this work is
to identify candidate genes for the regulation of specific metabolic pathways
important for stress adaptation and to create improved cultivars.
64
Session 6
AUTOPHAGY MACHINERY CONTROLS NITROGEN REMOBILIZATION TO THE
SEEDS IN ARABIDOPSIS THALIANA
Céline Masclaux-Daubresse1, Anne Guiboileau1, Liliana Avila-Ospina1, Kohki
Yoshimoto1
1INRA UMR1318, Institut Jean-Pierre Bourgin, RD10, F-78000 Versailles, France
Email:
celine.masclaux@versailles.inra.fr
Processes allowing the recycling of organic nitrogen and export to young leaves
and seeds are important determinants of plant yield, especially when plants are
nitrate limited. Because autophagy is induced during leaf ageing and in response to
nitrogen starvation, its role in nitrogen remobilization has been suspected. To
investigate the role of autophagy in nitrogen remobilization, several
autophagy-defective (atg) Arabidopsis mutants were grown under low and high
nitrate supplies and labeled with 15NO3- at vegetative stage in order to determine
15N partitioning in seeds at harvest in a pulse/chase experiment(1). Results showed
that nitrogen remobilization efficiency was significantly lower in all the atg mutants
irrespective to biomass defects, harvest index reduction, leaf senescence
phenotypes and whatever nitrogen conditions(2). It was also observed that atg
mutants accumulate larger amount of ammonium, amino acid and proteins in their
leaves than wild type and are depleted in sugars. Over accumulation of proteins in
atg mutants occurred despite higher endopeptidase and carboxypeptidase
activities can be measured in mutants. The specific over accumulation of the RPS6,
RPL13 ribosomal proteins, catalase and glutamate dehydrogenase proteins, and
the accumulation of peptides putatively identified as degradation products of
Rubisco large subunit and GS2 led us to conclude that incomplete chloroplast
protein degradation results from autophagy defects and that protein degradation
through autophagy might be selective(3).
1. Masclaux-Daubresse C, Chardon F (2011) Exploring nitrogen remobilization
for seed filling using natural variation in Arabidopsis thaliana. J Exp Bot 62:
2131-2142.
2. Guiboileau A et al. (2012) Autophagy machinery controls nitrogen
remobilization at the whole-plant level under both limiting and ample nitrate
conditions in Arabidopsis. New Phytol 194: 732-740.
3. Guiboileau et al. (2013) Physiological and metabolic consequences of
autophagy defisciency for the management of nitrogen and protein resources in
Arabidopsis leaves depending on nitrate availability. New Phytol doi:
10.1111/nph.12307.
65
Session 6
GENOTYPIC DIVERSITY FOR ROOT PLASTICITY AND N UPTAKE
Vanessa J Melino1, Gabriele M Fiene1, Sigrid H Heuer1
1Australian Centre for Plant Functional Genomics
Email:
vanessa.melino@acpfg.com.au
A comparative study of root development of Mexican and Australian wheat
germplasm developed from 1966 to 2005 was undertaken to characterise their
response to nitrogen (N) deficiency. Previous studies comparing wheat germplasm
have shown that N uptake increases in parallel to yield; both traits following the year
of cultivar release (Ortiz-Monasterio et al., 1997; Sadras & Lawson, 2013). We were
interested in assessing how these breeding programs have influenced root
development and their efficiency for N uptake. We further screened these cultivars
for root plasticity, a trait which is beneficial for nutrient foraging in heterogeneous
soils. This genotypic screen for root traits using historic CIMMYT cultivars
(Opata-85, Ciano-79, Jupateco-73 and Inia-66) and modern South Australian
cultivars (Kukri, RAC875 and Gladius) has revealed three major types of seedling
root responses to N. Cultivars Kukri and Inia-66 showed the fastest rate of root
development in N-deficient soils, which was accounted for by an early (6-9 days)
increase in root biomass with increased total length and root surface area (RSA) of
both seminal and lateral roots. The root system of cultivars RAC875 and Opata-85
increased at a slower rate than the first group with a preference to enhance lateral
root length and RSA, rather than seminal roots. The third group, which includes
Gladius and Jupateco-73, were slow to respond to N deficiency, increasing length
and RSA of lateral roots only by 17 days. Despite this distinction of root types in
response to N, a correlation with plant N accumulation was not found. Instead, the
amount of N accumulated in the plant tissue of historic cultivars was equivalent
under both N treatments. In contrast, modern cultivars accumulated significantly
more N when grown in N-sufficient conditions. These findings suggest that
breeding programs have influenced the N uptake system of wheat varieties.
66
Session 6
IMPROVING NITROGEN USE EFFICIENCY IN CROPS FOR
SUSTAINABLE AGRICULTURE
Bertrand J Hirel1, Nardjis N Amiour1, Patrick A Armengaud1, Rafael M Canas2,
Céline Dargel-Graffin1, Gilles M Clément1, Lenaïg N Guillard1, Isabelle N Quillere1,
Thérèse C Terce-Laforgue1
1Institut National de la Recherche Agronomique, 2University of Malaga
Email:
hirel@versailles.inra.fr
In this review, we present the recent developments and future prospects of
improving nitrogen use efficiency (NUE) in crops using various complementary
approaches. These include conventional breeding and molecular genetics, in
addition to alternative farming techniques based on organic nitrogen (N) nutrition.
Whatever the mode of N fertilisation, an increased knowledge of the mechanisms
controlling plant N economy is essential for improving NUE and for reducing
excessive input of fertilisers, while maintaining an acceptable yield and sufficient
profit margin for the farmers. Using plants grown under agronomic conditions, it is
now possible to develop further whole plant agronomic and physiological studies.
These can be combined with gene, protein and metabolite profiling to build up a
comprehensive picture depicting the different steps of N uptake, assimilation and
recycling to produce either biomass in vegetative organs or proteins in storage
organs. We provide a critical overview as to how our understanding of the
agro-ecophysiological, physiological and molecular controls of N assimilation in
crops, under varying environmental conditions, has been improved. We have used
combined approaches, based on agronomic studies, whole plant physiology,
quantitative genetics, forward and reverse genetics and the emerging systems
biology. Long-term sustainability may require a gradual transition from synthetic N
inputs to legume-based crop rotation, including continuous cover cropping
systems, where these may be possible in certain areas of the world, depending on
climatic conditions. Current knowledge and prospects for future agronomic
development and application for breeding crops adapted to lower mineral fertiliser
input and to alternative farming techniques are explored, whilst taking into account
the constraints of both the current world economic situation and the environment.
67
Session 6
HIGH-THROUPUT PHENOTYPING OF NITROGEN RESPONSE AND USE IN
WHEAT WITH LEMNATEC SCANALYZER 3D
Mamoru Okamoto1, Sanjiv Satija1, Jingwen Tiong1, Ramya Sampath1, Sayuri
Watanabe1, Sigrid Heuer1
1Australian Centre for Plant Functional Genomics, Waite Research Institute,
University of Adelaide, Adelaide, SA, 5064, Australia.
Email:
mamoru.okamoto@acpfg.com.au
High-throughput phenotyping of nitrogen (N) response and N-use efficiency (NUE)
in crop plants is still a developing genre. Because the methods could be variable
depending on target traits, growth conditions and species, standardizing
phenotyping protocols for N-related traits is challenging. The aim of the project is to
establish a high-throughput phenotyping method for quantifying NUE in wheat
which could be applied to different species and labs/facilities. We have measured
growth response of 8 different wheat cultivars from Australia and Mexico with
several N treatments using the Plant Accelerator, the automated non-destructive
high-throughput imaging facility, equipped with the LemnaTec Scanalyzer 3D. Daily
images were taken from early tillering to heading stage of wheat plants grown in
pots. The visible light (RGB) image was used for biomass estimation, and RGB
and/or fluorescence images were employed to assess leaf colour changes during
the growth period. Based on the image analysis, a typical N response curve in
biomass was observed among all cultivars tested (N treatment: 10−450 mg N kg-1
soil). At the highest N treatment (450 mg N) only Mace, an Australian modern
cultivar, kept a positive correlation between growth and N input, whereas other
Australian cultivars did not positively respond. This finding corresponded with the
final grain yield with only Mace increasing grain yield with increasing N. The set of
historic Mexican varieties from CIMMYT revealed a trend showing that newer
varieties grew faster and larger compared with older varieties at a given N
concentration indicating an improvement in N use through selection by breeders.
N-deficiency symptoms were assessed by leaf colour image analysis revealing that
the degree of deficiency symptoms varied between the cultivars. These results
indicate that high-throughput phenotyping during the mid-growth stage could be
useful to identify genetic variations in N use in wheat, and potentially in other
species.
68
Session 6
PLASTICITY IN THE SHOOT BRANCHING REGULATORY NETWORK
Maaike de Jong1, Tanya Waldie1, Rachel Borrows1, Raj Pasam1, Sally Ward1,
Paula Kover2, Ottoline Leyser1
1Sainsbury Laboratory, University of Cambridge, 2Department of Biology and
Biochemistry, University of Bath
Email:
maaike.dejong@slcu.cam.ac.uk
Plants can modulate their developmental program depending on the prevailing
environmental conditions. A good example of such plasticity is the degree of shoot
branching where both developmental and environmental inputs are integrated by a
network of hormonal signals. This network systemically transmits the information,
which is locally interpreted to regulate axillary bud outgrowth. To understand the
molecular basis for dynamic variation in branch number, we examined quantitatively
different versions of the shoot branching regulatory network. Branching data were
collected from Arabidopsis Multiparent Advanced Generation Intercross (MAGIC)
lines 1 grown under low or high nitrogen conditions and used to identify Quantitative
Trait Loci (QTL) for branch number and branching plasticity. We also collected
branching data from genotyped natural Arabidopsis accessions 2,3. Comparing the
data sets revealed interesting trait correlations between branch number, branch
number plasticity and flowering time, and showed that the magnitude of the
architectural plasticity depends on two alternative life history strategies. Currently,
we are working on the identification of the genes that underlie these QTL and are
characterising these genes to determine their role in the shoot branching regulatory
network.
References:
1 Kover et al., (2009) A multiparent advanced generation inter-cross to fine-map
Quantitative Traits in Arabidopsis thaliana. PLoS Genetics, 5: e1000551.
2 Cao et al., (2011) Whole-genome sequencing of multiple Arabidopsis thaliana
populations. Nature Genetics, 43(10): 956-963.
3 Atwell et al., (2010) Genome-wide association study of 107 phenotypes in
Arabidopsis thaliana inbred lines. Nature 465: 627-631.
69
SESSION 7
NITROGEN NUTRITION IN
PLANT AND BACTERIAL
SYSTEMS
Chair: Bertrand Hirel
Session 7
REGULATION OF THE MEVALONATE PATHWAY BY SYMBIOTIC
RECEPTOR-LIKE KINASES AND ITS ROLE IN EARLY SYMBIOTIC SIGNALING
Muthusubramanian Venkateshwaran1, Dhileepkumar Jayaraman1, Brendan K.
Riely2, Estibaliz Larrainzar2, Douglas Cook2, Jean-Michel Ané1.
1Department of Agronomy, University of Wisconsin Madison, 2Department of Plant
Pathology, University of California Davis
Email:
jane@wisc.edu
HMGRs (3-hydroxy-3-methylglutaryl coenzyme A reductases) are key enzymes in
the mevalonate pathway controlling isoprenoid biosynthesis. Surprisingly, one of
these enzymes (HMGR1) was found to interact with the symbiotic receptor-like
kinase DMI2 and is required for legume nodulation in the model legume Medicago
truncatula. Using split-ubiquitin yeast-two hybrid, interactions between HMGR1 and
two other symbiotic receptor-like kinases, NFP and LYK3, were also found. In vitro
kinase assays revealed that HMGR1 is phosphorylated by DMI2 but not by NFP or
LYK3. Mass spectrometry was used to characterize phosphorylation sites in the
linker region of HMGR1, a region which is highly variable between different HMGR
isoforms. Enzymatic assays revealed that HMGR1 activity is affected by interaction
with DMI2. Mimicking phosphorylation by serine to aspartic acid substitutions at the
phosphorylation sites also affected HMGR1 enzymatic activity. HMGR1-silenced
roots were impaired for nuclear calcium spiking and symbiotic gene expression.
Reciprocally, application of mevalonate, the product of HMGR1 activity, was
sufficient to induce calcium spiking and symbiotic gene expression in wild-type and
HMGR1-silenced roots. Mevalonate was able to induce nuclear calcium spiking and
ENOD11 expression in dmi2 but not in dmi1 mutants. These results indicate that
HMGR1 plays an early role in the symbiotic signaling cascade after DMI2 and
before the nuclear cation channel DMI1 and calcium spiking. We hypothesize that
HMGR1 connects signaling events at the plasma membrane level to nuclear ones
by generating second messengers controlling downstream symbiotic signaling.
71
Session 7
PLANT SIGNALING DURING SUGARCANE COLONIZATION WITH ENDOPHYTIC
NITROGEN-FIXING BACTERIA
Anna Carolina J.S. Bomfim1, Thais Louise G. de Carvalho1, Rodrigo M. Saraiva1,
Lívia S. Vargas1, Emília B. Pires1, José I. Baldani2, Paulo C.G. Ferreira1, Clicia G.
Grativol1, Adriana S. Hemerly1.
1Laboratório de Biologia Molecular de Plantas, Instituto de Bioquímica Médica,
Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, 21.941-590 ,
Rio de Janeiro, RJ, Brazil, 2Embrapa Agrobiologia, BR465, Km47, 23851-970,
Seropédica, RJ, Brazil
Email:
hemerly@bioqmed.ufrj.br
The associations that occur between sugarcane and other grasses with
nitrogen-fixing endophytic bacteria have raised a large interest in their use in
agriculture, in view of the positive effects on root development, and the increase in
biomass and productivity. Promotion of plant growth by the endophytic
nitrogen-fixing bacteria might be mediated by providing nitrogen trough Biological
Nitrogen Fixation (BNF) and hormones. In Brazil, BNF plays a fundamental role in
sugarcane cultivation by reduction of the use of nitrogen fertilizers, making Brazilian
sugarcane culture more competitive in global markets. The fact that distinct
sugarcane genotypes have different rates of BNF suggests that plant genetic
factors might be controlling the processes of bacteria recognition, colonization
and/or nitrogen fixation. It has been demonstrated that BNF efficiency is also
dependent on specific soil conditions where plant and bacteria association is
established, such as nitrogen fertility and water content. Our group has been
studying plant signaling mechanisms involved in the establishment of this particular
type of association with nitrogen-fixing bacteria, aiming to investigate the role of
plant genotype and soil conditions in the efficiency of the association. Next
generation sequencing technologies are being applied to compare expression
profile in two sugarcane BNF contrasting genotypes and in response to soil
conditions, such as nitrogen sources and water deficit. An integrated differential
transcriptome was generated and it provided an overview of sugarcane metabolism,
growth and development controlled by nitrogen, water and endophytic
nitrogen-fixing bacteria during a successful association. All together, the data
suggest that plant genotype, nitrogen and water soil conditions control regulatory
networks that are important during the establishment of the beneficial association.
This work was supported by INCT, CNPq, FAPERJ & CAPES.
