Phosphate starvation signaling: a threesome controls systemic Pi

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COPLBI-558; NO OF PAGES 5
Available online at www.sciencedirect.com
Phosphate starvation signaling: a threesome controls
systemic Pi homeostasis
Peter Doerner
Systemic signaling between roots and shoots is required to
maintain mineral nutrient homeostasis for optimal metabolism
under varying environmental conditions. Recent work has
revealed molecular components of a signaling module that
controls systemic phosphate homeostasis, modulates uptake
and transport in Arabidopsis. This module comprises PHO2, a
protein that controls protein stability, the phloem-mobile
microRNA-399 and a ribo-regulator that squelches the activity
of miR399 towards PHO2 by a novel mechanism. This advance
is a significant step for the design of future rational approaches
to improve crop phosphate use efficiency.
Addresses
Institute of Molecular Plant Science, School of Biological Sciences,
Daniel Rutherford Building, King’s Buildings, University of Edinburgh,
Edinburgh EH9 3JH, Scotland, United Kingdom
Corresponding author: Doerner, Peter (Peter.Doerner@ed.ac.uk)
Long-distance and systemic signaling
When roots grow into a low Pi patch, or the root system
overall encounters Pi-deficient conditions, Pi must be
provided to sustain meristematic and physiological activities in this organ. This is because plants can only acquire
new Pi resources by growth that brings roots into contact
with previously unexploited soil. In the metabolically
highly active cells of the root apex, Pi levels must be
maintained at high levels. Steady state cytoplasmic concentrations between 5 and 15 mM have been reported [3].
This is not primarily because large amounts of Pi are
consumed, i.e. incorporated into newly synthesized
DNA, RNA and membranes; but rather because Pi functions analogously to a currency, and is required for most
biochemical transactions in the cell to proceed. During
the early stages of an episode of Pi starvation, this demand
is satisfied by Pi translocation from shoot tissues via the
phloem.
Current Opinion in Plant Biology 2008, 11:1–5
This review comes from a themed issue on
Cell signalling and Gene regulation
Edited by Jason Reed and Bonnie Bartel
1369-5266/$ – see front matter
# 2008 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.pbi.2008.05.006
Introduction
Phosphate [Pi] is an essential macronutrient for plants
that is generally in short supply as it is difficult to
assimilate. Pi is distributed very heterogeneously in the
soil matrix, which conditions specific behavioral patterns
for root growth. These have been recently reviewed [1]
and will not be discussed in detail here. Phosphate
distribution within the plant is also heterogeneous at
different scales and follows cellular patterns of physiological demand; as a generalization, under Pi replete
conditions: shoot tissues accumulate more Pi than root
tissues, metabolically active cells more than less active
cells, and the vacuole and plastids more than the cytoplasm [2,3]. Such non-uniform distribution requires
extensive and sophisticated regulatory mechanisms that
allow plants to maintain Pi homeostasis required for
efficient metabolism, specifically in non-equilibrium conditions when acquisition rates vary. This review will focus
on recent advances in our understanding of systemic Piresponsive signaling mechanisms and highlight some of
the major unresolved questions.
www.sciencedirect.com
Precise regulation of net Pi flux from shoot to root in these
conditions is critical as careful budgeting of limiting
resources has consequences for competitive success vis
a` vis other plants competing for the same limiting
resources, and overall fitness to permit completion of
the individual’s lifecycle. Recent work has uncovered a
regulatory module involved in the systemic control of Pi
allocation (Figure 1) that hard-wires this cost–benefit
analysis. A family of phloem-mobile microRNAs
(miR399a-f), their target gene PHO2, and a family of
regulatory, non-coding RNAs, the IPS/At4-like genes,
form a circuit in which the balance of microRNA and
ribo-regulator abundance fine-tune PHO2 activity.
Regulated proteolysis in Pi signaling
Phosphate 2 (PHO2) was first identified by mutational
approaches, by virtue of the phenotype of the pho2
mutant, which overaccumulates Pi in shoots of
plants grown in Pi-replete conditions [4]. PHO2
encodes an E2 ubiquitin conjugase-related enzyme
(UBC24), and is expressed in shoot and root tissues
[5,6]. In shoots, histochemical analysis indicated
expression in leaf vasculature, and in roots, expression
was observed in all vascular cell types, except the
mature xylem [5].
