Investigations on Mitochondrial Pleomorphy and

Investigations on Mitochondrial Pleomorphy and Interactions with the Endoplasmic
Reticulum and Peroxisomes
by
Erica-Ashley Jaipargas
A Thesis
presented to
The University of Guelph
In partial fulfilment of requirements
for the degree of
Master of Science
in
Molecular and Cellular Biology
Guelph, Ontario, Canada
© Erica-Ashley Jaipargas, August, 2015
ABSTRACT
INVESTIGATIONS ON MITOCHONDRIA PLEOMORPHY AND INTERACTIONS
WITH THE ENDOPLASMIC RETICULUM AND PEROXISOMES
Erica-Ashley Jaipargas
University of Guelph, 2015
Advisor:
Dr. Jaideep Mathur
Mitochondria are pleomorphic organelles capable of constant fusion and fission. Live
fluorescent microscopy was employed to investigate mitochondrial pleomorphy during
fluctuations in light, sugar and O2. Light and sugar are shown to induce fission whereas a greento-red photo-convertible mEos fluorescent protein targeted to mitochondria (mitoEos) reveals
that hypoxia induces fusion, leading to giant mitochondria. The endoplasmic reticulum (ER) is
shown to be a mediator of mitochondrial fission and acts as a mould for morphological
transitions displayed by mitochondria. Simultaneous live-imaging also reveals sustained
mitochondria-peroxisome interactions, which are apparent in anisotropy1, a cell wall mutant
shown here to be light sensitive. Triple transgenic lines were created to differentially label
mitochondria, peroxisomes and the ER and show that the ER cages the other organelles
including chloroplasts during these sustained interactions. Together, simultaneous live-imaging
of mitochondria, peroxisomes and the ER using double and triple Arabidopsis thaliana
transgenics further illuminate organelle pleomorphy and interactivity.
ACKNOWLEDGEMENTS
I would like to thank Drs. George Harauz and Andrew Bendall for taking me into their
labs during my undergraduate degree when I had minimal lab experience. It was under their
supervision that my passion for research truly commenced. I would also like to thank my
colleagues and the close friends I have made in the Molecular and Cellular Biology Department
and my committee members, Drs. Joseph Colasanti and Robert Mullen for their guidance and
inputs.
To my family. You taught me perseverance and to always have a greater outlook, both of
which are qualities I could not have gotten through my degree without. And of course, Lucy.
Thank you for sitting by my side every day while I was writing. You truly are Man’s best friend.
Most of all, I would like to thank the members of our lab, past and present. You became
quite the extended family over the years. I would especially like to thank Alena Mammone for
climbing walls with me, both literally and figuratively, Kiah Barton for the pep-talks, her inputs
and for going on coffee runs with me, Cole Anderson for his never ending “Fun Facts” and
optimism, Michael Wozny for knowing when I needed to hear a bit of Billie Holliday in the lab,
and of course Neeta for always making sure we were fed and for all of the behind the scenes
work that makes our research possible.
Last, but definitely not least, my supervisor Dr. Jaideep Mathur. In the second year of my
undergraduate degree, you illuminated the dynamic wonders of the cell for me. Thank you for
taking me into your lab in my fourth year and for sharing such wisdom whenever we sat together
on the microscope trying to decode mitochondria. The work we have done together is truly
something I will never forget.
I would like to acknowledge the Natural Sciences and Engineering Research Council of
Canada, the Canada Foundation for Innovation, and the Molecular and Cellular Biology
department of the University of Guelph for their funding.
iii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS .................................................................................................... iii
LIST OF FIGURES ............................................................................................................... vii
LIST OF TABLES .................................................................................................................. ix
LIST OF SUPPLEMENTARY MOVIES ................................................................................x
LIST OF ABBREVIATIONS ................................................................................................. xi
CHAPTER 1 INTRODUCTION ............................................................................................1
1.1
Mitochondrial pleomorphy and function ........................................................................1
1.1.1
Mitochondrial ultrastructure: Orthodox and condensed states..................................2
1.1.2
The electron transport chain and ATP synthesis ......................................................3
1.2
Triggers and consequences of mitochondrial fusion and fission .....................................5
1.3
ROS production in plants ...............................................................................................6
1.4
Mitochondria and peroxisome pleomorphy in response to ROS .....................................9
1.5
Mechanisms of mitochondrial fusion and fission ......................................................... 13
1.6
Live imaging of organelles .......................................................................................... 15
1.7
Objectives ................................................................................................................... 16
1.8
Hypotheses .................................................................................................................. 17
CHAPTER 2 MATERIALS AND METHODS.................................................................... 18
2.1
Cloning of fluorescent protein-fusion constructs .......................................................... 18
2.2
Creating transgenic Arabidopsis lines .......................................................................... 21
2.2.1
Selection of stable lines ........................................................................................ 21
2.3
Growth conditions ....................................................................................................... 22
2.4
Colorimetric sugar quantification ................................................................................. 22
2.4.1
Sample preparation ............................................................................................... 23
2.4.2
Quantification ....................................................................................................... 23
2.5
Assessing light-induced stress ..................................................................................... 23
2.5.1
Induction of High Light Stress .............................................................................. 23
2.5.2
3,3-diaminobenzidine Staining .............................................................................. 24
2.5.3
H2O2 Responsive Fluorescent Probe ..................................................................... 24
iv
2.6
Assessing anisotropy1 mutant susceptibility to stress................................................... 24
2.6.1
2.7
Criteria for estimating interactions ........................................................................ 25
Microscopic visualization ............................................................................................ 26
2.7.1
Epi-fluorescent microscopy .................................................................................. 26
2.7.2
Confocal Laser Scanning Microscopy ................................................................... 26
2.8
Statistical analysis ....................................................................................................... 27
CHAPTER 3 RESULTS ....................................................................................................... 28
3.1
Identifying conditions to induce transitions between mitochondrial morphologies ....... 28
3.1.1
Mitochondria are predominantly small when grown with exogenous sugar ........... 28
3.1.2
Light induces mitochondrial fission ...................................................................... 30
3.1.3
Mitochondria elongate and become giant under O2 limited conditions .................. 30
3.2
The endoplasmic reticulum mediates mitochondrial pleomorphy ................................. 33