72
Session 7
A MEMBRANE LOCALISED BHLH TRANSCRIPTION FACTOR INVOLVED IN
LEGUME NODULE DEVELOPMENT AND AMMONIUM TRANSPORT
Brent N Kaiser1, David M Chiasson1, Danielle Mazurkiewicz1, Manijeh
Mohammadidehcheshmeh1, Patrick C Loughlin2, Elena Fedorova3, Mamoru
Okamoto1, Elizabeth McLean4, Ton Bisseling3, Anthony DM Glass5, Sally Smith1,
Stephen D Tyerman1, David Day6.
1School of Agriculture Food and Wine, The University of Adelaide, 2School of
Biological Sciences, The University of Sydney, 3Laboratory of Molecular Biology,
Wageningen University, 4School of Plant Biology, The University of Western
Australia, 5Department of Botany, The University of British Columbia, 6Flinders
University
Email:
brent.kaiser@adelaide.edu.au
GmSAT1 is a membrane bound basic helix-loop-helix DNA-binding transcription
factor localised to the symbiosome membrane, plasma membrane, endoplasmic
reticulum and nucleus of infected nodule cells. GmSAT1 expression is enhanced in
nodules relative to roots with the onset of nitrogen fixation. Loss of GmSAT1 activity
using RNAi roots (sat1) reduces nodule fitness and nodule number. In yeast cells,
we have shown that overexpression of GmSAT1 results in the transcriptional
activation of a novel class of plasma membrane localised low-affinity ammonium
channel (AMF1). AMF1 homologs exist in soybean and other plant species where
they often share chromosomal microsynteny to SAT1 loci. To elucidate function of a
plant AMF1 homolog, we analysed GmAMF3, a nodule enhanced AMF1. Using
both yeast and Xenopus laevis oocytes, we show that GmAMF3 is a low-affinity
ammonium channel. Promoter analysis using a GUS reporter shows that GmAMF3
is expressed specifically in nodule parenchyma cells and vascular tissues that
encircle the infected region of the nodule; while in roots GmAMF3 expression is
restricted to the fasicular cambium cell layer separating the xylem and phloem.
Collectively these results show that GmSAT1 participates in a membrane-based TF
signalling cascade that influences the rhizobia symbiotic interaction in soybean.
GmSAT1 has identified a novel mechanism for ammonium transport (AMF1) that is
most likely involved in legume nodule and root ammonium transport.
73
Session 7
SCREENING FOR AMINO ACID EXPORTERS AND THEIR REGULATORS IN
ARABIDOPSIS AND SOYBEAN
Rejane C Pratelli1, Guillaume L Pilot1.
1Virginia Tech
Email:
pratelli@vt.edu
Amino acid metabolic pathways lie at the crossroads of nitrogen and carbon
metabolisms, and serve as precursors of proteins and secondary metabolites. As
the main carriers of organic nitrogen, amino acids are transported through the plant
via both xylem and phloem. Control of fluxes is thought to result from the
modulation of metabolic and transport activities, and identifying the key regulators is
a primary target of crop improvement. In particular, the control of protein content in
soybean seeds is of high importance and yet attempts at modifying the seed
loading have failed. Only a dozen of the 60 amino acid importers identified in
Arabidopsis genome are characterized so far. Among a probably equally large
number of exporters, only two (BAT1 and SiAR1) have been studied to date. The
mechanisms that underlie the modulation of the transporters’ activity are also largely
unknown.pratelli@vt.edu.doc While regulation at the transcript level can be inferred
from expression databases, regulation at the protein level is undocumented. The
Glutamine DUmper1 protein may be one of these elusive regulators. This plant
specific, one transmembrane domain protein, although unlikely to mediate export
by itself, causes an increased efflux of amino acids from plant cells when
overexpressed. This phenotype is dependent on the activity of the E3 ubiquitin
ligase LOG2, suggesting ubiquitination steps control amino acid export activities.
Since BAT1 and SiAR1 interact neither with GDU1 nor LOG2, we initiated a large
scale screening aimed at identifying other amino acid exporters. The amino acid
export activity of 400 selected putative transporters was tested in a yeast assay,
leading to the identification of 2 new families of amino acid exporters. In a parallel
approach, we identified a putative soybean transporter that is associated with high
protein content in seeds. All these proteins are currently under investigation.
74
Session 7
MOLECULAR MECHANISMS CONTROLLING FUNCTIONAL
ASSOCIATION BETWEEN ARABIDOPSIS THALIANA AND
SINORHIZOBIUM MELILOTI BACTERIUM
Tatiana Kraiser1, Diana Gras1, Bernardo González2, Rodrigo A. Gutiérrez1.
1FONDAP Center for Genome Regulation. Millennium Nucleus for Plant Functional
Genomics. Departamento de Genética Molecular y Microbiología. Facultad de
Ciencias Biológicas. P. Universidad Católica de Chile. Santiago, Chile, 2Facultad de
Ingeniería y Ciencias. Universidad Adolfo Ibáñez.
Email:
tdkraise@uc.cl
Nitrogen acquisition in plants by the association with nitrogen fixing bacteria (NFB)
have been mostly studied in legumes. In those plants, regulatory mechanisms are
essential for a successful association and nodule development. In the case of
non-legume plants unable to form nodules, it is unknown the extent to which they
can or cannot establish functional associations with NFB and the molecular
regulatory mechanisms involved. Our goal was to evaluate a functional association
between Arabidopsis thaliana and NFB to develop a model system in which to
study and identify molecular mechanisms underlying non-legumes and NFB
association. We found A. thaliana can functionally associate with Sinorhizobium
meliloti RMP110 contributing to plant growth under N-limiting conditions. We
showed the growth-promoting effect to be at least partly dependent on nitrogen
fixation by genetically inactivating the nitrogenase complex in the bacterium.
Analysis of plant gene expression under different nitrogen regimes in the presence
or absence of NFB identified candidate plant genes. Arabidopsis mutant lines
verified the importance of these genes for NFB-mediated plant growth promotion.
We propose Arabidopsis and S. meliloti as an excellent model system to identifying
key regulatory networks in non-legumes and NFB association.
Acknowledgements: Howard Hughes Medical Institute, Millennium
Nucleus P10-062-F, FONDAP 1509007, FONDECYT 1100698 and CONICYT PhD
scholarship 21080821.
75
POSTER SESSIONS
SESSION 1
NITROGEN SIGNALING
Session 1
QUANTITATIVE PHOSPHOPROTEOME ANALYSIS OF THE PRIMARY NITRATE
RESPONSE IN ARABIDOPSIS ROOTS
Andrea Vega1, José M. Álvarez1, Eleodoro J. Riveras1, Zhouxin Shen2, Steven P.
Briggs2, Rodrigo A. Gutiérrez1.
1Center for Genome Regulation. Millennium Nucleus Center for Plant Functional
Genomics. Departamento de Ciencias Vegetales. Departamento de Ciencias
Vegetales. Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad
Católica de Chile. 2Division of Biological Sciences, University of California, San
Diego, U.S.A.
Email:
avegac@uc.cl
Nitrate is one the most important nitrogen sources in agriculture. Despite its role
as a nutrient, nitrate can also act as a signaling molecule that modulates gene
expression of a wide range of processes, including root growth and development.
Although transcriptional responses in response to nitrate have been characterized
by a number of groups, the nitrate-signaling pathway is yet to be discovered. Most
signaling pathways involved post-translational modifications of key components.
Among these, protein phosphorylation is one of the most abundant, affecting
protein-protein interactions and thus providing a framework for signaling networks.
As a first step to identify potential regulatory factors involved in early signaling
events in response to nitrate treatments, we performed quantitative time-course
analyses of the Arabidopsis root phosphoproteome in response to nitrate by liquid
chromatography with tandem mass spectrometry detection (LC MS/MS). This
large-scale approach allowed us to identify peptides with changes in their
phosphorylation level as early as 5 min after nitrate treatments (fast responses). The
nature of the proteins identify differ significantly from genes implicated in
transcriptome studies. The new proteins found implicated in nitrate responses
include mainly signaling associated proteins, kinases and transcription factors. Our
data provide new insights into the phosphoproteome of Arabidopsis roots and
identify putative novel components of the nitrate-signaling pathway.
Acknowledgements: Milenio-P10-062-F, Fondap-15090007, Howard Hughes
Medical Institute, Fondecyt-11110095.
78
Session 1
MISINTERPRETATION: A LEGUME MUTANT ENTERS RHIZOBIAL SYMBIOSIS
WHEN SUPPLIED WITH NITROGEN
Dong Wang1, Minsoo Kim1, Chrsitopher S Waters1,
1University of Massachusetts Amherst
Email:
dongw@biochem.umass.edu
Legumes have the remarkable ability to enter a symbiosis with nitrogen-fixing
rhizobia in the root nodule, where atmospheric dinitrogen is converted into
ammonia. In this mutualistic relationship, the host plant provides not only the
carbon source, but also the pink pigmented leghemoglobin protein in the nodule to
sequester free oxygen molecules, which are toxic to the bacterial nitrogenase
enzyme. Because the nitrogen-fixing symbiosis demands heavy host investment, it
is under strict control: wild type plants produce a limited number of nodules,
presumably just sufficient for the host’s nitrogen needs, before blocking additional
nodule organogenesis. High levels of ammonium or nitrate in the soil can prevent
nodule formation altogether. Many symbiotic mutants form nonfunctional nodules
continuously without producing leghemoglobin, indicating that the host monitors the
productivity of each newly formed nodule and sanctions unproductive ones. The
identity of this “molecular gauge” has been unknown. We recently identified a
nitrogen-fixing deficient mutant from Medicago truncatula that turns on
leghemoglobin production conditionally: leghemoglobin is synthesized in nodules
only when the medium is supplemented with a low level of nitrogen exogenously
(below the amount needed to block nodule formation). These nodules are
nonfunctional, as they again lose leghemoglobin as the externally nitrogen becomes
depleted. This phenotype suggests that the mutant plant cannot distinguish
between different sources of nitrogen, and opts for the symbiotic as a default
response to nitrogen. We will provide up-to-update characterization of this mutant,
and describe our efforts to identify the causal gene.
79
Session 1
THE ROLE OF CA+2 IN THE NITRATE SIGNALING PATHWAY IN ARABIDOPSIS
THALIANA ROOTS
Eleodoro J Riveras1, José M. Álvarez1, Carolina Oses1, Karem P. Tamayo1,
Rodrigo A. Gutierrez1.
1Center for Genome Regulation. Millennium Nucleus Center for Plant Functional
Genomics. Departamento de Genética Molecular y Microbiología. Pontificia
Universidad Católica de Chile.
Email:
ejrivera@uc.cl
Nitrate is the main nitrogen source in agriculture soils. Besides its role as a
nutrient, nitrate act as a potent signal that control global gene expression. However,
the signal transduction pathway involved in the nitrate response still remains elusive.
It is known that calcium is an essential second messenger in signal transduction in
plants. Nonetheless, its role mediating the response to nitrate has not been
addressed. As a first step to determine if calcium is involved in the nitrate response,
we tested whether nitrate produced an increased in cytoplasmic calcium
concentration. We demonstrated that nitrate treatments produce a transient
increase in cytoplasmic calcium. Moreover, a pharmacological inhibitor of
phospholipase C (PLC) affected the increase of cytoplasmic calcium. We also
evaluated the expression of sentinel nitrate responding genes in Arabidopsis roots
in the presence of calcium channel blockers and a pharmacological inhibitor of PLC.
We found that both calcium and PLC are necessary for the expression of such
genes in response to nitrate treatments. With this work, we identify a new signaling
pathway involving calcium and PLC that modulates changes in gene expression in
response to nitrate.
Acknowledgment: MilenioP10-062-F, Fondap1509007, Fondecyt110698, HHMI,
Beca AT-24121649 and CONICYT doctoral fellowship grants.
80
Session 1
NITROGEN SIGNALING BY GLUTAMATE SYNTHESIZED BY GDH TRANSGENES
IN THE CYTOPLASM IMPROVES CROP GROWTH
David A Lightfoot
Southern Illinois University
Email: ga4082@siu.edu
New crop plants suited to growth in semi-arid environments will be fundamental
to the future of agriculture. The interactions between nitrogen supply and water
availability that determine yield and quality in crops grown in semi-arid environments
are being elucidated. Maize (Zea mays L.) and other crop plants have altered
transcript and metabolic profiles caused by in planta expression of the bacterial
glutamate dehydrogenase (EC 1.4.1.2), a modified gdhA. The change in glutamate
concentration in the cytoplasmic pool has profound effects on plant metabolisms.
The metabolic changes resulted in phenotypic changes that included increases in
mean plant biomass production in dry soils, tolerance to the herbicide
phosphinothricin, tolerance to both severe and mild water deficit. Leaves and grain
had higher nutritional value and higher yield indicating improved NUE and WUE.
Comparisons of transgenic and non transgenic maize under drip irrigation showed a
11% increase in WUE and 9% increase in NUE across a range of water delivery
rates. The variation caused by the transgene was greater than that found in a set of
maize and soybean germplasm tested. Resistance to rotting necrotrophs including
carcinogenicAspergillus flavus contaminations was noted. Sporulation of A. flavus
was inhibited and the abundance of 747 fungal and 395 maize grain transcripts
were altered suggesting the GDH maize was not supporting normal fungal growth
because of metabolic and compositional alterations. Cancer incidences due to toxin
contamination can potentially be reduced by 50% by GDH. There were about 283
metabolites in roots, 98 metabolites in leaves and 56 metabolites in grain that
changed abundances including some increases in nutritionally valuable amino
acids. The altered metabolites and proteins provided biomarkers for these valuable
traits. See US patents 5,998,700; 6,329,573; and 8,383,887.
81
Session 1
POSSIBLE ROLE OF GLUTAMINE SYNTHETASE OF THE PROKARYOTIC TYPE
(GSI-LIKE) IN NITROGEN SIGNALING IN MEDICAGO TRUNCATULA
Liliana S Silva1, José N Leitão1, Ana R Seabra1, Helena G Carvalho1.
1Instituto de Biologia Molecular e Celular, Rua do Campo Alegre, 823. 4150-180
Porto, Portugal
Email:
mhcarval@ibmc.up.pt
Genes encoding glutamine synthetase of the prokaryotic type (GSI-like) are
widespread in higher plants, but their function is currently unknown. Interestingly, it
has been reported that the expression of GSI-like genes responds to alterations in
nitrogen metabolism in root nodules of Medicago truncatula (Ruffel et al. 2008, Plant
Physiol, 146, 1-16; Seabra et al. 2012, MPMI, 25, 976–992), suggesting a function
related to N sensing and/or signaling. To gain insights into the possible role of
GSI-like proteins, we characterized the GSI-like gene family of M.truncatula and
investigated the functionality of the encoded proteins.The genome of M. truncatula
contains two GSI–like genes, MtGSIa and MtGSIb, encoding polypeptides of 454
and 474 amino acids, respectively. The proteins share homology with the
Aspergillus nidulans FluG, which has been implicated in important signaling
pathways during fungal conidiogenesis in response to N starvation, further
suggesting a role related to N signaling. The two M.truncatula GSI-like proteins were
expressed in E.coli and functional studies indicate that they do not retain GS
activity. The expression of the two MtGSI-like genes was evaluated by qRT-PCR
and western blot in different organs of the plant and in response to different N
regimens. These studies revealed that the two genes are preferentially expressed in
roots and root nodules and are upregulated by NH4+. Localization of gene
expression was evaluated in transgenic plants expressing
MtGSI-like-promoter-gusA fusions and revealed a specific and strong expression in
the vascular bundles of both roots and nodules and in uninfected cells of root
nodules. Taken together, the tissue-specific pattern of expression, the differential
response to distinct N regimens and the finding that GSI-like proteins do not retain
GS activity support an involvement of GSI-like proteins in N signaling.This work was
supported by FCT project PTDC/BIA-PLA/2291/2012
82
Session 1
PARTICIPATION OF NITRIC OXIDE AND CELL WALL INVERTASE IN
INHIBITION OF PHOTOASSIMILATE TRANSLOCATION FROM LEAVES
UNDER INCREASED NITRATE NUTRITION
Svetlana N Batasheva1, Liliya I Shamova2, Guzel A Salyakhova1, Larisa A
Khamidullina1, Golsoyar G Bakirova1, Vladimir I Chikov1.