PHO2 is thus the second genetic function involved in
responses to Pi-starvation that is mechanistically linked to
ubiquitin or similar modification systems. The SIZ1 gene,
shown earlier to modify the putative transcription factor
Phosphate starvation Response 1 (PHR1) in vitro, encodes
a SUMO E3 ligase [7]. However, SIZ1 is not specific for Pi
Please cite this article in press as: Doerner P, Phosphate starvation signaling: a threesome controls systemic Pi homeostasis, Curr Opin Plant Biol (2008), doi:10.1016/j.pbi.2008.05.006
2 Cell signalling and Gene regulation
signaling, as it is involved in regulating responses to many
types of abiotic stress.
Reciprocal grafting studies showed that a pho2 genotype
in roots is sufficient to mimic the whole plant pho2
Figure 1
phenotype [6], suggesting that UBC24 primarily acts
in root tissues. When phosphate is available, low PHO2
activity leads to increased expression of two rootexpressed phosphate transporter genes, Pht1;8 and
Pht1;9 [5,6] and also of at least two members of the
IPS/At4 gene family [6]. Overexpression of the two
high-affinity transporter genes is most likely responsible
for most of the pho2 phenotype, as RNAi-mediated inhibition of their expression in the pho2 background suppresses the accumulation of excess shoot phosphate [6].
It appears likely that UBC24 protein functions by targeting genes involved in controlling the expression of the
two transporters and other affected genes for degradation.
Negative control of Pi-responsive gene expression has
previously been reported [8], and could involve the
degradation of a transcriptional repressor or, alternatively,
control the stability of a transcriptional activator for Pht1;8
and Pht1;9 expression in the presence of Pi. The mechanisms by which UBC24 affects systemic Pi distribution
remain to be elucidated in detail.
microRNA 399 as long-distance signal
Several microRNAs have recently been shown to be
involved in regulating plant mineral nutrient homeostasis.
These include: miR395, which is regulated by sulfur [9];
miR398, regulated by copper [10], which may also play an
indirect role in the growth response to perceived Pi
limitation, as a multi-copper oxidase plays a critical role
in this response in Arabidopsis [11]; and miR399, which is
regulated by phosphate [12].
Schematic illustration of the regulatory module that controls systemic Pi
homeostasis. The regulatory module, comprising PHO2, miR399 and
IPS/At4 genes controls the activity of PHO2 in roots to regulate the
allocation of Pi to the shoot. High PHO2 activity (1) which occurs when
the plant perceives adequate Pi supply, results in low levels of Pht1;8
and Pht1;9 phosphate transporter expression. Likewise, levels of
miR399 expression (2) and IPS/At4 (3) expression are low in high
phosphate, but in contrast to miR399, IPS/At4 expression is suppressed
further by UBC24 activity in the presence of high Pi [6]. This keeps the
regulatory module in a highly Pi-limitation responsive state. In a high Pi
environment, phosphate is transported to mature and young leaves.
When the plant, specifically the shoot, experiences Pi limitation, miR399
expression is strongly stimulated, particularly in shoot tissues (4). Mature
miR399 is then translocated via the phloem to the root system. Here (5),
binding of cognate sites in the 50 UTR of PHO2 transcripts to miR399charged silencing complexes leads to the degradation of PHO2 mRNA,
resulting in low UBC24 protein levels. This leads to increased Pht1;8 and
Pht1;9 expression, facilitating increased Pi uptake and transport to the
shoot. In low phosphate, Pi in shoot tissues is mobilized from mature
leaves to young leaves (4) and also to roots to sustain root meristem
activity for the longest time possible. As the Pi starvation response
progresses, expression of IPS/At4 is induced. These ribo-regulators (5)
inhibit the action of miR399-charged silencing complexes on PHO2
mRNA, and thus allow UBC24 levels to adjust to the dynamic balance of
supply and demand in the plant more rapidly.
miR399 is strongly and specifically induced by perceived
Pi limitation with parallel >1000-fold induction of the
primary transcript and mature miR399d and miR399f
steady state levels [5,6,12,13]. miR399 is expressed
in vascular tissues, specifically in companion cells and
phloem [5]. PHO2 has an unusually long 50 UTR that
contains five target sites for miR399 [6,12,14], and
miR399 binding to these sites leads to scission of the
PHO2 transcript [15]. A further predicted, but not yet
validated, miR399 target is the Pi transporter, Pht1;7,
which is expressed in roots and floral tissues [16]. Forced
expression of miR399 phenocopies the PHO2 loss-offunction phenotype of elevated shoot Pi accumulation
[6,14], suggesting that PHO2 is the primary target of
the miR399 family.