3.2.1
The endoplasmic reticulum is compact in light grown plants................................. 34
3.2.2
The endoplasmic reticulum acts as a mould for mitochondrial shape ..................... 35
3.2.3
The endoplasmic reticulum aids in mitochondrial fragmentation ........................... 36
3.2.4
Mitochondrial beading and the endoplasmic reticulum .......................................... 36
3.3
The ER cages mitochondria, plastids and peroxisomes ................................................ 38
3.4
Investigation of mitochondria and peroxisome interactions .......................................... 39
3.4.1
Mitochondria and peroxisome morphology in response to H2O2 ........................... 39
3.4.2
Peroxisome morphology in response to light, sugar and low O 2 conditions ........... 39
3.4.3
Mitochondria and peroxisomes have sustained interactions ................................... 42
3.5
The anisotropy1 mutant response to light..................................................................... 43
3.5.1
Production of light-induced H2O2 is more rapid in any1 than wild type ................. 44
3.5.2
Mitochondria-peroxisome interactions in any1...................................................... 47
3.6
Investigation of mitochondria fission machinery .......................................................... 47
3.6.1
DAL as possible mechanism of hierarchical fission between mitochondria and
peroxisomes.......................................................................................................... 47
CHAPTER 4 DISCUSSION ................................................................................................. 50
4.1
Mitochondria morphology in response to light, sugar and O2 limitated conditions ....... 50
4.1.1
Morphological differences between mitochondria in light and dark grown plants .. 50
4.1.2
Mitochondria morphology under O2 deplete conditions......................................... 54
4.2
Mitochondria and endoplasmic reticulum interactions ................................................. 56
4.3
Mitochondria and peroxisome interactions ................................................................... 57
v
CONCLUSIONS ..................................................................................................................... 61
FUTURE DIRECTIONS ........................................................................................................ 62
REFERENCES ........................................................................................................................ 64
APPENDIX I
SUMMARY OF MITOCHONDRIAL MORPHOLOGY DISCUSSED .... 75
APPENDIX II MUTANTS AND TRANSGENIC LINES ..................................................... 77
APPENDIX III PRIMER INFORMATION ....................................................................... 80
APPENDIX IV DESCRIPTIONS OF SUPPLEMENTAL MOVIES................................. 81
vi
LIST OF FIGURES
Figure 1.1 Mitochondria structure and inner membrane composition ...........................................4
Figure 1.2 Mitochondrial orthodox and condensed state transitions in response to ADP and ATP
injections ..................................................................................................................5
Figure 1.3 Triggers and consequences of mitochondrial fusion and fission ..................................7
Figure 1.4 Key metabolic and ROS links between mitochondria, peroxisomes and chloroplasts 10
Figure 1.5 Sequential events leading to peroxisome and mitochondria fission reveals
morphological similarities in the division pathway. ................................................. 12
Figure 1.6 Fluorescent labelling of the ER for live imaging ....................................................... 16
Figure 2.1 Core vectors used for clonings.. ................................................................................ 19
Figure 2.2 Criteria for classifying a sustained interaction........................................................... 25
Figure 3.1 Effects of light, dark and sugar on mitochondria length.. .......................................... 29
Figure 3.2 Morphological transition of small to giant mitochondria in response to oxygen
depletion during lengthy imaging ............................................................................ 31
Figure 3.3 Fusion of giant mitochondria .................................................................................... 32
Figure 3.4 Giant mitochondria formed by plant submergence in water or mineral oil................. 33
Figure 3.5 Average ER polygon size comparison between light and dark conditions. ................ 34
Figure 3.6 Mitochondria are embedded in the ER ...................................................................... 35
Figure 3.7 ER-mediated fission and beading of elongated mitochondria .................................... 37
Figure 3.8 ER cages mitochondria, peroxisomes and chloroplasts ............................................. 38
Figure 3.9 Effects of H2O2 on mitochondria and peroxisome morphology ................................. 40
Figure 3.10 H2O2 production in response to short HL irradiation ............................................... 42
Figure 3.11 Morphological comparisons of mitochondria and peroxisomes in response to light,
sugar and hypoxia. .................................................................................................. 41
Figure 3.12 Sustained interactions between mitochondria and peroxisomes ............................... 44
Figure 3.13 Comparisons of wild type and any1 cell shape and arrangement ............................. 45
vii
Figure 3.14 Comparisons of wild type (WT) and any1 responses to light................................... 46
Figure 3.15 Localization of FIS1A and FIS1B to mitochondria, peroxisomes and plastids. ........ 49
Figure 4.1 Influence of light, sugar and energy status on mitochondrial shape ........................... 52
Figure 4.2 The effects of dark, hypoxia and starvation on respiration and mitochondria
morphology............................................................................................................. 53
Figure 4.3 Proposed model for orthodox to condensed state transition to explain fusion-fission
activity in response to HL ....................................................................................... 55
Figure 4.4 Proposed models to explain ER-mediated mitochondrial pleomorphy ....................... 58
Figure 4.5 Model to explain effects of light on mitochondria, peroxisomes and plastids ............ 61
Figure I-1 Effects of light, dark and sugar on mitochondrial morphology .................................. 75
Figure I-2 Morphological effects of O2 depletion on mitochondria ............................................ 76
viii
LIST OF TABLES
Table 1.1 Factors influencing mitochondrial morphology ............................................................2
Table 1.2 Common ROS and their product and removal mechanism ...........................................8
Table 1.3 Proteins involved in mitochondrial fusion and fission ................................................ 13
Table 2.1 Emissions used to simultaneously visualize multiple FP ............................................ 27
Table I-1 Cytosolic sugar, light and oxygen effects on mitochondrial morphology .................... 76
Table II-1 Arabidopsis transgenics and mutants used and discussed in this thesis ...................... 77
Table II-2 References for mutants and probes used .................................................................... 79
Table III-1 Primer sequences ..................................................................................................... 80
ix
LIST OF SUPPLEMENTARY MOVIES
Movie 3.1 Mitochondrial elongation during water submergence
Movie 3.2 Giant mitochondrial fusion
Movie 3.3 ER moulding a giant mitochondrion
Movie 3.4 ER-mediated mitochondrial fission
Movie 3.5 ER caging chloroplasts, mitochondria and peroxisomes
Movie 3.6 Sustained mitochondria-peroxisome interactions
Movie 3.7 Fragmented mitochondria clustering a peroxule
Movie 3.8 Abundant peroxules in any1
All movies were acquired with 3.945s between frames and are shown at 5 frames per second. A
detailed description of each movie can be found in Appendix IV.