1Kazan Institute of Biochemistry and Biophysics, RAS, 2Kazan State Unversity
Email:
sbatasheva@mail.ru
In plants fertilized with high doses of nitrates, the translocation of photosynthetic
products from leaves is decreased compared to that in control or urea fed plants. It
was shown that the main transport product of photosynthesis, sucrose, is intensely
hydrolyzed in the plant extracellular space (apoplast) under high nitrate nutrition, but
the mechanism of this increased hydrolysis of sucrose is still unknown. In this work
it was shown that potassium nitrate increased the sucrose hydrolyzing activity of
cell wall fraction isolated from an apoplastc plant, flax (Linum usitassimum), and a
symplastic plant, basil (Ocimum basilicum). Cell wall invertase contains SH-groups
that can be a target of nitric oxide (NO), proably formed from nitrate. Modification of
proteins with NO is now proven to play a key role in regulation of many processes
both in plants and animals. To test the hypothesis that nitate can act through NO
binding to invertase protein, the classical molecular dynamics study (Gromacs, 3ns
unconstrained runs) of invertase-sucrose and NO-modified (cysteine 204 mutated
to nitrocystein) complexes was employed. The energy of invertase-sucrose
interaction was crudely estimated in both cases, and the increase of Eint in 10
kcal/mole in case of nitrosylated invertase enzyme complex with sucrose,
compared to the native non-modified enzyme-substrate complex, might indicate
the better affinity of the enzyme to the substrate. In flax shoots, the influence of NO
donor, sodium nitroprusside, fed through the transpiration water stream, on
photoassimilate translocation was similar to that of potassium nitrate. When
potassium nitrate was introduced into flax shoots together with NO scavenger,
cPTIO, no inhibition of photoassimilate translocation was observed compared to
control. These observations support the idea that the influence of nitrate on
photoassimialte translocation can be mediated by nitric oxide. The work was
supported by RFFI grant 12-04-31677.
83
Session 1
ANALYSIS OF AMMONIUM-SENSITIVE PATHWAY VIA CHL1 IN
ARABIDOPSIS THALIANA
Takushi Hachiya1, Hitoshi Sakakibara1.
1RIKEN Center for Sustainable Resource Science
Email:
sakaki@riken.jp
CHL1 (NRT1.1) is a dual-affinity nitrate transporter and a nitrate sensor that plays
a role in nitrate-dependent signaling pathway. We have revealed that chl1 mutants
show enhanced tolerance to toxic ammonium in the absence of nitrate. This
indicates a nitrate-independent function of CHL1 and an existence of
ammonium-sensitive mechanism via CHL1. The aim of this study is to clarify the
mechanism. First, we verified whether the ammonium-sensitive mechanism could
be distinct from the nitrate-dependent signaling pathway via CHL1. This
nitrate-dependent pathway is modulated by phosphorylation status of CHL1,
unaffeted by P492L mutation of CHL1 and followed by NRT2.1, another candidate
for nitrate sensor. However, the ammonium-tolerance was not affected by the
phosphorylation status of CHL1, enhanced by the P492L mutation and not
mediated via NRT2.1. Thus, a novel pathway via CHL1 is related to the
ammonium-tolerance. Next, we explored the candidate genes which function
downstream of CHL1 in the ammonium-sensitive pathway. Concentrated
ammonium causes apoplastic acidification, and therefore, ammonium toxicity is
partly an acidic stress. We found that an elevation of media pH diminished
enhanced ammonium tolerance of chl1 mutants. Thus, we focused on a master
regulator for acidic stress responses, a zinc finger transcription factor STOP1
(SENSITIVE TO PROTON RHIZOTOXICITY1). Our genetic evidence suggests that
STOP1 would mediate the ammonium-sensitive pathway via CHL1.
84
SESSION 2
AMMONIUM TRANSPORT
AND ASSIMILATION
Session 2
INVOLVEMENT OF A MYB FACTOR IN THE REGULATION OF
PHENYLALANINE PATHWAY IN MARITIME PINE
Blanca Craven-Bartle1, Maria Belen Pascual1, Francisco M Canovas1,
Concepcion Avila1.
1Universidad de Malaga
Email:
cavila@uma.es
Wood is traditionally among the most important commercial products because of
the high demand that exits for its derivatives. Trees, including conifers, divert large
quantities of carbon into the biosynthesis of phenylpropanoids, particularly to
generate lignin. Although lignin and other phenolic compounds do not contain
nitrogen, phenylalanine metabolism is required to channel photosynthesis-derived
carbon to phenylpropanoid biosynthesis. The phenylpropane skeleton required for
lignin biosynthesis is provided by the deamination of phenylalanine in the reaction
catalysed by the enzyme phenylalanine ammonia-lyase (PAL). This reaction is
quantitatively important in trees because lignin biosynthesis is required for wood
formation, and it releases large quantities of ammonium. An efficient and
coordinated pathway for the amination of prephenate and the deamination of
phenylalanine should be operative in lignifying cells to provide phenylalanine for
lignin biosynthesis, and to re-assimilate ammonium.
We hypothesized that one way to ensure efficient photosynthetic carbon
channeling for lignin and other phenylpropanoid biosynthesis, together with nitrogen
recycling, would be to couple both processes in time and in space by
transcriptionally regulating the genes involved in phenylalanine biosynthesis and
use.
The experiments described in this communication attempt to test this hypothesis.
To this end, we have isolated the promoter region of the three genes involved in the
phenylalanine pathway in Pinus pinaster: PAL, GS1b and PAT. We have conducted
both in vitro and in vivo studies using three different Myb transcription factors:
PtMyb1, PtMyb4 from P. taeda and PpMyb8 from P. pinaster. We have studied the
possible coupling in space and time of gene products for the operative
co-regulation of both processes in pine trees, and have proven that Myb8 is a
potential candidate to be the transcriptional regulator of phenylalanine metabolism
in P. pinaster vascular cells.
86
Session 2
DECIPHERING THE PRIMARY METABOLIC PATHWAY OF PURINE
NUCLEOTIDE CATABOLISM
Claus-Peter Witte1, Kathleen Dahncke1, Nieves Medina Escobar1,
1Freie Universitaet Berlin, Dahlem Centre of Plant Sciences, Plant Biochemistry
Email:
cpwitte@zedat.fu-berlin.de
In natural ecosystems, plants frequently grow under nitrogen limitation. Not only
effective uptake and assimilation mechanisms but also biochemical pathways for
internal reallocation of nitrogen are required for efficient utilization of this scarce
resource. Using bioinformatic approaches coupled with biochemical studies and
metabolic analyses of mutants, we have identified several enzymes which are
involved in the generation of ammonia from nitrogen stored in purine nucleotides.
This presentation will highlight new findings in nucleotide catabolism and then focus
on guanosine deaminase (GSDA), a novel plant-specific enzyme which catalyzes
the deamination of guanosine to xanthosine and ammonia. Surprisingly, metabolite
and phenotypic analyses of several Arabidopsis single and double mutants revealed
that purine nucleotides dedicated for degradation are channeled to great extent
through GSDA. Xanthosine is exclusively generated by GSDA in vivo. Possible
implications for the biosynthesis of purine alkaloids (caffeine and theobromine) in
coffee and tea and ureides in the nodules of tropical legumes are discussed. In
summary, our data indicate that in plants a linear pathway of GMP catabolism is
operative which differs from what is known from mammals and microbes.
87
Session 2
UNIQUE PROPERTIES OF SULFITE REDUCTASE HOMOLOGS FOR NITRITE
AND SULFITE REDUCTION IN RED ALGAE CYANIDIOSCHYZON MEROLAE.
Sekine Kohsuke1, JuYaen Kim2, Toshiharu Hase2, Naoki Sato3.
1Komaba Organization for Educational Excellence, University of Tokyo, 2Institute
for Protein Research, Osaka University, 3Graduate School of Arts and Sciences,
University of Tokyo
Email:
kosk@koskn.net
Plant nitrite reductase (NiR) and sulfite reductase (SiR) have common features in
structure and function. Both enzymes are generally distinguished in terms of
preferences for nitrite and sulfite as substrates. The Cyanidioschyzon merolae
genome contains two genes encoding SiR homologs, termed CmSiRA and
CmSiRB, but no gene for NiR homolog. We characterized catalytic properties of
CmSiRA and CmSiRB prepared and purified as recombinant enzymes in E. coli. The
turnover numbers of CmSiRB in nitrite reduction and sulfite reduction were 5-fold
higher and 45-fold lower, respectively, than those of maize SiR. Therefore we
concluded that CmSiRB is an unusual enzyme with little activity for sulfite reduction,
but enhanced activity for nitrite reduction, indicating physiological role of CmSiRB is
nitrite reduction. CmSiRA showed a high sulfite reductase activity comparable with
maize SiR and also significant nitrite reductase activity comparable with CmSiRB.
These results suggest that CmSiRA plays a dual role as NiR and SiR in a C.
merolae. This assumption is supported by phenotype of CmSiRB-null mutant,
which is able to grow in medium containing nitrate as a sole nitrogen source. We are
now engaging in site-directed mutation study of CmSiRB to explore the structural
basis for discrimination of the two enzyme activities.
88
Session 2
RICE NOVEL PROTEIN KINASE INVOLVED IN REGULATION OF
AMMONIUM UPTAKE INTO ROOTS UNDER HIGH CONCENTRATION
OF EXTERNAL AMMONIUM
Toshihiko Hayakawa1, Mitsuhiro Obara2, Akiko Taniai1, Yuki Sawa1, Jin Ishizawa1,
Soichi Kojima1, Tomoyuki Yamaya1.
1Graduate School of Agricultural Science, Tohoku University, 2Japan International
Research Center for Agricultural Sciences
Email:
toshi@biochem.tohoku.ac.jp
Down-regulation of the high-affinity transport system (HATS) for ammonium
uptake into plant roots in response to increasing ammonium supply is an important
event for preventing ammonium-toxicity in plants. Ammonium transporter1 (AMT1)
is responsible for HATS; however, molecular mechanisms of down-regulation of
HATS are largely unknown. In this study, we have explored novel participants in
modulation of ammonium uptake and use in roots of ammonium-preferring paddy
rice. A rice gene that encodes a novel protein kinase, designated OsACTPK1,
showed much higher expression in roots of rice seedlings grown under sufficient
than low ammonium applications. Two rice homozygous mutants lacking
OsACTPK1 were established through insertion of retrotransposon Tos17 into
distinct exons of the gene. Under sufficient ammonium, seedlings of these mutants
exhibited ammonium-hypersensitivity in root growth with accompanying increased
accumulation of ammonium and amino acids, especially glutamine and asparagine,
and promotion of shoot growth, while under excess ammonium, a severe plant
growth inhibition. Under sufficient ammonium, a higher level of ammonium influx by
impairment of a proper decrease in Vmax of HATS activity was found in roots of
mutants compared to control plants, although transcript expression of AMT1 genes
was down-regulated in both mutant and control roots. Concurrently, cytosolic
glutamine synthetase1 and NADH-glutamate synthase, key enzymes for the primary
ammonium assimilation, were more accumulated in roots of mutants than control
roots. These results indicate that OsACTPK1 is directly or indirectly involved in
down-regulation of root HATS activity to prevent cytosolic ammonium overload in
response to an ascending external ammonium.
89
SESSION 3
NITRATE TRANSPORT AND
ALLOCATION
Session 3
THE TRANSITION FROM MATERNAL TO EXTERNAL NITROGEN
SOURCES IN MAIZE SEEDLINGS
Kasra Sabermanesh1, Luke Reid Holtham1, Jessey George1, Ute Roessner2, Berin
Boughton2, Sigrid Heuer1, Mark Tester1, Darren Plett1, Trevor Garnett1.
1Australian Centre for Plant Functional Genomics, Waite Research Institute,
University of Adelaide, Adelaide, SA, 5064, Australia; School of Agriculture, Food
and Wine, Waite Research Institute, University of Adelaide, Adelaide, SA, 5064,
Australia, 2Australian Centre for Plant Functional Genomics, School of Botany, The
University of Melbourne, Parkville, Vic., 3010, Australia; Metabolomics Australia
School of Botany, University of Melbourne, Vic., 3010, Australia
Email:
kasra.sabermanesh@acpfg.com.au
During seedling growth, seed reserves rapidly deplete and as demand for nitrogen
(N) rises, seedlings must capture N externally to maintain growth and yield. This
important transition provides us with an ideal system to dissect the regulation of N
uptake. Here, two maize lines (B73 and Mo17), were grown hydroponically at low
(0.5mM) and adequate (2.5mM) NO3- and traits involved in N uptake and
metabolism were quantified with high time resolution across the transition from
seed N usage to external N capture. Results showed that in low N, 18 days after
sowing (DAS), Mo17 was better able to maintain N uptake and shoot growth than
B73. Most of the seed N reserves were depleted as early as 5 DAS. As seedlings
grew, the initial shoot %N halved by 7 DAS, whilst root N% remained constant.
Although shoot N% was initially similar across lines, Mo17 maintained higher shoot
N% than B73. High concentrations of seed-derived free amino acids rapidly diluted
across both lines, in roots and shoots, even though root N% remained constant.
Interestingly, despite Mo17 having higher starting concentrations of seed N and free
amino acids in tissues compared to B73, treatment differences in key free amino
acids, including alanine and glutamine, arose in the shoots of Mo17 earlier than
B73. Dilution of shoot N% and free amino acids began to stabilise at 7 DAS across
both lines. This corresponded with a rapid rise in root N uptake capacity and
transcripts of the genes encoding putative nitrate transporters NRT2.1 and NRT2.2
correlated with this. By investigating the transition from seed use to external N
capture, we identified critical time-points where N uptake begins and rises to meet
demand. Further investigation into these time-points will help understand the
molecular basis for the regulation of N uptake capacity in plants.