A recent study reported key observations that indicate
how miR399 functions in systemic signaling: shootexpressed miR399 is translocated to the root, miR399 is
present in phloem exudates, and high levels of shoot
miR399 expression suffice to repress PHO2 in roots [13].
Together, these properties strongly indicate that miR399
functions as systemic signal to inform root cells of the
perceived state of Pi homeostasis in the shoot by controlling PHO2 levels in the root.
Phosphate starvation signaling Doerner 3
A role for non-coding RNAs
A decade ago, a new class of Pi starvation-induced transcripts was discovered, in tomato and Medicago, with the
unusual feature that they did not encode long open
reading frames [17,18]. Subsequently, related ‘Induced
by Pi Starvation’ (IPS) genes were identified in Arabidopsis [19,20], but their function remained poorly understood. In low-Pi conditions, these genes are expressed in
shoot and root tissues, with particularly high levels of
expression in mature root vascular tissues ([20,21],
Thacker and Doerner, unpublished).
Two recent papers have now uncovered exciting functional features of these genes in the regulation of Pi
starvation responses: the loss-of-function of one such
homolog in Arabidopsis, At4, resulted in altered shoot
to root Pi ratios [21], suggesting a possible involvement
in the PHO2-miR399 regulatory loop. Strikingly, it was
found that a 23 base sequence that is highly conserved in
all IPS1-like genes was completely complementary to
miRNA399, save for two to three critical bases in the
middle [21,22]. This mismatch is precisely where
the miRNA guided cleavage reaction occurs, and in plant
microRNA targets, these bases are normally completely
complementary [23]. Overexpression of IPS1 resulted in
higher levels of PHO2 accumulation and lower steadystate shoot Pi levels [22], but interestingly, did not result
in degradation of the IPS1 transcript. However, these
effects were lost when a modified version of IPS1 was
expressed that was entirely complementary to miR399
[22].
This ability to modulate the activity of miR399 suggested
a novel regulatory paradigm, for which the term ‘target
mimicry’ was coined: an RNA with incomplete central
complementarity functions analogously to a competitive
enzyme inhibitor, effectively sequestering miR399charged silencing complexes in a state in which they
cannot act on PHO2 transcripts [22]. It is not yet clear
what the biological significance of this additional level of
control is. At4, one member of the IPS family, is induced
only gradually and late after onset of perceived Pi
starvation (Lai and Doerner, unpublished), raising the
possibility that target mimicry functions to dampen oscillations in Pi distribution caused by the antagonism between miR399 and PHO2, to prevent the system from
becoming stuck in a detrimental state of long-term low
PHO2 and high miR399 activity, or perhaps to de-sensitize
the response to limiting Pi in the course of adaptation in
which the balance of supply and demand are dynamic
[24]. These models are consistent with the observation
that the expression of at least two IPS/At4 gene family
members is modulated by PHO2 [6].
Evolution of signaling mechanisms
It is interesting to consider the evolution of this
signaling network: the genomes of single-celled algae
(Chlamydomonas, Ostreococcus) or simple multi-cellular
algae (Volvox) lack UBC genes closely-related to PHO2,
while the bryophyte (Physcomitrella) and lycophyte
(Selaginella) genomes have two or more closely homologous genes (Lai and Doerner, unpublished). However,
their function has not yet been characterized and so it is
still unclear whether they are involved in controlling Pi
homeostasis in basal plants as well. PHO2 homologs have
been identified in monocots and eudicots [6], but a clear
gymnosperm homolog has not yet been identified. By
contrast, there is no evidence for miR399 in bryophytes or
lycophytes [25,26,27], but as miR399 is not expressed
under all growth conditions, it could have been missed in
these studies. However, miR399 has been clearly identified in core and basal eudicots and monocots [28]. IPS1like genes have been identified in core eudicots
[17,18,20], but their identification in other species is more
challenging, as the conserved sequences are short and
they lack a canonical structure, such as the stem-loop
found in miRNA precursors, which could be used to
identify candidate loci.
Together, these observations suggest that the basic function ascribed to PHO2 – the feedback control of Pi
assimilation – evolved first, possibly as the result of a
selective advantage to prevent toxic effects associated
with over-accumulation of Pi [4]. However, as plants
evolved vascular systems, and also extended their lifespan, feedback controls restricted to the cellular level
limited the latitude with which Pi homeostasis could be
regulated for the whole plant. Thus, the evolution of
systemic, RNA-based communication such as miR399and IPS1/At4-mediated mechanisms to precisely and
differentially regulate Pi homeostasis at the organ level,
likely provided huge selective advantages.