x
LIST OF ABBREVIATIONS
ADP
adenosine diphosphate
AIM
Agrobacterium infiltration media
any1
anisotropy1
AOX
alternative oxidase
AsA-GSH
ascorbate-glutathione cycle
ATP
adenosine triphosphate
C
cytochrome c complex
Caf4p
CCR4p-associated factor 4
[Ca2+]m
matrix calcium
CAT
catalase
cDNA
complementary DNA
CesA
cellulose synthase
CLSM
confocal laser scanning microscopy
Complex I
NADH dehydrogenase
Complex II
succinate dehydrogenase
Complex III
cytochrome c reductase
Complex IV
cytochrome c oxidase
DAB
3,3-diaminobenzidine
DAL1/DAL2 Drosophila inhibitor of apoptosis-like protein 1/2
DLP
Dynamin-Like Protein
Dnm
Dynamin
DRP
Dynamin Related Protein
ELM1
Elongated Mitochondria1
ER
endoplasmic reticulum
ETC
electron transport chain
FAD
oxidized flavin adenine dinucleotide
FADH2
reduced flavin adenine dinucleotide
FIS1
Fission1
FP
fluorescent protein
xi
Fzo1
fuzzy onions homolog 1
gDNA
genomic DNA
GFP
Green Fluorescent Protein
GOX
glyoclate oxidase
GTP
guanosine-5’-triphosphate
H2O2
hydrogen peroxide
HL
high light
HO˙
hydroxyl radicals
IMM
inner mitochondrial membrane
IMS
intermembrane space
LB
Luria broth
MAL
maltose
MAPL RING MAPL-Really Interesting New Gene finger domain
MAPL
Mitochondria-Anchored Protein Ligase
MCS
membrane contact site
Mdv1
Mitochondrial Division Protein 1
MES
2-(N-morpholino)ethanesulfonic acid
Mfn1/Mfn2
Mitofusin1/2
Mgm1
Mitochondrial Genome Maintenance
MS
Murashige and Skoog medium
NAD+
oxidized nicotinamide adenine dinucleotide
NADH
reduced nicotinamide adenine dinucleotide
NOS
reactive nitrogen species
1
singlet oxygen
O2
O2˙ –
superoxide anion radicals
OAA
oxaloacetate
OMM
outer mitochondrial membrane
Opa1
Optic Atrophy1
p35S
Cauliflower Mosaic Virus 35S promoter
PYR
pyruvate
PSI
photosystem I
xii
PSII
photosystem II
Q10
coenzyme Q10
r
radius of curvature
RFP
Red Fluorescent Protein
ROS
reactive oxygen species
Rubisco
ribulose-1,5 bisphosphate carboxylase-oxygenase
SEM
scanning electron microscopy
SOD
superoxide dismutase
TCA cycle
tricarboxylic acid cycle
TEM
transmission electron microscopy
TPR
tetratricopeptide repeat
tr
trichomes
Ugo1
Ugo (Japanese for fusion)
WT
wild type
YEB
Yeast Extract Broth
YFP
Yellow Fluorescent Protein
ΔΨm
membrane potential
xiii
CHAPTER 1
INTRODUCTION
1.1
MITOCHONDRIAL PLEOMORPHY AND FUNCTION
Sui generis: in a class of its own; unique. This phrase was used to describe mitochondria
just over a century ago by Cavers (1914) because of the plethora of appearances they can
display. Mitochondria are typically long and filamentous in fibroblast cells, whereas hepatocyte
cells have a predominance of spherical or ovoid mitochondria (Youle and van der Bliek 2012). In
yeast cells, they usually form a network consisting of 1-10 mitochondria (Hoffman and Avers
1973; Stevens 1981; Nunnari et al. 1997). In most plant cells however, they are typically small,
spherical or rod shaped (Logan 2010), although the first published image of mitochondria in
plants showed them to be elongated ‘fila’ (Meves 1904). This organelle can also exhibit
pleomorphy within one particular cell under observation; the same mitochondrion could be
spherical and stretch into a small rod one moment or become elongated the next (Cavers 1914;
Logan and Leaver 2000). Flemming (1882) first called this truly dynamic organelle ‘fila’ as they
appeared thread-like, but Altman (1890) described them as granules that resembled bacteria,
calling them ‘plastosomes’. Since then, they have been called bioblasts, chondriomiten,
chondriosomes, chromidia, plastokonts etc., all describing a particular form of the same
1
organelle. For this reason, Benda (1897) justly assigned the name ‘mitochondria’ (Gr. mitos =
thread, chondrion = granule), in acceptance that this organelle was capable of a wide range of
appearances. Although many factors, stresses (Table 1.1) and cell types, functions, and
requirements seem to dictate the appearance this organelle assumes, there is a clear link between
the energy and metabolic status of a cell and the pleomorphy that mitochondria exhibit. This is
especially apparent at the ultrastructural level.