91
Session 3
SYSTEMATIC RESPONSES TO N SUPPLY & DEMAND IN MAIZE – A TWO
COMPONENT CONTROL MODEL GOVERNING NO3- UPTAKE CAPACITY
Luke Reid Holtham1, Kasra Sabermanesh1, Jessey George1, Ute Roessner2, Berin
Boughton2, Sigrid Heuer1, Mark Tester1, Mamoru Okamoto1, Darren Plett1, Trevor
Garnett1,
1Australian Centre for Plant Functional Genomics, Waite Research Institute,
University of Adelaide, Adelaide, SA, 5064, Australia; School of Agriculture, Food
and Wine, Waite Research Institute, University of Adelaide, Adelaide, SA, 5064,
Australia, 2Australian Centre for Plant Functional Genomics, School of Botany, The
University of Melbourne, Parkville, Vic., 3010, Australia; Metabolomics Australia
School of Botany, University of Melbourne, Vic., 3010, Australia
Email:
luke.holtham@acpfg.com.au
Given the inherently low N uptake efficiency of cereals we believe a better
understanding of the N uptake process will help to identify the factors limiting N
uptake efficiency and overall cereal nitrogen use efficiency (NUE). In order to
examine this, uptake capacity and transcript levels of putative high- (NRT2) and
low-affinity (NRT1) NO3- transporter genes were profiled across the lifecycle of
dwarf maize plants grown at reduced (0.5mM) and adequate (2.5mM) NO3-. Even
under constant adequate N, uptake capacity varied greatly across the lifecycle. A
reduction in NO3- supply led to a dramatic increase in NO3- uptake capacity. The
changes in uptake capacity were correlated with changes in NRT2 transcript levels.
These observations led to a proposed two-component model of NO3- uptake
capacity regulation involving both short term post-translational regulation and longer
term transcriptional regulation responding to tissue amino acid levels (Garnett et al.,
2013*). In order to further refine this model, reveal key points of control, and identify
candidate control genes, a fine time resolution and more comprehensive lifecycle
study with dwarf maize has been carried out. A wider separation of NO3- treatments
(0.5mM & 5mM) were used in order to test the suggested two-component model.
Some plants were switched from high (5mM) to low (0.5mM) NO3- mid-growth to
analyse how the NO3- uptake system and plant growth adapt. In this study a subset
was also removed, starved and then re-exposed to NO3- to assess the responses
observed in the lifecycle study in the context of the widely published primary nitrate
response. Transcriptomic, metabolomic and flux analysis were used to quantify
plant responses. The results to be discussed give important insights into the way
NO3- uptake capacity is controlled in response to supply and demand.
* Garnett et al. (2013) New Phytologist 198: 82-94
92
Session 3
CHARACTERIZATION OF THE NITRITE-SPECIFIC TRANSPORTER
FROM MARINE CYANOBACTERIA
Shin-ichi Maeda1, Tatsuo Omata1.
Graduate School of Bioagricultural Sciences, Nagoya university, Japan
Email:
maeda@agr.nagoya-u.ac.jp
For assimilation of nitrate, most of the freshwater cyanobacteria have an
ATP-Binding Cassette (ABC)-type nitrate/nitrite transporter, whereas marine
cyanobacteria of the Synechococcus group have a Major Facilitator Superfamily
(MFS)-type nitrate/nitrite transporter NrtP. Although most marine cyanobacteria of
the Prochlorococcus group do not assimilate nitrate or nitrite, some retain nitrite
reductase (NiR). Interestingly, the marine Synechococcus species and the
NiR-containing Prochlorococcus strains have focA, a gene encoding a transporter
similar to the bacterial formate transporter and the green algal nitrite transporter. To
determine the function of focA, we expressed the gene from three marine
cyanobacterial strains in a mutant of the freshwater cyanobacterium
Synechococcus elongatus that is defective in nitrite transport activity due to
inactivation of the ABC-type cyanate/nitrite- and the ABC-type nitrate/nitrite
transporters. Expression of focA from Synechococcus sp. PCC7002 was found to
allow the mutant to take up nitrite with a Km value of 8 microM and to grow on low
concentrations of nitrite. The nitrite uptake activity was not inhibited by nitrate,
cyanate, nor formate, suggesting that the FocA protein specifically transports nitrite.
93
Session 3
THE NITRATE TRANSPORTER NRT2.5 PLAYS A MAJOR ROLE IN NITRATE
ACQUISITION IN NITROGEN-STARVED ADULT ARABIDOPSIS
Takatoshi Kiba1, Lina Lezhneva2, Ana-Belen Feria-Bourellier2, Florence Lafouge2,
Stephanie Boutet-Mercey2, Nino Niccolo2, Hitoshi Sakakibara1, Francoise DanielVedele2, Anne Krapp2.
1RIKEN Center for Sustainable Resource Science, 2Institut Jean-Pierre Bourgin,
Institut National de la Recherche Agronomique-AgroParisTech
Email:
tkiba@psc.riken.jp
Plants often encounter nitrogen (N)-limited environments in nature. To survive
under such environment, N acquisition and utilization must be properly regulated.
Among seven NRT2 family nitrate transporter genes in Arabidopsis, two genes are
N-starvation inducible. One is NRT2.4, which we have reported previously to be a
high-affinity nitrate transporter playing a role both in roots and shoots of N-starved
plants (Kiba et al. 2012), and another is NRT2.5. The transcript level of NRT2.5
increases steadily and 10 days after the onset of starvation, it becomes the most
abundant transcripts among NRT2 family genes both in roots and shoots of adult
plants. To assess the localization of NRT2.5 expression, transgenic plants harboring
proNRT2.5:GUS:3’-NRT2.5 fusion gene were analyzed. GUS staining was observed
predominantly in the epidermal cell of lateral roots and in higher order leaf veins.
Overexpression of NRT2.5 in the high-affinity nitrate uptake-deficient nrt2.1-2.2
mutant partly restores nitrate uptake, while nrt2.5 T-DNA insertion mutant and RNAi
plants show decreased high-affinity nitrate uptake activity in N-starved adult plants.
These results indicate that NRT2.5 is a nitrate transporter playing a role in nitrate
acquisition in N-starved adult plants. Furthermore, growth and nitrate uptake
analyses of a series of multiple mutants between nrt2.1-2.2, nrt2.4 and nrt2.5
suggest that NRT2.5 acts in concert with NRT2.1, NRT2.2 and NRT2.4 to optimize
uptake and phloem loading of nitrate in the root and shoot, respectively, for
N-starvation adaptation. We also found that orthologs of NRT2.5 in Brachypodium,
maize, sorghum, and castor bean show N-starvation inducible pattern both in roots
and shoots, pointing to the possibility that the physiological role of this gene is
conserved among angiosperms.
94
Session 3
FUNCTIONAL CHARACTERIZATION OF MAIZE NRT1 TRANSPORT PROTEINS
IN XENOPUS OOCYTES
Zhengyu Wen1, Julie Dechorgnat1, Kanwarpal S Dhugga2, J Antoni Rafalski3,
Stephen D Tyerman1, Brent N Kaiser1.
1School of Agriculture, Food and Wine, University of Adelaide, Urrbrae, SA, 5064,
Australia, 2Pioneer HiBred, Johnston, Iowa, 50131, USA , 3DuPont Crop Genetics,
Wilmington, Delaware, 19803, USA
Email:
a1205876@adelaide.edu.au
Uptake of nitrate (NO3-) into plant root cells and its redistribution between cells
and tissues is predominantly mediated by nitrate transport proteins (NRTs).
Agricultural crops grown in soils where NO3- is the predominant N form are highly
dependent on the functional activity of NRT proteins for access and ultimately
utilization of soil N. In this study we have initiated a process to define the NO3transport network operating in the agriculturally important crop plant, Zea mays
(Maize). In the first instance we have analysed the functional activity of two
members of the NRT1 class of NO3- transporters (NRT1;1a, NRT1;1b) expressed in
roots using both chemical and electrical techniques in a Xenopus laevis oocyte
expression system. Both proteins were capable of accumulating 15NO3- into
cRNA-injected oocytes in a pH dependent manner, where low pH (5.5) increased
NO3- uptake. We also observed that both ZmNRT1;1a and ZmNRT1;1b were able
to accumulate chloride (36Cl) but that only ZmNRT1;1b showed strong competition
by external NO3-. On the other hand, ZmNRT1;1a was less selective as competing
anions including chloride, iodide and bromide reduced 15NO3- and 36Cl uptake.
Our data suggests, both ZmNRT1.1a and ZmNRT1.1b are proton-coupled nitrate
transporters that also facilitate the transport of Cl-. ZmNRT1;1b is more highly
selective for NO3- over Cl-, while ZmNRT1;1a displays a broader anion substrate
range.
95
SESSION 4
GENOMICS AND SYSTEMS
BIOLOGY
Session 4
SYSTEMS APPROACHES MAP REGULATORY NETWORKS DOWNSTREAM OF
THE AUXIN RECEPTOR AFB3 IN THE NITRATE RESPONSE OF ARABIDOPSIS
THALIANA ROOTS.
Elena A. Vidal1, Tomás C. Moyano1, Eleodoro J. Riveras1, Orlando ContrerasLópez1, Rodrigo A. Gutiérrez1
1FONDAP Center for Genome Regulation, Millennium Nucleus for Plant Functional
Genomics, Departamento de Genética Molecular y Microbiología, Pontificia
Universidad Católica de Chile.
Email:
eavidal@uc.cl
Auxin is a key phytohormone regulating central processes in plants. Although the
mechanism by which auxin triggers changes in gene expression is well understood,
little is known about the specific role of the individual members of the TIR1/AFB
auxin receptors, Aux/IAA repressors, and ARF transcription factors and/or molecular
pathways acting downstream leading to plant responses to the environment. We
previously reported a role for AFB3 in coordinating primary and lateral root growth
to nitrate availability. In this work, we used an integrated genomics, bioinformatics,
and molecular genetics approach to dissect regulatory networks acting downstream
of AFB3 that are activated by nitrate in roots. We found that the NAC4 transcription
factor is a key regulatory element controlling a nitrate-responsive network, and that
nac4 mutants have altered lateral root growth but normal primary root growth in
response to nitrate. This finding suggests that AFB3 is able to activate two
independent pathways to control root system architecture. Our systems approach
has unraveled key components of the AFB3 regulatory network leading to changes
in lateral root growth in response to nitrate.
97
Session 4
SYSTEMS BIOLOGY ANALYSIS OF TRANSCRIPTOME DATA PROVIDES NEW
HYPOTHESIS ABOUT ARABIDOPSIS ROOT RESPONSE TO NITRATE
TREATMENTS.
Javier C. Canales1, Tomas C. Moyano1, Eva M. Villarroel1, Rodrigo A. Gutiérrez1
1Departamento de Genética Molecular y Microbiología, Facultad de Ciencias
Biológicas, Pontificia Universidad Católica de Chile, Santiago 8331010, Chile
Email:
jcanales@bio.puc.cl
Nitrogen (N) is an essential macronutrient for plant growth and development.
Plants adapt to changes in N availability partly by changes in global gene
expression. We integrated publicly available root microarray data under contrasting
nitrate conditions to identify new genes and functions important for adaptive nitrate
responses in Arabidopsis roots. More than two thousand genes exhibited changes
in expression in response to nitrate treatments in Arabidopsis thaliana root organs.
Global regulation of gene expression by nitrate depends largely on the experimental
context. However, despite significant differences from experiment to experiment in
the identity of regulated genes, there is a robust nitrate response of specific
biological functions. Integrative gene network analysis uncovered relationships
between nitrate-responsive genes and eleven highly co-expressed gene clusters
(modules). Four of these gene network modules have robust nitrate responsive
functions such as transport, signaling and metabolism. Network analysis
hypothesized G2-like transcription factors are key regulatory factors controlling
transport and signaling functions. Our meta-analysis highlights the role of biological
processes not studied before in the context of the nitrate response such as root hair
development and provides testable hypothesis to advance our understanding of
nitrate responses in plants.
98
Session 4
TRANSCRIPTOMICS OF PLASTIDIC GS DEFICIENCY IN THE MODEL
LEGUME LOTUS JAPONICUS.
Marco Betti1, Carmen M Pérez-Delgado1, Margarita García-Calderón1, Antonio J
Márquez1
1Departamento de Bioquímica Vegetal y Biología Molecular, Facultad de Química,
Universidad de Sevilla, Calle Prof. García González 1, 41012 Sevilla, Spain.
Email:
mbetti@us.es
Plants have a remarkable ability to cope with highly variable environmental
stresses. The response of plant cells to different kinds of abiotic stresses includes
an extensive reprogramming of the transcriptome, with both shared and
stimulus-specific components. Recent works from our group using the model
legume Lotus japonicus demonstrated that the plastidic isoform of glutamine
synthetase (GS2) was involved in the response to different kinds of abiotic stresses.
These studies made use of the Ljgln2-2 mutant, which lacks of GS2, in order to
demonstrate the importance of this key enzyme in stress response (1-2). In this
work, we will carry out a comparative study of the transcriptional response of the
Ljgln2-2 mutant to drought stress under non-photorespiratory conditions together
with the stress situation produced as a result of impairment of the photorespiratory
cycle. In both cases, the stress conditions imposed caused massive transcriptomic
changes in the mutant that were not observed in the case of WT plants.
Transcriptome analysis showed that these two stress conditions triggered several
common responses in the mutant. A general repression of the genes involved in
carbon fixation and in the biosynthesis of photosynthetic pigments was observed
under both stress conditions. Moreover, genes involved in the response to oxidative
stress and in the biosynthesis of secondary metabolites (mainly flavonoids) were
among the most modulated by either drought or impaired photorespiration. Several
differences in the transcriptome were also observed among the two different stress
situations analyzed. A detailed comparative study of the two stress transcriptomes
will be presented using different bioinformatic tools.
Acknowledgements
Consejería de Economía, Innovación y Ciencia, Junta de Andalucía
(P10-CVI-6368, BIO163).
References
1) Díaz et al. (2010). New Phytol., 188: 1001-1013
2) Pérez-Delgado et al. (2013) Plant Physiol., DOI: 10.1104/pp.113.217216
99
Session 4
REGULATORY GENE NETWORK INTEGRATING NITROGEN AND CARBON
SIGNALING TO CONTROL ROOT NITRATE TRANSPORT.
Sandrine Ruffel1, Pascal Tillard1, Cécile Fizames1, Alain Gojon1, Rodrigo
Gutierrez2, Laurence Lejay1.
1INRA BPMP Montpellier France , 2Pontificia Universidad Católica de Chile
Email:
sandrine.ruffel@supagro.inra.fr
In Arabidopsis, the root NO3- transporter encoded by the NRT2.1 gene has a key
role for NO3- uptake at low concentration range in soil, as it accounts for up to 75
percent of the total activity of the high-affinity NO3- transport system. Unfortunately,
a wide range of regulatory mechanisms locking the NO3- transport level limits the
manipulation of a central gene like NRT2.1 as target for nitrogen use efficiency
improvement. Indeed, the level of NO3- transport is highly integrated and adjusted
to a combination of external and internal nutritional signals, allowing a proper
coordination of metabolism, development and growth. Thus, our objective is to
understand the molecular mechanisms leading to such integrative response by
deciphering the regulatory network governing root NO3- transporter expression and
activity. In order to build such gene regulatory network, a systems biology approach
starting with the integration of transcriptomic data with qualitative data about gene
interaction (e.g., transcription factors – putative target genes) has been undertaken.
A set of environmental conditions leading to variations of nitrogen and carbon
supply that triggers variations of NRT2.1 mRNA accumulation has been selected.
Whole genome transcriptomic response associated to this NRT2.1 variation has
been obtained and integrated through the VirtualPlant platform. A reduced gene
network including 3 transcription factors putatively controlling NRT2.1 expression
has been defined. A genetic approach has been chosen to validate this inferred
gene network, to understand possible interactions between these transcription
factors belonging to different families and, to determine the other physiological
functions that they control. This approach should allow us to iteratively build a
network whose predictive power will be improved at each cycle. The predictions
may help to anticipate and modulate NO3- uptake response to future environmental
conditions.