Conclusions and future challenges
Recent advances indicate that control of protein stability
by PHO2 plays a pivotal role in regulating systemic Pi
homeostasis. Its activity in roots is regulated by the
balance of miR399 abundance, which is determined by
its systemic, phloem-mediated movement from shoot to
root; and the abundance of ribo-regulators of the IPS1/At4
family that antagonize miR399 activity. All members of
this regulatory triad are also expressed in shoots and it will
be interesting to determine whether they have an
immediate function in these tissues as well.
Many unresolved questions must be addressed before our
knowledge of these regulatory circuits can be put to use to
improve crop plant nutrient use efficiency. The main
focus will be on the mechanisms that function upstream
and downstream of the PHO2 regulatory module. The big
prize will go to the answer of the question how Pi is
perceived, and how many such mechanisms exist. One or
more of these mechanisms are upstream activators of
miR399 and IPS1/At4 gene expression. Another important
4 Cell signalling and Gene regulation
gap in our knowledge are the substrates of PHO2, specifically those that mediate Pht1;8 and Pht1;9 over-expression. Furthermore, although we know that miR399 is
translocated in the phloem, the mechanisms that determine how it enters the phloem and whether it is transported as a naked RNA, associated with specific carriers or
as miR399-primed silencing complex are not yet known.
Lastly, we must examine, at the tissue and cellular level,
Pi flux in source and sink tissues to better grasp the how
the balance of supply and demand is achieved.
Acknowledgements
I thank Veronique Vitart, Gwyneth Ingram and members of the Doerner lab
for critical reading of the manuscript. Research in my lab on phosphate
signaling is supported by the Darwin Trust, The Leverhulme Trust, and
the Royal Society.
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
of special interest
of outstanding interest
1. Desnos T: Root branching responses to phosphate and nitrate.
Curr Opin Plant Biol 2008, 11:82-87.
An excellent recent review on the mechanisms that couple the perception
of low Pi and N to adaptive growth behavior.
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Bieleski R: Phosphate pools, phosphate transport and
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Mimura T: Regulation of phosphate transport and homeostasis
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Together with ref. [6], this paper reports that the PHO2 gene encodes
UBC24. Over-expression of miR399 re-capitulates the pho2 phenotype,
independently confirming results also obtained in ref. [6]. A detailed
analysis showed that the greater Pi uptake in pho2 and miR399 overexpressors is owing to an increased Vmax. Their analysis shows that PHO2
and miR399 expression are co-localized in the vascular cylinder.
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Bari R, Datt Pant B, Stitt M, Scheible WR: PHO2, microRNA399,
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Together with ref. [5], this paper reports the molecular cloning of the
PHO2 gene. It is shown by reciprocal crosses that a pho2 phenotype in
roots is required to generate the pho2 phenotype of excess Pi accumulation in shoots. It is shown that miR399 induction in response to low Pi is
significantly diminished in the phr1 mutant background, indicating that
the PHO2/miR399 regulatory module is genetically downstream of this
putative transcription factor. Besides extensive micro-array analysis of
responses to low Pi in various genetic backgrounds, the paper also shows
that PHO2 homologs are present in the genomes of various mono- and
dicot species.
7.
Miura K, Rus A, Sharkhuu A, Yokoi S, Karthikeyan AS,
Raghothama KG, Baek D, Koo YD, Jin JB, Bressan RA et al.: The
Arabidopsis SUMO E3 ligase SIZ1 controls phosphate
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Multi-copper oxidases are identified as crucial components of signaling
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behavior in plants that experience Pi starvation.
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phosphate-starvation response in Arabidopsis. Curr Biol 2005,
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The first experimental validation that expression of members of the
miR399 family is induced by low Pi. The authors demonstrate that
different UBC24 transcript levels in low and high Pi are post-transcriptionally mediated by the binding sites for miR399 in its 50 UTR. This paper
also shows that over-expression of miR399 enhances Pi accumulation in
transgenic lines.
13. Pant BD, Buhtz A, Kehr J, Scheible WR: MicroRNA399 is a long distance signal for the regulation of plant phosphate
homeostasis. Plant J 2008, 53:731-738.