Table 1.1 Factors influencing mitochondrial morphology
Factor
Influence on Shape
References
Glutamate
Increase roundness
Rintoul et al. 2003
Calcium
Increase roundness
Boustany et al. 2002; Stolz
and Bereiter-Hahn 1987
Matrix accumulation - beading
Glucose
Fragmentation
Increase roundness
Hackenbrock 1966
H2O2
Increase roundness
Yoshinaga et al. 2005
NO
Fragmentation
Barsoum et al. 2006
CO2
Fragmentation
Lewis and Lewis 1914
Riboflavin deficiency
Enlargement
Tandler et al. 1969; BereiterHahn and Voth 1994
ATP
Orthodox ultrastructure
Novikoff and Holtzman 1976;
Bereiter-Hahn and Voth 1994
ADP
Condensed ultrastructure
Novikoff and Holtzman 1976;
Bereiter-Hahn and Voth 1994
1.1.1 Mitochondrial ultrastructure: Orthodox and condensed states
Mitochondria can exist in two general conformational states: condensed and orthodox
(Fig. 1.1B). The balance between these states is influenced by changing environmental
conditions such as fluctuations in oxygen, carbon and metabolite availability (Hackenbrock
1968; Hackenbrock 1971; Novikoff and Holtzman 1976; Bereiter-Hahn and Vöth 1994; Logan
and Leaver 2000). In the condensed state, the intracristal spaces are dilated which condenses the
matrix. This state is seen when adenosine diphosphate (ADP) levels are high and the respiratory
pathways are active (Fig. 1.2). But when adenosine triphosphate (ATP) levels increase,
respiration and ADP phosphorylation are signalled to stop. In this resting state, the intracristal
2
spaces become narrow again, allowing the matrix to expand (Novikoff and Holtzman 1976;
Bereiter-Hahn and Vöth 1994). This is the orthodox state. In cells with high energy functions,
such as muscle cells, cristae are closely packed and more abundant (Novikoff and Holtzman
1976), most likely because of the greater requirement for active respiration (Fig. 1.2). The
rearrangement of the inner mitochondrial membrane (IMM) is seen in response to fluctuating
ATP/ADP levels because this is where the electron transport chain (ETC) is located and where
oxidative phosphorylation takes place in mitochondria.
1.1.2 The electron transport chain and ATP synthesis
Cellular respiration consists of several pathways whereby nutrients are metabolized to
produce ATP. During glycolysis, glucose is catabolized to produce pyruvate, reduced
nicotinamide adenine dinucleotide (NADH) and ATP. Pyruvate is further metabolized by the
tricarboxylic acid (TCA) cycle. This cycle also oxidizes NAD+ and FADH to NADH and
FADH2, respectively. Available NADH and FADH2 enter the oxidative phosphorylation pathway
where they are reduced by the ETC (Fig. 1.1C). The electrons released are transported down a
chain of complexes in the IMM until they are finally accepted by O2. As the electrons are being
transported, the complexes also pump H+ from the matrix into the intermembrane space (IMS).
This creates an electrochemical gradient where the membrane potential (ΔΨm) is negative on the
matrix side but positive on the IMS side (Hoefnagel et al. 1998; Brookes et al. 2004). This
gradient is used by ATP synthase to phosphorylate ADP to ATP. The more energy utilized by
the cells, the more rapidly ATP is converted back to ADP. This increase in ADP level
subsequently permits a cycle whereby the rate of electron transport, oxygen consumption and
ATP production increases again (Novikoff and Holtzman 1976). However, as this oxidative
phosphorylation process becomes more frequent, higher levels of reactive oxygen species (ROS)
are produced by the mitochondrial complexes. This leads to damage of lipids, proteins, and
mtDNA.
3
A
B
OMM
Condense
Orthodox
IMM
IMS
Matrix
Cristae
C
4H+
4H+
2H+
4H+
C
I
IV
III
Q10
Intermembrane
space (+ΔΨm )
IMM
ATP
Synthase
II
Matrix (-ΔΨm)
ATP
NADH
+
-
NAD + 2e
FADH2
+
ADP
½ O2 + 2H+
-
FAD + 2e
H2O
Figure 1.1 Mitochondria structure and inner membrane composition. (A) Mitochondria are
semi-autonomous organelles with two membranes. The IMM encapsulates the matrix. It is enclosed
by the OMM, which creates a space between them called the IMS. The folds created by the IMM
are cristae. (B) When the intracristal spaces become dilated, thereby condensing the matrix, a
mitochondrion is said to be in the condensed state. If the intracristal spaces are narrow, the matrix
expands. This is the orthodox state. (C) The IMM is home to the ETC. NADH and FADH 2 are
reduced by complex I and II, respectively. The 2e - released from each reaction are transported from
complex to complex. In the process, an electrochemical gradient is created as H + are pumped out of
the matrix by complexes I, III and IV. This gradient is used to drive the production of ATP by ATP
synthase. Here, ADP is phosphorylated to ATP. The condensed state is electron dense and is seen
when ADP levels are high. Here, respiration is taking place to phosphorylate ADP to ATP. The
matrix is electron dense as the gradient is being created to drive ATP synthesis. When ADP levels
are low however, ATP levels are high, respiration and phosphorylation cease and the orthodox state
is assumed. Hence, the condensed state can be associated with active respiration and the orthodox
state with inactive respiration and high ATP levels. OMM = outer mitochondrial membrane; IMM =
inner mitochondrial membrane; IMS = intermembrane space; ETC = electron transport chain;
complex I = NADH dehydrogenase; complex II = succinate dehydrogenase; complex III =
cytochrome c reductase; complex IV = cytochrome c oxidase; Q10 = coenzyme Q10; C = cytochrome
c complex; dotted arrows = path of 2e- released from the reduction of each NADH and FADH 2
before being accepted by O2; dashed arrows = transport of H+. Compiled from Hackenbrock 1968;
Hackenbrock et al. 1971 (source of TEMs); Bereiter-Hahn and Vöth 1994; Hoefnagel et al. 1998;
Brookes et al. 2004.