100
Session 4
INTEGRATED RNA-SEQ AND SRNA-SEQ ANALYSIS IDENTIFIES NOVEL
NITRATE-RESPONSIVE GENES IN ARABIDOPSIS THALIANA ROOTS
Elena A Vidal1, Tomás C Moyano1, Gabriel Krouk2, Manpreet S Katari3, Milos
Tanurdz R Tanurdzic4, W Richard McCombie4, Gloria M Coruzzi3, Rodrigo A
Gutiérrez1.
1FONDAP Center for Genome Regulation. Millennium Nucleus Center for Plant
Functional Genomics, Departamento de Genética Molecular y Microbiología,
Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago
8331010, Chile, 2Biochimie et Physiologie Moléculaire des Plantes, UMR 5004
CNRS/INRA/SupAgro-M/UM2, Institut de Biologie Intégrative des Plantes- Claude
Grignon, Montpellier, France; Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY 10003, USA , 3Center for
Genomics and Systems Biology, Department of Biology, New York University, New
York, NY 10003, USA, 4Cold Spring Harbor Laboratory, New York, USA
Email:
tcmoyano@uc.cl
Background Nitrate and other nitrogen metabolites can act as signals that
regulate global gene expression in plants. Adaptive changes in plant morphology
and physiology triggered by changes in nitrate availability are partly explained by
these changes in gene expression. Despite several genome-wide efforts to identify
nitrate-regulated genes, no comprehensive study of the Arabidopsis root
transcriptome under contrasting nitrate conditions has been carried out. Results In
this work, we employed the Illumina high throughput sequencing technology to
perform an integrated analysis of the poly-A + enriched and the small RNA fractions
of the Arabidopsis thaliana root transcriptome in response to nitrate treatments. Our
sequencing strategy identified new nitrate-regulated genes including 40 genes not
represented in the ATH1 Affymetrix GeneChip, a novel nitrate-responsive antisense
transcript and a new nitrate responsive miRNA/TARGET module consisting of a
novel microRNA, miR5640 and its target, AtPPC3. Conclusions Sequencing of small
RNAs and mRNAs uncovered new genes, and enabled us to develop new
hypotheses for nitrate regulation and coordination of C and N metabolism.
101
SESSION 5
NITROGEN INTERACTIONS
WITH OTHER
NUTRIENTS/SIGNALS
Session 5
INTERACTION BETWEEN NITRATE AND WATER CONDUCTIVITY IN MAIZE
Julie Dechorgnat1, Rebecca K Vandeleur1, Karen L Francis1, Kanwarpal S
Dhugga2, J Antoni Rafalski3, Steve D Tyerman1, Brent N Kaiser1.
1School of Agriculture, Food and Wine, University of Adelaide, Urrbrae, SA, 5064,
Australia, 2Pioneer HiBred, Johnston, Iowa, 50131, USA, 3DuPont Crop Genetics,
Wilmington, Delaware, 19803, USA
Email:
julie.dechorgnat@adelaide.edu.au
Nitrogen (N) uptake by plant roots is linked to the flow of water through the plant,
which provides the driving force for mass flow delivery of dissolved N to the root
surface. Previous studies have indicated a positive interaction between nitrate
supply and water conductance in roots across multiple plant species (Gorska et al.,
2010, Plant Soil). To characterise this interaction further, we examined how external
N influences water conductivity across a range of maize inbreds. In general, we
observed that N availability (starvation or re-supply of N after starvation) influenced
root hydraulic conductivity either positively or negatively depending on the inbred
tested and the respective N treatment. More detailed analysis across two
contrasting maize inbreds (a temperate line B73, and a tropical line F44) revealed a
strong contrast in root hydraulic conductivity to the presence or absence of
externally applied nitrate. Under N starvation, the hydraulic conductance of B73
roots increased compared to non-starved roots. In contrast, the F44 root system
showed a decrease in root hydraulic conductance under N starvation. This
difference in behaviour is correlated with an increased uptake rate and tissue
accumulation of nitrate in F44 plants compared to B73 plants. These findings
highlight a strong interdependence between nitrate and water conductivity in maize
roots and indicates a genotype dependent mechanism regulating water conductivity
relative to a nitrate signal.
103
Session 5
CYTOKININ AND NITRATE SIGNALING PATHWAYS INDUCE PRIMARY ROOT
GROWTH IN ARABIDOPSIS THALIANA.
Pamela A. Naulin1, Karem P. Tamayo1, Diana E. Gras1, Andrea Vega12, Javiera de
la Cruz1, Rodrigo A. Gutiérrez1.
1FONDAP Center for Genome Regulation, Millennium Nucleus Center for Plant
Functional Genomics, Departamento de Genética Molecular y Microbiología,
Pontificia Universidad Católica de Chile, Santiago, Chile, 2Pontificia Universidad
Católica de Chile, Santiago, Chile.Departamento de Ciencias Vegetales, Facultad de
Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile, Santiago,
Chile
Email:
ktamayo@bio.puc.cl
Nitrate can act as a potent signal to control growth and development in plants.
However, the mechanisms by which nitrate exert its signaling role to control
developmental processes are still poorly understood. We show that nitrate
stimulates primary root growth mainly by stimulating meristem activity via cytokinin
signaling. Citokinin perception and biosynthesis mutants exhibited shorter roots
when grown with nitrate as the only nitrogen source. Histological analysis of the root
tip revealed decreased cell division and elongation in the ahk2/ahk4 double mutants
as compared to wild-type plants under nitrate regime. Cytokinin perception mutants
and wild-type plants grown under nitrate conditions are indistinguishable at day 4
both at the molecular and histological level. However, 10 days after germination
there is no detectable meristem activity in the ahk2/ahk4 double mutant and global
analysis of gene expression revealed large changes in gene expression in the
ahk2/ahk4 double mutant as compared to wild-type plants. Transcriptomics
analysis identified important core cell cycle genes that may explain the observed
phenotypes. Our results provide strong evidence linking nitrate and cytokinin
signaling for the control of active cell division and elongation in the root tip under
nitrate conditions.
ACKNOWLEDGEMENTS
FONDECYT (1100698), ANR/CONICYT 007 and Millennium Nucleus for Plant
Functional Genomics (P06-009-F and P10-062-F).
104
Session 5
UBIQUITIN LIGASE ATL31 REGULATES PLANT GROWTH VIA 14-3-3
DEGRADATION IN RESPONSE TO C/N NUTRIENT CONDITION
Takeo Sato1, Shoki Aoyama1, Shigetaka Yasuda1, Shugo Maekawa1, Yoichiro
Fukao2, Junji Yamaguchi1.
1Faculty of Science, Hokkaido University, 2Plant Global Educational Project, Nara
Institute of Science and Technology
Email:
t-satou@sci.hokudai.ac.jp
Nutrient availability, in particularly the balance of carbon (C) and nitrogen (N) is one
of the most important factors for regulating plant metabolism and development.
However detailed molecular mechanisms mediating C/N signaling are not well
understood in higher plants. We isolated a novel ubiquitin ligase, ATL31, which
functions in the C/N nutrient response in Arabidopsis thaliana. In this study,
proteomics and biochemical analysis demonstrated that the ATL31 targets 14-3-3
proteins for ubiquitination and regulates the 14-3-3s stabilities in response to C/N
status. In addition, now we are further evaluating the physiological function of
ATL31 proteins with modified CO2 and nitrogen condition. We will report the
detailed biochemical and physiological functions of the ATL31 as the essential
regulator of C/N nutrient response.
105
Session 5
TOWARDS A BETTER UNDERSTANDING OF THE PHYSIOLOGICAL
FUNCTION OF PLANT GLUTAMATE DEHYDROGENASE
Therese C Terce-Laforgue1, Magali M Bedu1, Celine L Dargel-Graffin1, Frederic JP
Dubois2, Yves A Gibon3, Francesco M Restivo4, Bertrand J Hirel1.
1Adaptation des Plantes à leur Environnement, Unité Mixte de Recherche 1318,
Institut Jean-Pierre Bourgin, Institut National de la Recherche Agronomique (INRA),
Centre de Versailles-Grignon, RD 10, 78026 Versailles cedex, France, 2Equipe
d’Accueil Ecologie et Dynamique des Systèmes Antropisés (EDYSAN), Agroécologie, Ecophysiologie et Biologie Integrative (AEB), Faculté des Sciences, 33 rue Saint
Leu, 80039 Amiens cedex 1, France, 3Biologie du Fruit et Pathologie, Unité Mixte
Recherche 1332, Institut National de la Recherche Agronomique (INRA), Centre de
Bordeaux-Aquitaine, BP81, 71 avenue Edouard Bourlaux, 33883 Villenave d’Ornon
cedex, France, 4Dipartimento di Bioscienze, Università di Parma, Parco Area delle
Scienze 11/A, 43100 Parma, Italy
Email:
therese.laforgue@versailles.inra.fr
The enzyme glutamate dehydrogenase (GDH, EC 1.4.1.2) is in vitro able to
incorporate ammonium into 2-oxoglutarate to form glutamate and to function in the
opposite direction to deaminate glutamate. It has been clearly demonstrated
previously by the means of labeling experiments that the deamination reaction
occurs in higher plant cells. In order to obtain a better understanding of the
physiological function of GDH in leaves, transgenic tobacco (Nicotiana tabacum L.)
plants were constructed, that overexpress two genes from Nicotiana plumbaginifolia
(GDHA and GDHB under the control of the CaMV 35S promoter) encoding the
alpha and beta subunits of GDH individually or simultaneously. A physiological
analysis of the transgenic plants was led to evaluate the impact of increased GDH
activity on the plant growth and on the main metabolites representative of C and N
metabolism. In the transgenic plants, the GDH protein accumulated in the
mitochondria of mesophyll cells and in the mitochondria of the phloem companion
cells (CCs), where the native enzyme is normally expressed. Major changes in
carbon and nitrogen metabolite accumulation and a reduction in the growth of GDH
overexpressing lines were induced.
106
SESSION 6
NITROGEN-USE EFFICIENCY
AND ECOPHYSIOLOGICAL
ASPECTS OF NITROGEN
NUTRITION.
Session 6
DYNAMICS OF NITROGEN METABOLISM DEPENDS ON THE
ONTOGENETIC STAGES OF BROMELIADS
Cassia A Takahashi1, Helenice Mercier1.
1Laboratório de Fisiologia do Desenvolvimento Vegetal / Instituto de Biociências /
Universidade de São Paulo
Email:
cas-thi@uol.com.br
The stages of ontogenetic development of bromeliads can be an important
feature to be considered in the physiology studies. Young plants can be classified
as atmospheric, which absorb the nutrients as N sources mainly from dry
atmospheric deposition while the adult ones have a special structure formed by
closely imbricated leaves called tank. Organic debris and water from stem-flow or
fall-through can be accumulated inside the tank and used as nutrients by epiphytic
bromeliads. Organic or inorganic N sources from the debris decomposition can be
absorbed by foliar trichomes which cover the whole basal leaf blade. The main
objective of this study was to verify the existence of differences in the N assimilation
arising from the use of distinct N sources in epiphytic bromeliad Vriesea gigantea
with different stages of development. A nutrient solution, consisting 5 mM of total N,
was used. Three different forms of N sources were employed: NH4+, NO3- or urea.
Different portions of the leaves (apex, middle and base) and roots were harvested in
six different times and the samples were used in enzymatic assays of urease, NR,
GS and GDH and in determination of NO3-, urea and NH4+ endogenous contents.
According to the results, the roots of young bromeliads can have an important role
in the absorption of N from environment since the highest NR or urease activities
were detected in roots which were supplied with NO3- or urea respectively. When
the bromeliad develops a tank, the basal leaf portion can start to make a similar
function of the roots of young bromeliads since, in the base, the values of NR or
urease activities were as high as those observed in the roots of young plants.
Moreover, atmospheric bromeliads might absorb and assimilate NO3- faster than
urea while the tank bromeliad showed an inverted tendency. The tank development
might be a crucial moment when the bromeliads change their metabolism to absorb
and assimilate mainly the N from organic sources.
108
Session 6
EXPLORING NITRATE UPTAKE EFFICIENCY IN A CORE COLLECTION
COVERING MAIZE GENETIC DIVERSITY
Isabelle N Quillere1, Celine L Dargel-Graffin1, Joel J Talbotec1, Bertrand J Hirel1.
1Adaptation des Plantes à leur Environnement, Unité Mixte de Recherche 1318,
Institut Jean-Pierre Bourgin, Institut National de la Recherche Agronomique (INRA),
Centre de Versailles-Grignon, RD 10, 78026 Versailles cedex, France
Email:
Isabelle.Quillere@versailles.inra.fr
Nitrogen use efficiency (NUE) in crops can be defined as the grain yield per unit of
available nitrogen (N) already present in the soil and added as N fertilizer. Thus,
improving NUE in crops is a way of reducing both the cost and the detrimental
environmental effects associated with N fertilization. NUE is the product of N uptake
efficiency and N utilization efficiency. At high N input, variation in NUE was
explained by variation in N uptake capabilities. Generally, cereals such as maize are
inefficient at acquiring N from the soil. Thus, identifying genotypes that are more
efficient in capturing mineral N resources and identifying both the phenotypic traits
and the biological mechanisms controlling the ability of maize to take up N is of
major importance. To explore N uptake efficiency, a core collection of nineteen
inbred lines covering the genetic natural diversity of maize was grown in
hydroponics under non-limiting N supply. When the plants had 4 visible leaves,
NO3- uptake was measured using 15N labeled NO3-. In addition, N uptake of
mature plants grown in a greenhouse during the plant development cycle was
measured and compared to that of young plants grown under hydroponic
conditions. In parallel, the architecture of the root system including seminal and
nodal roots was analyzed using the WinRhizo software and the level of expression
of a number of NO3- transporters was measured using Real-time Q-PCR. We found
that within this natural population covering maize genetic diversity there is a very
strong variability for both roots architecture and NO3- uptake efficiency.
109
Session 6
SPECIFIC LEAF AREA AND FOLIAR NITROGEN CONTENT IN TREE SPECIES
OF LOWER MONTANE ATLANTIC RAIN FOREST ,CARAGUATATUBA, BRAZIL
Janaina G. Silva1, Marcos P.M. Aidar2.
1University of Campinas – UNICAMP, 2Institute of Botany, IBT
Email:
janainagomes@ig.com.br
Specific leaf area (SLA) is a measure related to growth rate, leaf longevity, carbon
and nitrogen assimilation. Among species is an important feature to determine their
competitive abilities. Tropical trees species are distinguished in different ecological
groups according growth attributes, shade tolerance and life span. Pioneer show
differences in leaf morphological and physiological attributes relative to non-pioneer
tree species. In this context, the aim of the study was to compare SLA and leaf N
content in 31 tree species belonging to different ecological groups in Lower
Montane Atlantic Rain Forest (Caraguatatuba, São Paulo, Brazil) and assessing the
effect of seasonality. Samples was taken during the winter and summer, tree
species chosen belong to 11 families and different ecological groups (Pionner - PS,
Early secondary – ESS, Early seconday leguminous - ESSL and Late secondary –
LSS). Leaf N content showed no significant differences between seasons. Only LSS
showed significant differences in SLA. PS, ESSL and ESS did not show differences
between seasons, so this can indicate that LSS can be influenced by seasonality
(lower precipitation and temperature during winter than summer). There is a positive
correlation between SLA and leaf N content, i.e., when higher SLA, there is a
tendency to higher leaf N content. SLA showed significant differences across
different ecological groups. PS had higher values in SLA compare with other
groups. This study produced results which corroborate the findings of studies
concerning SLA and ecological groups. LSS showed the lowest values of SLA. Leaf
N content showed differences among groups with ESSL showing the highest
values, followed by PS. These results show SLA and leaf N content are related to
ecological groups and the competitive abilities of species. Moreover LSS are
affected by seasonality.