The first demonstration that miR399 is phloem-mobile and that this
movement is sufficient to reduce PHO2 steady-state mRNA levels in
roots and elevate shoot Pi concentration, indicating that miR399 is a
critical systemic signal for control of Pi homeostasis.
14. Chiou T-J, Aung K, Lin S-I, Wu C-C, Chiang S-F, Sua C-L:
Regulation of phosphate homeostasis by microRNA in
Arabidopsis. Plant Cell 2006, 18:412-421.
This study, together with refs. [5,6,12] shows that overexpression of
miR399 downregulates PHO2 steady state levels and enhances Pi accumulation in shoots 5–6-fold. The authors show that pho2 plants are
specifically defective in mobilizing Pi from mature leaves to young and
growing leaves.
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phasing during trans-acting siRNA biogenesis in plants. Cell
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Medicago truncatula roots is suppressed in response to
colonization by vesicular-arbuscular mycorrhizal (VAM) fungi
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Involves phosphate translocation to the shoots. Plant Physiol
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20. Martin AC, del Pozo JC, Iglesias J, Rubio V, Solano R, de la Pena A,
Leyva A, Paz-Ares J: Influence of cytokinins on the expression
of phosphate starvation responsive genes in Arabidopsis.
Plant J 2000, 24:559-568.
21. Shin H, Shin H-S, Chen R, Harrison MJ: Loss of At4 function
impacts phosphate distribution between the roots and the
shoots during phosphate starvation. Plant J 2006, 45:712-726.
The first report that IPS/At4 genes are involved in controlling systemic
phosphate allocation. at4, a loss-of-function allele of At4, accumulates
excess Pi in the shoot. The authors also show that the 22–23 base
sequence conserved between all IPS/At4 genes interacts with a micro
RNA.
22. Franco-Zorrilla JM, Valli A, Todesco M, Mateos I, Puga MI, Rubio Somoza I, Leyva A, Weigel D, Garcia JA, Paz-Ares J: Target
mimicry provides a new mechanism for regulation of
microRNA activity. Nat Genet 2007, 39:1033-1037.
A thorough characterization of the mechanism by which IPS/At4 genes
modulate miR399 activity, thereby uncovering the novel regulatory paradigm, target mimicry. The paper conclusively shows that inhibition of
miR399-charged silencing complexes, and not saturation of miR399dependent scission is the mode of IPS/At4 gene action. Artificial target
Phosphate starvation signaling Doerner 5
mimics were generated for other miRNAs, showing that this mode of
regulation to fine-tune miRNA activity can be used in biotechnology for
other endogenous targets.
This paper extends the authors’ previous phylogenetic survey [25] to
focus more on the genomes of the moss Physcomitrella patens and the
lycopod Selaginella moellendorffii.
23. Jones-Rhoades MW, Bartel DP, Bartel B: MicroRNAS and their
regulatory roles in plants. Annu Rev Plant Biol 2006, 57:19-53.
27. Fattash I, Voss B, Reski R, Hess WR, Frank W: Evidence for the
rapid expansion of microRNA-mediated regulation in early
land plant evolution. BMC Plant Biol 2007, 7:13.
A systematic analysis of miRNAs in the moss Physcomitrella patens
uncovers novel miRNAs, including some that are differentially expressed
in the haploid and diploid generations.
24. Lai F, Thacker J, Li Y, Doerner P: Cell division activity determines
the magnitude of phosphate starvation responses in
Arabidopsis. Plant J 2007, 50:545-556.
25. Axtell MJ, Bartel DP: Antiquity of microRNAs and their targets in
land plants. Plant Cell 2005, 17:1658-1673.
A phylogenetic survey was used to examine plant miRNA conservation. It was shown that many plant miRNAs, and their targets, have
remained essentially unchanged since before the emergence of
flowering plants.
26. Axtell MJ, Snyder JA, Bartel DP: Common functions for diverse
small RNAs of land plants. Plant Cell 2007, 19:1750-1769.
28. Barakat A, Wall K, Leebens-Mack J, Wang YJ, Carlson JE,
Depamphilis CW: Large-scale identification of microRNAs from
a basal eudicot (Eschscholzia californica) and conservation in
flowering plants. Plant J 2007, 51:991-1003.
Using modern ultra-high throughput sequencing technologies the paper
reports the identification of many conserved and some novel miRNA
families from the analysis of a basal eudicot. Amongst others miR399 is
identified in the California poppy

Phosphate starvation signaling: a threesome controls systemic Pi

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