4
Inject:
Inject:
ADP
ATP
Respiration:
Respiration:
Active
Inactive
Intracristal spaces:
Intracristal spaces:
Expand
Narrow
State:
State:
Condensed
Orthodox
ATP:
ADP:
ATP:
ADP:
Produced
Phosphorylated
Used
Produced
Figure 1.2 Mitochondrial orthodox and condensed state transitions in response to ADP
and ATP injections. When cells are injected with ADP, the intracristal spaces dilate,
condensing the matrix. This is the condensed state. The cell is actively respiring, and ADP is
being phosphorylated into ATP. When cells are injected with ATP, however, the opposite is
seen. The cell no longer needs to respire, as ATP levels are high now. Respiration is signalled to
cease. The intracristal spaces are narrow, and the matrix can expand. This is the orthodox state,
which is typically seen in most cells. As ATP is utilized, it becomes dephosphorylated to ADP;
ATP levels drop. The cell must commence respiration to replenish ATP. The mitochondria
acquire the condensed state. Once levels are normal again, respiration stops. The orthodox state
is assumed. Based on Hackenbrock 1968; Hackenbrock et al. 1971; Novikoff and Holtzman
1976; Bereiter-Hahn and Vöth 1994.
1.2
TRIGGERS AND CONSEQUENCES OF MITOCHONDRIAL FUSION AND
FISSION
The pleomorphic nature of mitochondria is permitted by constant membrane fusion and
fission. Fusion and fission serve different purposes but the balance between both events
ultimately leads to cellular homeostasis. Fusion of the OMM and IMM of two or more
mitochondria allows complementation of damage caused by oxidative stress (Fig. 1.3). By
forming a continuous network of membranes, contents of the IMS and of the matrix of each
5
mitochondrion become mixed. As a result, solutes, metabolites, proteins and electrochemical
gradients are shared (Nakada et al. 2001; Skulachev 2001; Arimura et al. 2004; Chen et al. 2005;
Twig et al. 2006; Benard et al. 2009). This is an important process because it allows the effects
of damaged constituents to be diluted, thereby permitting recovery and continued functionality
(Chan 2006; Twig et al. 2008). Subsequent fission allows each mitochondrion to disperse within
the cell where the process can occur again if necessary. Thus, it comes as no surprise that
fragmented ‘plastosomes’ are found in cells of high energy demand such as muscle and near
flagella (Bereiter-Hahn and Vöth 1994). This is because the damage caused by higher rates of
respiration is more severe and this process of complementation must occur more rapidly.
1.3
ROS PRODUCTION IN PLANTS
A key signal responsible for stimulating oxidative phosphorylation in mitochondria is
calcium (Ca2+). If the concentration of matrix Ca2+ ([Ca2+]m) becomes elevated, oxidative
phosphorylation and ATP output are also upregulated (Brookes et al. 2004). [Ca2+]m is regulated
by several mechanisms such as the Ca2+ uniporter (UP), ryanodine receptor (RyR), the
permeability transition pore (PTP) and ΔΨm. Under normal conditions, Ca2+ is pumped into the
mitochondria through the PTP and signals the TCA cycle. This feeds electrons into the ETC
(Fig. 1.4). Under certain stress conditions that lead to increased [Ca2+]m, oxidative
phosphorylation occurs more rapidly causing an even greater increase in ATP synthesis. Such a
scenario is seen during a cell death response. This is because ROS, a signal for cell death and
apoptosis, is overproduced (Foyer and Noctor 2003; Apel and Hirt 2004; Brookes et al. 2004;
Mittler et al. 2004; Yoshinaga et al. 2005; Yu et al. 2008).
ROS are reactive derivatives of oxygen due to their partially reduced or activated states. It
includes singlet oxygen (1O2), superoxide anion (O2˙ˉ), hydroxyl radical (OH˙) and hydrogen
peroxide (H2O2) (Klotz 2002; Apel and Hirt 2004). ROS are involved in signaling and activating
transcription factors, but can be detrimental at high levels. Due to the highly reactive nature of
ROS, they can cause severe oxidative damage to proteins, lipids and DNA (Apel and Hirt 2004;
Bhattacharjee 2011), which is why the complexity and sheer abundance of the mechanisms
6
Fusion
Mixing of constituents
Fission
Triggered by stress:
 Low energy/starvation
Such as:
 (Damaged) proteins
Triggered by:
 ATP depletion
 (Damaged) mtDNA
 Overwhelming of ETC
 Depolarization of ΔΨm
 Solutes
Leading to:
 Inhibition of fission &
autophagic catabolism
Leading to:
 Complementation of damaged
mitochondrion
 High energy input
 Transmission of energy
Leading to:
 Increase in number &
dispersal of complemented
mitochondria and of energy
 Maximized oxidative
phosphorylation
 Quality control & reduced
stress
 Hyperpolarization of ΔΨm
 Sometimes mitophagy
Figure 1.3 Triggers and consequences of mitochondrial fusion and fission. Fusion and
fission activity constantly fluctuates as a cell attempts to achieve homeostasis. Under low
energy, such as during metabolic starvation, ATP levels are depleted and ΔΨm becomes
depolarized. Mitochondria undergo fusion to help repolarize the ΔΨm and maximize oxidative
phosphorylation. The mitochondria then undergo fission and disperse. This dispersal permits the
transfer of energy throughout the cell. When mitochondrial constituents such as proteins, lipids
and mtDNA become damaged (i.e. by oxidative damage via the ETC), mitochondria also fuse.
This allows the mixing of wild type and damaged constituents and complementation. The
mitochondrion then undergoes fission and the resulting mitochondria disperse. However, when
the damage is too much, the mitochondrion enters the autophagic pathway. Such a case is seen
during ROS-induced apoptosis. Red = damaged mitochondrion; green = normal, wild type
mitochondrion; yellow = fusion between damaged and normal mitochondria; ΔΨm = membrane
potential. Adapted from and based on Amchenkova et al. 1988; Bereiter-Hahn and Vöth 1994;
Legros et al. 2002; Yu et al. 2006; Twig et al. 2008; Youle and van der Bliek 2012.
involved in ROS regulation (Table 1.2) is not surprising; the detoxification of ROS is crucial for
maintaining cell survival.