110
Session 6
IMPROVED NITROGEN USE EFFICIENCY BY ROOT-SPECIFIC EXPRESSION
OF ALANINE AMINOTRANSFERASE
Jingwen Tiong1, Nenah MacKenzie2, Ramya Sampath1, Sayuri Watanabe1, Jean C
Kridl3, Brent N Kaiser2, Mamoru Okamoto1.
1Australian Centre for Plant Functional Genomics, Hartley Grove, Urrbrae, SA 5064,
Australia, 2School of Agriculture, Food and Wine, University of Adelaide, Urrbrae,
SA 5064, Australia, 3Arcadia Biosciences, 202 Cousteau Place, Suite 200, Davis,
California 95618, U.S.A.
Email:
jingwen.tiong@acpfg.com.au
Nitrogen is a limiting factor for cereal crop production. The ability to obtain
nitrogen (N) from fertilizer is a critical limiting step in the efficient use of N.
Development of crop plants with improved N uptake and utilization is therefore an
important aim in agricultural research. We adopted a transgenic approach as an
attempt to achieve this by expressing the root specific abiotic stress-inducible
OsAnt1 promoter-driven HvAlaAT (OsAnt1/HvAlaAT) in wheat, barley and rice. This
transformation resulted in a significant increase in biomass and grain yield in wheat
compared to control lines when grown under adequate N. Preliminary analysis also
showed that the expression of OsAnt1/HvAlaAT was upregulated significantly under
high N (5mM) compared to low N (1mM) in hydroponics. Collectively, these results
suggest that the expression of HvAlaAT in wheat could enhance N use efficiency
under adequate N conditions, resulting in better growth and yield. Additional tests
are underway to study the metabolomics and transcription profile in the wheat lines,
which will help determine the molecular characteristics contributing to the
phenotypes observed. Tissue N distribution of these lines will also be investigated
by using 15N pulse-chase labelling. In addition, 15N uptake studies will be
undertaken to determine N influx activity in the transgenic rice lines. Observations
from this research will help in the effort to enhance N use efficiency in crop plants.
111
Session 6
EFFECTS OF CO2 ENRICHMENT ON THE GROWTH AND METABOLISM
OF ARABIDOPSIS THALIANA UNDER THE CONSTANTLY
NITROGEN-LIMITED CONDITIONS
Nobuyuki Takatani1, Marie Mori1, Tetsuro Miyamoto1, Takatoshi Kiba2, Shin-ichi
Maeda3, Tatsuo Omata1.
1Graduate School of Bioagricultural Sciences, Nagoya University, 2RIKEN Plant
Science Center
Email:
takatani@agr.nagoya-u.ac.jp
CO2 concentration and nitrogen nutrients could become the limiting factors of
plant growth. It has been observed that elevated CO2 leads to stimulation of growth
under nitrogen-sufficient conditions. However, effects of elevated CO2 on the
growth under the "constantly nitrogen-limited conditions", which are relevant to
most natural habitats of plants, are still unclear because of difficulties in maintaining
such conditions in experiments. Here, we kept Arabidopsis thaliana under the
constantly nitrogen-limited conditions by growing the mutant with reduced nitrate
uptake activities on a medium containing nitrate as the sole nitrogen source and
examined the effects of elevated CO2. While the mutant growing under low-CO2
conditions (280 ppm) showed no visible phenotype as compared to the wild type,
the mutant growing under high-CO2 conditions (780 ppm) showed the well-known
symptoms found in nitrogen-starved plant, e. g., a decreased shoot/root ratio, a
reduced nitrate content and an accumulation of anthocyanin. An increased
chlorophyll content was, however, contradictory to the known responses to
nitrogen-deficiency. This symptom is considered to be a specific response to
elevated CO2 in plants kept under the constantly nitrogen-limited conditions. The
metabolite profile showed that the levels of the most metabolites, e. g., amino acids
and TCA cycle intermediates, were affected by elevated CO2. However, these
alterations were commonly observed in the wild type and the mutant, suggesting
that the mutant under the constantly nitrogen-limited conditions maintained the
nitrogen and carbon metabolism in the same way as the nitrogen-sufficient plants.
112
Session 6
HIGH YIELD AND HIGH PROTEIN IN WHEAT: IS IT PREDICTABLE IN A
MEDITERRANEAN ENVIRONMENT?
Trevor P Garnett1, Julian Taylor2, Rob Wheeler3, Vanessa Melino1, Sigrid Heuer1.
1Australian Centre for Plant Functional Genomics, Waite Research Institute,
University of Adelaide, Adelaide, South Australia, 5064, AUSTRALIA, 2School of
Agriculture, Food and Wine, Waite Research Institute, University of Adelaide,
Adelaide, South Australia, 5064, AUSTRALIA, 3South Australian Research and
Development Institute, Waite Campus, Adelaide, South Australia, 5064, AUSTRALIA
Email:
trevor.garnett@acpfg.com.au
Improved nitrogen use efficiency (NUE), whilst maintaining high yield, is a goal for
most cereal breeders. For wheat, improved NUE cannot compromise grain protein
as the latter is used in quality assessment and pricing of wheat. High grain yields are
typically associated with reduced grain protein and timely fertiliser applications
before grain filling are aimed at alleviating this. In water limited Mediterranean
environments, maximising grain protein whilst maintaining grain yield by increasing
nitrogen (N) fertiliser application is further complicated because too much N can
lead to excessive biomass growth and “haying off”, resulting in pinched grains.
Germplasm that maintain grain protein with high yield would help address this
problem. Researchers aiming to identify such germplasm have utilised the grain
protein deviation; genotypes above the negative regression line of grain protein
plotted against grain yield. However, these efforts are hampered by environmental
effects masking genetic effects. To understand the relationship between grain
protein and grain yield, we have analysed wheat variety evaluation trials carried out
in South Australia, a Mediterranean farming region of Australia with average yields of
2.5 t/ha. Trials were carried out over 26 sites spread throughout the region with 37
varieties grown over 4 years. There were large differences between sites but the
classic negative relationship reported in the literature was only observed on less
than a quarter. The relationship obtained by averaging all sites did not accurately
reflect those relationships seen at individual sites making it difficult to find varieties
that consistently maintained high protein with high yield. These results will be
discussed in the context of the wider literature relating to this area.
113
Session 6
NITRATE AND AMMONIUM UPTAKE IN SUGARCANE: AN IN VITRO
APPROACH TO CHARACTERISE NITROGEN USE EFFICIENCY
Elliosha Hajari1, S. Snyman1, M. P. Watt2.
1School of Life Sciences, University of KwaZulu-Natal, Westville campus, Private
Bag X54001, Durban, 4000, South Africa. South African Sugarcane Research
Institute, Private Bag X02, Mount Edgecombe, KwaZulu-Natal, 4300, South Africa,
2South African Sugarcane Research Institute, Private Bag X02, Mount Edgecombe,
KwaZulu-Natal, 4300, South Africa
Email:
elliosha.hajari@sugar.org.za
At present, the assessment of N-use efficiency of sugarcane cultivars is based on
field and pot trials. As this takes 9 to 24 months, the aims were to establish an in
vitro protocol and investigate its merit in determining N-use characteristics of
cultivars from the breeding programme. In vitro plants were multiplied from
meristems and starved of N for 4 days prior to NO3--N and/or NH4+-N (20 mM total
N) supply. Up to 85% of N was used in 7 days with a positive correlation between
fresh mass and N uptake. Hence, all other studies employed plants of 0.27 - 0.3 g
fresh mass, with sampling at 3 and 7 days. N uptake was then investigated at high
and low N (20 and 2 mM total N, respectively). By day 3, plants used significantly
more NH4+-N than NO3--N (520.38 vs. 357.22 µmoles g-1 DM) when N was
provided as combined NH4+-N and NO3--N at 10 mM each. However, when N was
supplied singly as either 20 mM NO3--N or 20 mM NH4+-N, the plants used similar
amounts (849.06 µmoles NO3--N g-1 DM and 859.96 µmoles NH4+-N g-1 DM).
Similar trends were observed at day 7 and at low N. Characterisation of N uptake
kinetics at 2 mM N, revealed a constitutive high affinity transport system (HATS) for
NH4+-N, while the NO3--N HATS was induced after 3 h. Plants had a similar Vmax
for each form (26.21 and 28.71 µmoles g-1 DM h-1 for NO3--N and NH4+-N,
respectively) but a higher affinity for NO3--N (Km of 0.02 mM for NO3--N vs. 0.06
mM for NH4+-N). At 20 mM N, plants exhibited a significantly higher Vmax for
NO3--N (28.66 µmoles g-1 DM h-1) than NH4+-N (19.51 µmoles g-1 DM h-1), but a
higher affinity for the latter (Km of 2.08 mM for NH4+-N vs. 7.38 mM for NO3--N).
Ongoing work includes: N uptake kinetics at 4 mM N; N-use efficiency in vitro; and
preliminary screening of five cultivars. These results will be used to further define the
in vitro protocol and ascertain if varietal differences in N use can be discerned in
vitro, and if they correlate with those obtained from pot and field trials.
114
Session 6
EXPLORING NATURAL VARIATION OF ARABIDOPSIS ROOT SYSTEM
ARCHITECTURE IN RESPONSE TO NITRATE
Jérôme De Pessemier1, Fabien Chardon2, Pascal Tillard3, Philippe Nacry3,
Christian Hermans1
1 Laboratory of Plant Physiology and Molecular Genetics, Université Libre de
Bruxelles, Brussels, Belgium, 2 Institut Jean-Pierre Bourguin, INRA, UMR 1318,
Versailles, France, 3 Université de Montpellier II, Biochimie & Physiologie Moléculaire des Plantes, UMR CNRS-INRA-SupAgro, Montpellier, France
Email: jedepess@ulb.ac.be
Arabidopsis thaliana has a broad geographical distribution and consequently is
subject to varying environments which makes it a useful model for studying
adaptation and selection. The natural populations which grow on a wide range of
soil conditions could provide a rich source of genetic diversity to explore potentially
adaptive differences in root architecture in response to nitrate availability. We have
already described the root phenotypes (biomass production, primary root length,
lateral root number, total lateral root length, lateral root density) of a core collection
of 24 accessions grown in vitro upon contrasted nitrate supplies (10 µM and 10 mM
NO3-). That study illustrated that natural variation existed within Arabidopsis for root
traits, which were primarily genetically determined (Mech. Dev. 130: 40-53).
Nonetheless, differences between accessions were somewhat more pronounced at
low than at moderate N supplies. In addition, nitrate uptake was measured using
15N tracer in order to corroborate if accessions with a highly branched root system
have higher uptake efficiency. However no robust correlation was found between
root traits and N uptake. The identification of root morphology ideotypes in the N
response was the foundation for further analysis of quantitative traits for root
morphology. Currently, we are fine-mapping several QTL intervals, which were
defined by screening recombinant inbred lines (sets of ~160 lines) generated from
the cross between contrasted (Bur, Cvi, Jea and Tsu) and reference (Col)
accessions. In a recent study, a larger data set was generated with ~350 accessions
from the HapMap collection. That data set represents a solid basis for genome-wide
association (GWA) mapping strategy, in order to identify genes and alleles
responsible for the natural variation of root traits. The initial GWA output supports
the nomination of a number of loci identified in the cell cycle and hormonal
pathways.
115
Session 6
GENETIC FACTORS INFLUENCING ROOT MORPHOLOGY IN RESPONSE
TO NITRATE SUPPLY IN BRASSICACEAE MODEL AND CROP SPECIES
Jérôme De Pessemier1, Ian Bancroft2, Daniel R. Bush3, Nathalie Verbruggen1,
Christian Hermans1
1Laboratory of Plant Physiology and Molecular Genetics, Université Libre de
Bruxelles, Brussels, Belgium, 2Department of Biology, University of York, Heslington, United Kingdom, 3Department of Biology, Colorado State University, Fort
Collins, Colorado, USA
Email: chermans@ulb.ac.be
Modifying root architecture to capture nutrients more efficiently may represent a
sustainable solution to maintain crop productivity whilst reducing fertilizer input. The
goal of our research is to first discover the physiological and molecular mechanisms
that underpin Nitrogen Use Efficiency (NUE) in the model species Arabidopsis and
then to exploit this knowledge to improve closely related Brassica crops using a
model-to-crop framework. Understanding how lateral roots are initiated and how
they emerge from the parent root is vitally important for improving crop yields. This
holds particularly true for the influence of nitrogen (in particular nitrate species)
supply on root architecture. We are trying to gain better knowledge about
mechanisms of lateral root growth stimulation or repression by nitrate availability.
Forward genetic dissections and natural screens are currently used to identify key
genes that shape root system architecture, in order to eventually draw strategies to
improve nutrient uptake. (i) We have identified Arabidopsis mutants, whose root
phenotype is conditional on the nitrate supply (absence of lateral root elongation
exerted by high nitrate). By using positional cloning, we successfully identified the
mutations in CHITINASE-LIKE 1 (Plant Physiol 152: 904-917; 157: 1313-1326) and
other genes (unpublished results). (ii) We are screening diversity panels which
represent structured samplings of the diversity across the gene pools of oilseed
rape and its progenitor species. We have recently phenotyped ~100 BnASSYST
lines and observed that genetic variation also exists for root biomass allocation and
root morphological traits in the crop response to nitrate supply as in the model
species. We will then proceed to genome wide-association for root traits with
genetic maps currently developed, helped with the aligning syntenic chromosomic
regions of Arabidopsis identified in (i).
116
Session 6
IDENTIFICATION OF NOVEL MOLECULAR FACTORS AFFECTING
NITROGEN USE EFFICIENCY IN ARABIDOPSIS THALIANA
Viviana Araus1, Elena A. Vidal1, Tomas Puelma1, Andrea Vega1, Rodrigo
Gutiérrez1
Center for Genome Regulation. Millennium Nucleus Center for Plant Functional
Genomics. Departamento de Genética Molecular y Microbiología. Pontificia
Universidad Católica de Chile.