A fundamental difference between animals and plants is a plant’s ability to carry out
photosynthesis through chloroplasts. Photosystem I (PSI) and PSII of thylakoid membranes
within chloroplasts are considered the primary sites of ROS production. Light drives the
photosynthetic process in which O2 is continuously produced by photosynthetic electron
transport in chloroplasts (Fig. 1.4). The photo-reduction of O2 by PSI then yields O2˙ˉ (the
Mehler reaction; Mehler 1951; Asada 1999; 2006). This is because PSI electron carriers have
7
negative electrochemical potentials and therefore the electrons are leaked to O2 (Bhattacharjee
2011). O2˙ˉ can be further photoreduced to H2O2. When there is excess photochemical or light
energy, the generation of O2˙ˉ and H2O2 is heightened. H2O2 then actually diffuses out of the
chloroplast membranes into the cytosol. It can also travel short distances into peroxisomes (Table
1.2; Foyer and Noctor 2003; Bhattacharjee 2011).
Table 1.2 Common ROS and their removal
ROS
Removal Mechanism (Product)
Removal Mechanisms
found in
Superoxide (O2˙ˉ)
Superoxide dismutase (H2O2)
C, Cyt, M, P
Hydrogen peroxide (H2O2)
Catalase (H2O)
Peroxidase (H2O)
Ascorbate/glutathione cycle (H2O)
Glutathione peroxidase (H2O)
Singlet oxygen (1O2)
Carotenes and tocopherol (O2)
M, P
P
C, Cyt?, M, P
C, Cyt, ER, M
C
Diffusion
Distance
30nm
1μm
30nm
C = chloroplasts; Cyt = cytosol; M = mitochondria; P = peroxisomes; ER = endoplasmic
reticulum. Adapted from Bhattacharjee 2011.
Peroxisomes, initially termed microbodies (Rhodin 1954), are organelles that play crucial
roles in eukaryotic cells. They are involved in fatty acid β-oxidation and photorespiration
(Beevers 1979). Through its resident antioxidant enzymes such as peroxidase, superoxide
dismutase (SOD) and catalase, this organelle plays an important role in detoxifying aerobic cells
of free radicals by aiding in the scavenging of ROS and reactive nitrogen species (NOS) (de
Duve and Baudhuin 1966; Foyer and Noctor 2003; del Río et al. 2006; Bhattacharjee 2011).
When H2O2 enters peroxisomes, it is reduced by catalase to H2O (Foyer and Noctor 2003; Asada
2006). Another pathway in which chloroplasts and peroxisomes are linked is by the Calvin cycle.
During the Calvin cycle in chloroplasts, glycolate is produced through an oxygenation reaction
(Fig. 1.4). Glycolate is translocated from chloroplasts to peroxisomes where it is converted to
glyoxylate by glycolate oxidase (GOX). In the process, H2O2 is generated again but is also
reduced by catalase (Fig. 1.4; Foyer and Noctor 2003).
Mitochondria are also producers of ROS due to the ETC (Halliwell and Gutteridge 1999;
Chen et al. 2003; Foyer and Noctor 2003; Apel and Hirt 2004; del Río et al. 2006; Bhattacharjee
2011). For example, O2˙ˉ is produced during the very first steps of the ETC when NADH
8
dehydrogenase (complex I) and succinate dehydrogenase (complex II) reduce NADH and
FADH2, respectively. O2˙ˉ then goes to the mitochondrial matrix-antioxidant enzyme system,
such as alternative oxidase (AOX) and catalase where it is inactivated (Chen et al. 2003; Foyer
and Noctor 2003; Bhattacharjee 2011). Cytochrome c reductase (complex III) is another major
producer of O2˙ˉ in mitochondria, however. Here, O2˙ˉ is released to the IMS rather than to the
matrix. SOD is found in the IMS and converts O2˙ˉ to H2O2 (Foyer and Noctor 2003). At high
concentrations, H2O2 can diffuse through the OMM into the cytosol as well (Chen et al. 2003).
The H2O2 released can diffuse through the peroxisomal membrane where it can also be reduced
to H2O. These metabolic and biochemical pathways are not the only way peroxisomes and
mitochondria are linked.
1.4
MITOCHONDRIA AND PEROXISOME PLEOMORPHY IN RESPONSE TO ROS
Imaging of peroxisomes in living cells has revealed them to be pleomorphic as well, with
spherical, tubular and beaded forms being observed (Fig. 1.5A). It has also provided an
appreciation of their dynamic nature and altered motility during ROS stress. Sinclair et al. (2009)
used a copper chloride/ascorbate mixture to produce hydroxyl radicals. After 1-2min of
exposure, peroxisomes, which are spherical under normal physiological conditions in plant cells,
became immotile and elongated after 1min. Elongated peroxisomes then became beaded and
resumed motility around 2h after the copper chloride/ascorbate mixture was washed off. The
beaded state was followed by fission and then dispersal of the new peroxisome counterparts. The
authors also demonstrated that UV irradiation, which induces ROS production, of single
Arabidopsis thaliana (Arabidopsis) hypocotyl cells caused all peroxisomes to elongate after 90s.
Neighbouring cells however, which received lower, peripheral exposure to UV, displayed
spherical peroxisomes with thin projections known as peroxules.
9
H2 O2
Lipids
Gene Expression
& Silencing
H2 O2
Lipids
Glycerate
Pyruvate
O2 H O
2 2
H2 O2
SOD
Glycolate
H2 O2
GOX
NH
4+
CAT,
AsAGSH
ETC H2O
Glycine
H2 O + O 2
O2. –
NADH, FADH2
CAT
Serine
TCA
Cycle
O2
Glycerate
Ca2+
MAL
P-Glycolate
OAA
PYR
Light
H2 O
ETC
Calvin
Cycle
½ O2
e–
PSII
1
O2
e–
PSI
e–
DHAP
NADPH
O2. –
Carotenoids,
α-tocophenol
SOD
O2
O2
H2 O2
Ascorbate
Peroxidase
H2 O
Figure 1.4 Key metabolic and ROS links between mitochondria, peroxisomes and
chloroplasts. Key pathways of ROS production and metabolic exchange are shown to
rationalize the close, physical contact between the organelles. Chloroplasts=blue;
peroxisome=yellow; mitochondria=green; ER= red; nucleus=magenta; white=cytoplasm.
CAT=catalase; GOX=glycolate oxidase; SOD=superoxide dismutase; AsA-GSH=ascorbateglutathione cycle; TCA= tricarboxylic acid cycle; PYR=pyruvate; OAA=oxaloacetate;
MAL=maltose. Based on Fredrick and Newcomb 1969 (source of TEM); Hoefnagel et al. 1998;
Foyer and Noctor 2003; Apel and Hirt 2004; Brookes et al. 2004; Bhattacharjee 2011; Gechev et
al. 2011.
Named for their morphological resemblance to plastid stromules, peroxules are thin, shortlived tubular projections from a main spherical to ovate peroxisome body (Scott et al. 2007;
Sinclair et al. 2009; Delille et al. 2010; Barton et al. 2014). In contrast to completely elongated
10
peroxisomes, it has never been shown that peroxules undergo fission. These elastic peroxules
may therefore be a mere "stress threshold sensing mechanism” (Sinclair et al. 2009; Mathur et al.
2012). They may extend and retract in response to low fluctuations of ROS, but once levels
exceed a certain threshold, peroxisomal elongation and fission may be signalled in order to
increase peroxisomal numbers and dispersal throughout the stressed cell (Schrader et al. 1999;
Scott et al. 2007; Sinclair et al. 2009; Delille et al. 2010; Mathur et al. 2012; Barton et al. 2014).
This morphological progression leading to fission is also exhibited by mitochondria (Fig.
1.5B), which become extensively fragmented in response to high ROS levels during mitophagy,
apoptosis and cell death (Ingerman et al. 2005; Yoshinaga et al. 2005; Jendrach et al. 2008; Yu et
al. 2006; 2008; Frank et al. 2012). Mitochondria have also been reported to have thin projections
called terminal matrixules. Additionally, when a tubular mitochondrion divides, it becomes
constricted causing it to appear as beads-on-a-string; medial matrixules were described as the
string portion (Logan et al. 2004; Logan 2006). Although the exact roles of peroxules and
matrixules are not fully understood, key proteins involved in the fission process of elongated
peroxisomes and mitochondria are known. Furthermore, it has become very evident that apart
from the biochemical and metabolic links and commonalities in morphological processions
during fission, they also share common fission machineries.
11
A
Low ROS
Contorted
DRP3A, DRP3B
FIS1A, FIS1B
Fission
Beaded
Enlarged
Elongated
High ROS
B
ROS
DRP3A, DRP3B
FIS1A, FIS1B
Increased ROS
Figure 1.5 Sequential events leading to peroxisome and mitochondria fission reveals
morphological similarities in the division pathway. (A) Under low ROS levels, peroxules
extend and retract rapidly from the main peroxisome body. Upon high levels of ROS,
peroxisomes elongate. These elongated peroxisomes become beaded before finally undergoing
fission. (B) ROS also leads to mitochondria fission, whereby the same division factors, DRP3
and FIS1 constrict the membranes of the elongated form. Peroxisome division complied from
compiled from the reports of Tabak et al., 2006; Titorenko and Mullen, 2006; Scott et al., 2007;
Sinclair et al., 2009; Delille et al., 2010; Mathur et al., 2012; Barton et al., 2014. Mitochondria
division complied from Ingerman et al., 2005; Yoshinaga et al., 2005; Jendrach et al., 2008; Yu
et al., 2006, 2008; Frank et al., 2012.
12
1.5
MECHANISMS OF MITOCHONDRIAL FUSION AND FISSION
Several components of the fusion and fission machinery of mitochondria in yeast and
mammals have been described (Table 1.3). No plant orthologues for fusion have been identified
to date, but three have for fission: FISSION1 (FIS1), DYNAMIN RELATED PROTEIN3
(DRP3) and ELONGATED MITOCHODNRIA1 (ELM1). The former two are also involved in
peroxisomal fission and the latter is thought to be a functional homologue of the yeast
Mdv1/Caf4 fission proteins.
Table 1.3 Proteins involved in mitochondrial fusion and fission
Fusion/Fission
Component
(Yeast/Mammals/Plants)
Location
Function
Fission
DNM1/ DRP1 or DLP1/
DRP3A & DRP3B1,3
Cytosol & OMM
Fission of OMM
FIS1p/ hFIS1/ FIS12,3
OMM
Receptor for DNM1/ DRP1 or DLP1/
DRP3A & DRP3B, respectively
MDV1/ – / ELM14
Cytosol & OMM
Mediator between the two above fission
proteins
CAF4p/ – / –
Cytosol & OMM
MDV1 homolog; redundant function
FZO1/ MFN1 & MFN2/ –
OMM
Fusion of OMM
UGO1/ – / –
OMM
Coordinates OMM and IMM fusion
MGM1/ OPA1/ –
IMM & IMS
IMM fusion
Fusion
1
DRP3A and DRP3B are also referred to as ADL1 and ADL2, respectively
FIS1 is also referred to as BIGYIN
3
all three orthologues are also involved in peroxisomal fission
4
no sequence similarity, but proposed function of EML1 is similar to MDV1 and CAF4p
OMM = outer mitochondrial membrane; IMM = inner mitochondrial membrane; IMS = intermembrane
space. Adapted and compiled from Arimura et al. 2008; Westermann 2008.
2
Arabidopsis has two DRP3 homologs, DRP3A and DRP3B, which have partially
redundant functions and are involved in both mitochondria and peroxisome fission (Arimura and
Tsutsumi 2002; Arimura et al. 2004; Logan et al. 2004; Mano et al. 2004; Zhang and Hu 2009).
DRPs are GTPases that are recruited to mitochondria and peroxisome membranes by FIS1, form
spirals around membranes thereby tubulating and constricting them, and mechanochemically
13
mediate the fission and fusion of those membranes (Yoon et al. 2001; 2003; Kobayashi et al.