Email:
Nitrogen (N) is an essential macronutrient for plants and its availability is a key
factor determining plant growth and productivity. To meet the increasing food
demand, one of the main agricultural practices to increase yield is to use of
N-fertilizers. However, their massive use is limited by their high cost and their
important detrimental environmental impacts. A major challenge involves
indentifying the key factors determining crop nitrogen use efficiency (NUE). Despite
the importance of understanding these processes, little is known about the
molecular mechanisms regulating NUE. Toward this goal, we used a bioinformatics
tool to find genes involved in NUE in A. thaliana. The most connected gene in the
search was a scaffold protein that acts as a transcriptional co-regulator in A.
thaliana, NE1. We evaluated NUE in an overexpressor line of NE1 (35s::NE1) and we
found a significant decrease in the NUE, specifically for N-limiting condition. We
also evaluated the phenotype of 35s::NE1 and we found a decrease in primary root
length and biomass also in N-limiting condition. We have evaluated the expression
of other genes predicted with a functional connection with NE1 and we found that
NE1 represses NTR2.1 and Glutamine synthetase 2. Thus, NE1 might control NUE
by controlling nitrate uptake and availability in Arabidopsis.
Acknowledgment: Milenio-P10-062-F, Fondap-15090007, Fondecyt-1100698,
HHMI, Beca de Estudios de Doctorado CONICYT, Beca de apoyo a la tesis
doctoral 24121609.
117
SESSION 7
NITROGEN NUTRITION IN
PLANT AND BACTERIAL
SYSTEMS
Session 7
TRANSCRIPTOME CHANGES IN ROOTS OF BARLEY GROWN UNDER
DIFFERENT NITROGEN FERTILIZATION LEVELS AND EXPOSED TO HYPOXIA
Ana Clarissa Negrini12, Diana Garnica1, Benedict Long1, John Evans1, Brent N
Kaiser3, Harvey Millar4, Jean C Kridl5, Owen Atkin1
1Research School of Biology, Australian Nation University , 2Embrapa Vegetables,
3School of Agriculture Food and Wine, University of Adelaide, 4ARC CoE in Plant
Energy Biology, University of Western Australia, 5Arcadia Biosciences Inc. Davis,
CA USA
Email: ana.alves@anu.edu.au
Waterlogging due to soil flooding results in low soil-oxygen availability (hypoxia)
for the plant root system which leads to a reduction of plant growth and crop yield.
Plants respond to abiotic stresses such as hypoxia by transcriptional changes that
induce metabolic and physiological changes. In roots subjected to hypoxia, one of
the most relevant changes involves up-regulation of genes linked to pyruvate
metabolism which switches from oxidative to fermentative mode. The change is
associated with an increase in cytoplasmic acidosis and reduction of respiration.
Nitrate can improve the tolerance of plants to hypoxia, however the mechanism
behind this improved tolerance is not well understood. To gain insights into how
nitrate supply affects the response of barley plants to hypoxia at the transcriptome
level, RNA-seq gene expression profiling was performed. Barley cv. Golden
Promise was hydroponically grown in low and high nitrate for three weeks in an
ebb-and-flow system and subsequently transferred to a static solution system with
normal aeration for one day for acclimation. Plants were subjected to hypoxia by
sparging the nutrient solution with N2 for 6 hours with the oxygen concentration
maintained around 12% of saturation. The normoxia condition was achieved by
sparging with air for the same period. RNA-seq was performed with 3 replicates per
treatment (hypoxia and normoxia at low and high nitrate levels) and each replicate
was represented by a pool of 12 plants. The transcriptome changes associated with
hypoxia in roots of plants grown under the two different levels of nitrate will be
discussed.
119
Session 7
DIAZOTROPHIC BACTERIA IN SPECIFIC LEAF PORTIONS OF THE EPIPHYTIC
BROMELIAD GUZMANIA MONOSTACHIA
Carolina K Kleingesinds1, Marcos PM Aidar2, Helenice Mercier1,
1Instituto de Biociências, Universidade de São Paulo,São Paulo, SP, Brasil,
2Instituto de Botânica de São Paulo, São Paulo, SP, Brasil
Email:
ckk@usp.br
The distinct leaf portions of the tank epiphytic bromeliad Guzmania monostachia
(L.) Rusby ex Mez var. monostachia perform different functions. The apical leaf
portion has higher stomatal density, chlorophyll and carotenoids contents and also
higher incident photosynthetically active radiation. Consequently, this leaf portion is
more related to performing photosynthesis. On the other hand, basal portion show
higher trichome density, larger hydrenchyma thickness and higher nitrate reductase
activity, indicating that this region has higher capacity of water and nutrient
absorption. Since the epiphytic plants are subjected to intermittent supply of water
and nutrients, the association with microorganisms may play an important role. This
study aimed to verify whether nitrogen fixing bacteria (diazotrophic bacteria) are
present in distinct leaf portions (apical, middle and basal) and if they are in the
exterior or interior of the leaves. The leaves were collected in their natural
environment (CE, Brazil) and the material was sliced into the tree portions
mentioned. Afterwards, plant material was submitted to sonication to separate
bacteria from the leaf surface (epiphytic bacteria). Subsequently, each leaf portion
was submitted to antisepsis procedure for final maceration and investigation of
bacteria present inside the plant tissues (endophytic bacteria). Four different media
(NFb, JNFb, LGI e LGD) were used for bacterial growth. These culture media are
free of reduced nitrogen. Selection of diazotrophic bacteria was assessed by
acetylene reduction assay (ARA). The outcomes show a higher number of ARA+
epiphytic than endophytic colonies. In both circumstances we found larger number
of colonies in the basal portion, suggesting that diazotrophic bacteria are localized
mainly in this foliar region, where they might fix atmospheric nitrogen which might
be available to the bromeliad mainly during periods of nutritious starvation.
120
Session 7
TRANSCRIPTOMICS ANALYSIS OF LEAVES OF LOTUS JAPONICUS PLANTS
GROWN UNDER DIFFERENT NITROGEN REGIMES: DIFFERENTIAL
EXPRESSION OF GENES FOR AMMONIUM ASSIMILATION.
Carmen M Pérez-Delgado1, Tomás C Moyano2, Margarita García-Calderón1,
Antonio J Márquez1, Rodrigo A Gutiérrez2, Marco Betti1,
1Departamento de Bioquímica Vegetal y Biología Molecular, Facultad de Química,
Universidad de Sevilla, Calle Prof. García González 1, 41012 Sevilla, Spain,
2Departamento de Genética Molecular y Microbiología, Facultad de Ciencias
Biológicas, Pontificia Universidad Católica de Chile, 833101, Santiago, Chile.
Email: cmperez@us.es
Legume plants are able to grow using atmospheric N2 fixed by rhizobacteria in
root nodules or by using mineral sources of nitrogen. We evaluated the
consequences of different N sources on plant growth and gene expression in the
model legume Lotus japonicus. A comparative transcriptomic study was carried out
in leaves of plants grown with NO3-, NH4+ or NH4NO3 as N sources or under
conditions of biological nitrogen fixation (Nod). We identified 609 differentially
expressed genes when comparing mineral nutrition versus Nod conditions by Rank
Product analysis with FDR correction. These genes were analysed using the
Sungear software tool, which generalizes Venn diagrams to compare groups of
genes. The analysis showed that N and secondary metabolism (especially
phenylpropanoids) were the most differentially expressed pathways in N-supplied
plants. The expression of genes involved in NH4+ assimilation was measured by
qRT-PCR for the N regimes considered. Transcript levels for several of these genes
paralleled differences observed in plant growth among the different N sources.
Recent studies also indicate endogenous photorespiratory NH4+ has an important
effect on N genes transcription (1). To evaluate this, we grew plants under normal
(active photorespiration) or CO2 enriched atmosphere (suppressed
photorespiration) conditions and with different N regimes. Our results indicate Lotus
gene expression response to high CO2 depends on the N source. Further insights
into the interaction between photorespiration and the transcriptomic responses to N
will be presented using a L. japonicus mutant with an impaired photorespiratory
cycle. Acknowledgements: Consejería de Economía, Innovación y Ciencia, Junta de
Andalucía (P10-CVI-6368, BIO163). References: 1) Pérez-Delgado CM,
García-Calderón M, Sánchez DH, Udvardi MK, Kopka J, Márquez AJ, Betti M (2013)
Plant Physiol. 162 (http://dx.doi.org/10.1104/pp.113.217216)
121
Session 7
ISOLATION OF NITROGEN-FIXING BACTERIA FROM LUPIN AND THEIR
MICROENCAPSULATION BY SPRAY DRYING TECHNIQUE
Daniela C. Campos1, Francisca F. Acevedo2, Eduardo E. Morales3, Veronique V.
Amiard3, Milko A. Jorquera2.
1Biotecnologia, Facultad de Ciencias Agropecuarias y Forestales, Universidad de
La Frontera, Temuco, Chile, 2Scientific and Technological Bioresource Nucleus,
Universidad de La Frontera, Temuco, Chile , 3Centro de Genómica Nutricional Agro
Acuícola, Temuco, Chile
Email: d.campos02@ufromail.cl
Yellow lupin (Lupinus luteus) is considered a specie attractive for sustainable
agriculture of southern Chile because to its high protein content in seeds. The
nitrogen (N)-fixing bacteria (NFB) inoculation has proven to be a potential strategy of
sustainable N fertilization. However, the low survival and prevalence of NFB under
field conditions is the main limitation to use this technology in Chilean agriculture.
Thus, diverse polymers and microencapsulation techniques are being studied to
improve survival and prevalence of NFB in fields. Here, we isolated on yeast
mannitol (YM) agar diverse NFB strains from the rhizosphere of lupin plants grown in
southern Chile. Ten strains were identified by 16S rRNA gene sequencing and one
strain showing highest growth rate in YM broth was selected and evaluated its
microencapsulation by spray dying technique, using six different combinations of
alginate-maltodextrin polymers (1:14, 2:13, 2:28, 4:26, 0:30, and 0:15). Our results
showed the isolation of NFB belonging to genera Enterobacter, Stenotrophomonas,
Ochrobactrum, Klebsiella, Achromobacter and Pseudomonas. The results also
showed to Klebsiella sp. 14 as the strains with highest growth rate (0.8 absorbance
at 600 nm) after 7 h incubation compared to other strains (0.5-0.6 absorbance). On
the other hands, the combination 1:14 of alginate-maltodextrin polymers was
selected as wall material for microencapsulation because a decrease of bacterial
viability (10e+7 CFU g-1 powder) was not observed after microencapsulation. This
study reveals that NFB microencapsulation by spray drying technique using
alginate-maltodextrin mixture represents an economic, effective and scalable
alternative for application of NFB as sustainable N fertilization in lupin crops in
southern Chile. Acknowledgments: FIA PYT-2012-088 and Fondecyt no. 1120505.
122
Session 7
EFFECT OF BACTERIA ASSOCIATION IN WHEAT BIOMASS GROWN UNDER
DIFFERENT SOILS
Eliane Cristina Gruszka Vendruscolo1, Elisiane Inês Dall´Oglio Chaves2, Vanessa
Suzane Schneider1, Maiara Pasuch Camargo1, Joel Antonio Cordeiro de Abreu1,
Vandeir Francisco Guimarães2, Marise Fonseca dos Santos1,
1LABIOGEN-Universidade Federal do Paraná, 2Universidade Estadual do Oeste do
Paraná
Email: egvendru@gmail.com
Diazotrophs are well characterized by promoting biological nitrogen fixation (BNF)
and plant growth (PG), however it is known the dependence of the strain x genotype
interaction to observe gains by this association. The aim of this study was to
evaluate wheat cultivar CD120 associated with two bacterial isolates (UFPR-14 and
UFPR 87) obtained from plant baits in western Paraná according to BNF and PG.
Azospirilum brasilense AbV5 strain was used as control. The experiment was
conducted at the greenhouse, matching the presence /absence of bacteria, NPK
and ammonium sulphate in 14 treatments. The experiments were conducted in two
soil types: low fertility and high fertility. Seeds of wheat were inoculated with 106
células.mL-1, in pots containing 4.5 kg of soil. After 45 days of germination, the
seedlings were collected and analyzed according to the following parameters:
shoots fresh and dry weight, total nitrogen (TN) and microbial counting in terms of
epiphytic and endophytic bacteria (CFU). As results, it was observed the presence
of epiphytic bacteria in all treatments but the treatments with UFPR-14 strain
showed high number endophytically (27.103 CFU.mL-1). The soil fertility had a high
influence on wheat biomass. On high fertility (OM contents = 41.6 g.dm3), bacterial
inoculation did not improve TN or biomass, however, under low fertility conditions
(OM contents = 1.34 g.dm3) biomass was increased and the best response was got
with UFPR-14, increasing plant biomass in 59% compared to control. The results
showed positive responses in plant growth by association with plant growth
promoting bacteria, notably under low fertility conditions. Strains isolated by the
group suggest the use of UFPR-14 as biofertilizer, reducing costs, improving
productivity and agricultural sustainability.
123
Session 7
SOYBEAN-BRADYRHIZOBIUM JAPONICUM-SOYBEAN MOSAIC
VIRUS TRIPLE INTERACTION
Marianela S Rodriguez1, D D Peshev2, Fillip F Rolland2, W van Den Ende2, German
L Robert1, Nacira B Muñoz1, Rodrigo R Parola1, Hernán R Lascano13.
1Instituto de Fisiología y Recursos Genèticos Vegetales (IFRGV-CIAP-INTA).
Córdoba Argentina, 2Molecular Plant Biology. KU Leuven Belgium, 3Cátedra de
Fisiología Vegetal. Fac. Cs. Exact. Fis y Nat. UNC. Córdoba. Argentina
Email: hrlascano@hotmail.com
Legume-rhizobum symbiotic interaction is a very sensitive process ending in the
nodule formation where the interchanges of sugars from plant and fixed nitorgen
from the bacteria occur. The virus infection provokes marked alteration of plant
primary metabolisms, inducing changes in carbon-nitrogen relationships and
impairment on growth. The soybean culture has a great economic importance and
the environmental conditions that diminish its production are being intensively
studied. In the present work, the triple interaction among soybean- Bradyrhizobium
japonicum (Bj)- Soybean Mosaic Virus (SMV) was studied, evaluating leaf surface,
nodule number, ureides and sugars changes. Different sequences of inoculation
with Bj or infection with SMV were conducted. All the SMV infected plants showed
the typical chlorotic symptom. The strongest chlorotic symptom was observed in
previously virus infected plant and then inoculated with Bj. The virus infection also
provokes leaf growth decrease and changes in the levels of sugars (sucrose,
fructose, glucose, maltose and trehalose) and ureides from the appearance of the
symptom. In Bj inoculated plant, SMV infection also induces a significant decrease
in nodule number. However, the physiological alteration induced by SMV infection
was lower in plant previously inoculated with Bj. Interestingly, the Bj inoculated
plants showed trehalose increases in leaves, roots and nodules, however, a
previous virus infection inhibit the trehalose increases induced by Bj inoculation. Our
results suggest that legume-rhizobium symbiotic interaction could modulate the
virus-induced alteration on primary metabolism.
124
Session 7
UREIDE SYNTHESIS, ACCUMULATION AND TRANSPORT IN ARABIDOPSIS
PLANTS UNDER SALT AND OSMOTIC STRESS
Carlos I Lescano1, Carolina Martini2, Tomás M Tessi2, Claudio A González2,
Marcelo Desimone1,
1IMBIV-CONICET, 2Cátedra de Fisiología Vegetal. FCEFyN-UNC
Email: ignaciolescano@gmail.com
The ureides allantoin and allantoic acid play a central role in nitrogen transport in
nodulating tropical legumes. However, the complete enzyme set for ureide
synthesis and a family of ureide permeases are widely distributed in the plant
kingdom suggesting their participation in physiological processes not properly
characterized yet. In Arabidopsis, microarrays studies showed an upregulation of
ureides synthesis genes (xanthine dehydrogenase, uricase) during abiotic stresses.
On the contrary, allantoinase gene expression is strongly reduced after stress
suggesting that allantoin may accumulate in the cells.
We observed accumulation of allantoin in Arabidopsis plants under osmotic and
salt stresses. This effect was exacerbated in plants grown with ammonium as
nitrogen source and suppressed in the presence of sucrose as carbon source. The
analysis of two independent T-DNA insertion lines, causing knockout of allantoinase
(alla-1 and alla-2) showed constitutively elevated concentrations of allantoin, but a
noticeable morphological phenotype remained elusive. To determine the
physiological relevance of allantoinase gene repression on resistance to stress,
transgenic lines were generated on the genotype alla-1, in which the coding
sequence of allantoinase was introduced under the control of the stress inducible
promoter RD29A. As expected, alla-1 pRD29A::Aln plants were not able to
accumulate allantoin under stress conditions. The phenotype of KO and
RD29A:ALN plants was analysed under salt and osmotic stress conditions. In
addition, the phenotype of a KO mutant of AtUPS5 (ups5) was analysed. AtUPS5
transports allantoin, is expressed in the root cortex and endodermis and its
expression increases during salt and osmotic stress, suggesting a rol in
long-distance transport of allantoin during stress.
125
Session 7
EFFECT OF POST-EMERGENCE HERBICIDES ON BACTERIAL COMMUNITY
STRUCTURE IN SOIL TREATED WITH UREA
Marileo, L.G.1, Jorquera, M.A.2, Briceño, G.S.2,Demanet, R.F.2,Fernandez, T.M.1,
Mora, M.L.2, Palma,G.S.2
1Doctorado en Ciencias de Recursos Naturales,Universidad de La Frontera,
Temuco, Chile, 2Scientific and Technological Bioresource Nucleus (BIORENUFRO), Universidad de La Frontera, Temuco, Chile.
Email: l.marileo01@ufromail.cl
In southern Chile, pastures support the milk and beef production. Nitrogen (urea)
fertilization and post-emergence herbicide application are common practices to
increase the quality and yields of pastures. However, a significant amount of
herbicide residues are deposited soils, modifying their physico-chemical properties
and soil microbial populations. Here, we investigated the effect of post-emergence
herbicides on bacterial community structure in soil treated with urea. Soils were
treated with combinations of herbicides and urea in equivalent rate of 200 kg N
ha-1, MCPA (750 and 1500 g i.a. há-1) and flumetsulam (75 and 150 g i.a. há-1).
The herbicides were applied 24 h after urea and controls without urea and
herbicides were also carried out. Bacterial community structure was assessed by
denaturing gradient gel electrophoresis (DGGE) with specific primer sets for the
following genes: 16S rDNA, nifH (nitrogen fixation) and amoA (ammonification).
Differences in bacterial community structure were visualized by analysis of DGGE
gels with Phoretix 1D software and non−metric multidimensional scaling (MDS)
analysis using PAST freeware with Bray−Curtis similarity index. Based on DGGE
gels with 16S rDNA gene, the results showed MCPA induced changes in bacterial
communities compared with controls and soils treated with flumetsulam. Changes
in bacterial communities were also observed in soils with absence of urea, but with
higher doses of MCPA. In relation to nifH gene, there were not changes in soils
under any treatments as revealed by DGGE. The application of urea to soils with
higher doses of MCPA produced also changes in bacterial communities based on
DGGE gels with amoA gene. This study reveals that doses of MCPA affect in higher
degree the soil bacterial community structure (including potential ammonifying
bacteria) compared to flumetsulam. Acknowledgments: Fondecyt no. 1120467 and
1120505.
126
Session 7
EFFECT OF PHOSPHORUS AND NITROGEN FERTILIZATION ON THE COMPOSITION OF RHIZOBACTERIAL COMMUNITIES OF TWO ANDISOL PASTURES
Milko A Jorquera1, Oscar A Martinez2, Luis G Marileo3, Jacquelinne J Acuna3,
Surinder K Saggar4, Maria de la Luz Mora1
1Scientific and Technological Bioresource Nucleus (BIOREN-UFRO), Universidad
de La Frontera, Temuco, Chile, 2Instituto de Bioquimica y Microbiolog¡a, Facultad
de Ciencias, Universidad Austral de Chile, Valdivia, Chile, 3Doctorado en Ciencias
de Recursos Naturales, Universidad de La Frontera, Temuco, Chile , 4Ecosystems
& Global Change, Landcare Research, New Zealand
Email:
milko.jorquera@ufrontera.cl
The effect of nitrogen (N) and phosphorus (P) fertilization on composition of
rhizobacterial communities of volcanic soils (Andisol) from southern Chile at
molecular level is poorly understood. Therefore, this study attempts to investigate
the effect of long-term application of N (urea) with and without P (triple
superphosphate) fertilization on the composition (abundance, structure and
diversity) of rhizobacterial communities of two Andisol pastures from southern Chile.
Composition of rhizobacterial communities was evaluated by denaturing gradient
gel electrophoresis (PCR-DGGE) based on PCR amplification of 16S rRNA, rpoB,
nifH, amoA and alkaline phosphatase (ALP) genes. Differences in the composition of
rhizobacterial community structure were visualized by analysis of DGGE gels with
Phoretix 1D software and non-metric multidimensional scaling (MDS) analysis using
PAST freeware with Bray-Curtis similarity index. In addition the abundance of
rhizobacteria was evaluated by quantitative PCR (qPCR). The results showed that in
absence of P fertilization, moderate N fertilization (270 kg N ha-1 yr-1), significantly
changed the composition of rhizobacterial communities, but no significant
community change was observed with P fertilization (240 kg P ha-1 yr-1). At high N
fertilization (600 kg N ha-1 yr-1) rhizobacterial communities changed, even with P
fertilization (400 kg P ha-1 yr-1) compared with lower N fertilization (200 and 400 kg
N ha-1 yr-1) and unfertilized soils. The changes observed in rhizobacterial
communities coincide in N fertilized pastures with lower soil pH and higher pasture
yields. This study indicates that N-P application affect the soil bacterial populations
at molecular level and need to be taken in consideration to adequate fertilizer
practices in Chilean pastoral Andisols.
Acknowledgements: Fondecyt no. 1120505 and 1100625, and MEC-Conicyt no.
80100011.
127
Session 7
REAL TIME PCR QUANTIFICATION OF THE PLANT GROWTH PROMOTING
BACTERIA HERBASPIRILLUM SEROPEDICAE STRAIN SMR1 IN
INOCULATED MAIZE (ZEA MAYS) ROOTS
Tomás Pellizzaro1, Fernanda Amaral2, Jessica Cavalheiro Bueno1, Fabio C. Brod1,
Ana Carolina Arisi1,
1Universidade Federal de Santa Catarina
Email:
samotpp89@gmail.com
The plant growth promoting bacterium (PGPB) Herbaspirillum seropedicae SmR1
is an endophytic diazotroph found in association with several economic important
crops such as rice, maize and sugarcane. H. seropedicae SmR1 can promote the
plant growth through the production and secretion of phytormones, increasing plant
resistance to pathogens and supplying fixed nitrogen to the plant. Nevertheless, the
mechanisms involved in the colonization of the host plant by H.seropedicae SmR1
are not fully understood and methods to monitor the interaction between this PGPB
and crops are required. In this study, two primer pairs were designed in order to
quantify the PGPB H.seropedicae strain SmR1 in inoculated maize roots harvested
1, 4, 7 and 10 days after inoculation (DAI). The primer pairs were evaluated for
specificity by testing DNA samples of 12 other bacterial species. Ten standard
curves using serially dilutions of H.seropedicae SmR1 DNA samples were
performed for DNA quantification. Mean PCR efficiency was 91% and correlation
coefficient was 0.99, indicating that primer pair HERBAS1 can be used for qPCR
detection and quantification of H.seropedicae SmR1 in maize roots. Also, maize
genomic DNA was quantified using primer pair ZM1 and a relation between the
number of genome copies of H.seropedicae and maize was calculated. Limit of
detection of the bacteria was 101 copies (corresponding to 60.3 fg of bacterial DNA)
and the repeatability standard deviation (%RSDr) results were below 15%. The
results obtained for bacterial DNA quantification, confirmed the infection and
colonization of plant maize roots by H.seropedicae SmR1, and the bacterial DNA
copy number per gram of root increased from 107 copies (1 DAI) to 109 copies (10
DAI). Primer pair HERBAS1 presented able to quantify H.seropedicae SmR1 in
inoculated maize roots and can be used for monitoring the interaction.
128
Session 7
NITRATE-MEDIATED INHIBITION OF SOYBEAN-BRADYRHIZOBIUM JAPONIUM
INTERACTION: NITRATE REDUCTASE AND NITRIC OXIDE INVOLVEMENT
Gisela Calvaresi1, Nacira Muñoz 12, Claudio González1, Ramiro Lascano 12
1 Cátedra de Fisiología Vegetal. Fac. Cs. Exact. Fis y Nat. Universidad Nacional de
Córdoba. Avda Vélez Sarsfield 299. 5000. Córdoba. Argentina. 2 Inst. de Fisiología y
Recursos Genéticos Vegetales (IFRGV-CIAP-INTA). Camino a 60 Cuadras km 5 y
½. 5119. Córdoba. Argentina.
Email: hrlascano@hotmail.com
It is widely known that high nitrate concentration inhibit the legume-rizobia
interaction. In the present work we evaluate the participation of nitrate reductase
(NR) and the nitric oxide (NO) generation during the soybean- Bradyrhizobium early
events under inhibitory nitrate concentration. As a first morphological marker of the
legume-rhizobia interaction the root hairs deformation was evaluated, and a
markedly inhibition under high nitrate concentration was reported. As we previously
demonstrated under other experimental conditions (Muñoz et al, 2012), the root
hairs deformation percentage reduction showed a close correlation with apoplastic
superoxide generation decreases. The root nitrate reductase activity, mainly the
NADPH-dependent one, was induced on a nitrate dose-dependent manner. Nitrate
reductase activity has been postulated as one nitric oxide source in plant. Using the
fluorescence dye, we could registered increased level of NO under nodulation
inhibitory nitrate concentration. The addition of tungstate to the incubation medium,
an inhibitor of NR activity, inhibits the NO generation and partially restores the
apoplastic superoxide generation. Moreover, when the NO production is scavenged
with cPTIO the nodulation under inhibitory nitrate concentration was partially
recovered. Our results suggests that nodulation inhibitory nitrate concentration
induce NR activity and NO production, affection root hair aposplastic superoxide
production, root hair deformation and in consequence nodule formation.
129
Tour Information
Information & Bookings: nitrogen@ctsturismo.cl
Phone: (56-2) 2251 0400, Ext. 775
Visit to the cities of Puerto Montt and Puerto Varas
Tours depart daily for this 3-hour trip:
The visit begins at a site offering a panoramic view of Puerto Montt, and then heads
downtown through the residential district and government buildings to the Main
Square. Visitors can observe the city’s Cathedral, a monument to the German
settlers, and the church of the Jesuit. We then continue, in the direction of the town
of Chinquihue, through Urmeneta Street and Antonio Varas Street towards the
commercial and harbor zones. We return through the fish and seafood market of
Angelmó, with its colorful arts and crafts stands and head via the Pan-American
Highway to the city of Puerto Varas, with its beautiful lakefront drive along the
Llanquihue lake. We visit San Francisco Street and its parochial church (1917), the
Main Square named Vicente Perez Rosales, the Municipal Casino, and residential
streets lined with old houses some of which have been declared National
Monuments. We visit the promenade that lines the lakefront and then head towards
Philippi Hill to another viewpoint which offers stunning sights of the city of Puerto
Varas and its surroundings.
Fare per person is USD 35
(Minimum passengers required: 2)
Departure Time: 14:30 hrs.
Bookings have to be requested at least 48 hours before departure. Tours subject to
availability at the moment of confirmation.
Around the Llanquihue Lake & Osorno Volcano
Tours depart daily for this 8-hour trip which includes lunch and entrance fee to the
Frutillar Museum
The tour starts by taking the Pan-American Highway north to the town of
Llanquihue, an important agro-industrial center. From there our drive borders the
Llanquihue lake and passes through agricultural fields and salmon production
centers. Upon arriving at the town of Frutillar, we take a walk by the lakeside
promenade and tour the Museum of the Colonization. The trip then heads northeast
131
Tour Information
to Puerto Octay. Lunch is en-route. During the drive, passengers can appreciate the
beautiful architecture of its church ( 1911) in neo-gothic style as we continue
towards the east through Maitén beach, passing by Puerto Fonck, La Picada and
Blanco river. Near the foothill of the Osorno volcano, we will visit the town of
Cascadas and then ascend the Osorno Volcano for a spectacular panoramic view
of the Lake Region. We will arrival at the Centro de Ski y Montaña Volcán Osorno,
where we will have leisure time for photographs (Chair-lift fee not included). We
return to the hotel in the evening.
Fare per person USD 93
(Minimum passengers required: 3)
Departure Time: 09:00 hrs.
Petrohué Waterfalls-Todos los Santos Lake & Osorno Volcano
Tours depart daily for this 7-hour trip, which includes lunch:
We depart from your hotel and follow the Llanquihue lakeshore, a drive which offers
panoramic and beautiful views of the Osorno and Calbuco Volcanoes. We head
towards the Petrohué River waterfalls where we will have leisure time for
photographs and walks in the midst of exuberant vegetation. Our journey then
continues to lake Todos los Santos, also known as Esmerald Lake due to the colour
of its waters, which is framed by the eternal snows of the Osorno, Calbuco,
Tronador and Puntiagudo Volcanoes. We then head back towards Ensenada, and
up to the slopes of Osorno Volcano, arriving at the Volcan Osorno Ski and Mountain
Center. Here you will experience spectacular panoramic views of Llanquihue Lake
and volcanos of the region. (ski lift fee not included).
Fare per person USD 87
(Minimum passengers required: 3)
Departure Time: 09:30 hrs.
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Tour Information
Chiloe Island – Ancud
Tours depart Tuesday, Thursday and Saturdays for this 9-hour trip, which includes
lunch and museum entrance fee.
Tour starts by heading towards Pargua via the Pan-American Highway until we
reach the mainland’s end point. We then take a 20-minute ferry ride in order to
cross the Channel of Chacao to Puerto Chacao, a small agricultural village and the
gate to the Island of Chiloé. We continue towards the city of Ancud, with it's classic
chilote architecture of wood tiles, parks and promenades that embellish the
surroundings. We visit the San Antonio Fort (1770), last fort to fly the flag of Spain,
and end the trip at the viewpoint in Huaiquén Hill, which offers a panoramic view of
the city, the Chacao Channel, Cochinos Island and the mainland with its coast, cliffs
and the creek of Carelmapu.
Fare per person USD 93
(Minimum passengers required: 4)
Departure Time: 08:00 hrs.
Bookings have to be requested at least 48 hours before departure. Tours subject to
availability at the moment of confirmation.
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Pontificia Universidad Católica de Chile
Av. Libertador Bernardo O’Higgins 340
Phone : (56 - 02) 23541926
Fax: (56 - 02) 23542185
E-mail : nitrogen2013@bio.puc.cl