2007). Mitochondria and peroxisomes in the drp3a/apm1 mutant have an aberrant morphology
where they appear greatly elongated (Mano et al. 2004), which suggests that the actual fission of
the organelles, not the tubulation, is affected.
FIS1 has two homologues in plants as well: FIS1A and FIS1B (Scott et al. 2006; Zhang
and Hu 2008). Both have a C-terminal transmembrane domain that anchors them to the OMM
and peroxisome membrane and a C-terminal tail that extends into the matrix (Yoon et al. 2003;
Scott et al. 2006). The N-terminal region is exposed to the cytoplasm and has a tetratricopeptide
repeat (TPR)-like binding domain that can interact with Dlp1 (animal DRP3 homologue) in the
case of hFis1 and supposedly DRP3 in the case of plant FIS1 (Yu et al. 2005). In fis1p/hfis1
mutants (yeast and mammalian homologs, respectively), mitochondria form a net-like sheet, but
mitochondria in the plant fis1 mutant decrease in number and increase in area (Scott et al. 2006).
Overexpression of both FIS1A and FIS1B on the other hand, results in an increase in the number
of mitochondria and peroxisomes (Zhang and Hu 2008). These phenotypes can be explained by
linking FIS1 to a role in fission; knockouts of FIS1 mean DRP3 cannot bind to it to constrict and
facilitate the scission of mitochondria and peroxisomes, but overexpression makes it more
available, leading to increased fission.
The elongated mitochondrial phenotype of drp3a/apm1 is also seen in elm1 mutants.
ELM1 is thought to be a functional homolog of the mitochondrial fission proteins Mdv1 and
Caf4p in yeast. The mutants not only have elongated mitochondria, but they form extensive
branched networks, resembling a net-like sheet similar in appearance to the polygon network of
the endoplasmic reticulum (ER; Arimura et al. 2004; Friedman and Voeltz 2011). For these
reasons, ELM1 is proposed to be a mediator protein between FIS1 and DRP3 for mitochondrial
fission, but not peroxisomal fission as the peroxisome morphology in elm1 is unaffected
(Arimura et al. 2008).
It is interesting that mitochondria in elm1 form networks that resemble the ER network
because Friedman et al. (2011) showed that in yeast and mammalian COS-7 cells, the ER is
involved in the fission of mitochondria as well. The authors used three-dimensional
reconstruction to show that ER tubules constrict mitochondria at potential fission sites. The
14
helices formed by Dnm1 and Drp1 are smaller in diameter than that of a non-constricted
mitochondrion. The authors proposed that because of this, the ER wraps around mitochondria to
constrict them enough for Dnm1/Drp1 to fit around the tubular structure. This has not been
shown in plants to date, but given that the functions of FIS1 are DRP3 conserved, this interaction
between the ER and mitochondria may occur in plant cells as well and should be investigated.
1.6
LIVE IMAGING OF ORGANELLES
Small organic dyes can be used for antibody targeting of specific intracellular proteins,
however utilization of these requires fixation, deeming live-imaging impossible (Giepmans et al.
2006). There are dyes that can penetrate the living cell to visualize particular components, such
as MitoTracker dyes that stain for mitochondria (Invitrogen/Molecular Probes; Chazotte 2011).
However, these dyes are often toxic, therefore there is only a short period of time to conduct
live-imaging observations (Mathur 2007). But since the cloning of the Green Fluorescent Protein
(GFP) from the jellyfish Aequorea victoria (Prasher et al. 1992), this is no longer a major barrier,
at least not for studying Arabidopsis. This is because the fluorescent protein (FP) can be fused to
a specific nucleotide sequence and incorporated into the Arabidopsis genome for stable transgene
expression (Giepmans et al. 2006; Haseloff and Siemering 2006; Mathur 2007; 2010). For
example, GFP can be fused to an ER membrane peptide sequence, calnexin (CX; Fig. 1.6). The
cells produce this GFP-CX fusion normally and the result is a fluorescently labelled ER still
capable of movement and trafficking. There is now a wide spectrum of FP available and because
a FP can be fused to a specific nucleotide sequence and therefore targeted to a particular
organelle, more than one FP-fusion can be expressed simultaneously. As a result, a key attribute
to cell dynamics, organelle interactions, can be investigated using live fluorescent microscopy.
15
A
B
C
D
Figure 1.6 Fluorescent labelling of the ER for live imaging. (A-C)The most common FP are
shown here labelling the ER: RFP, YFP and GFP. They were N-terminally fused to the
signalling peptide of calnexin, an ER membrane specific protein. Fusion of the FP to a signal
peptide specific to a particular organelle permits differential labelling, whereby more than one
organelle can be fluorescently labelled, each with a different FP. (D) The green-to-red photoconvertible mEosFP was fused to HDEL, a specifically ER lumen protein. Short UV irradiation
of a small region of the organelle (right) photo-converts the GFP to RFP. As the contents of the
ER lumen mix, GFP and RFP mix as well. An intermediate colour would be seen.
1.7
OBJECTIVES
A living cell is quite a dynamic system. This is attributed to the constant movement of
organelles and trafficking of constituents between the organelles such as metabolites and
signalling compounds like ROS. Because of this, even though organelles are membrane bound
entities, the processes occurring within them are by no means isolated from the rest of the cell –
organelles are truly interactive. While moving throughout the cell and interacting with one
another, they display morphological rearrangements. Some of these morphological transitions
have been associated with cell stress and fluctuating inter- and intracellular environmental
conditions.
The main objectives of this thesis are to:
1) identify conditions to reproducibly induce morphological transitions of mitochondria and
peroxisomes to better understand how pleomorphy can be an indicator of cell stress;
16
2) better understand the mechanisms behind their pleomorphy; and
3) better understand organelle interactivity during light stress.
Double and triple Arabidopsis transgenics will be created and used to simultaneously
visualize fluorescently labelled mitochondria, peroxisomes and the ER using live imaging. The
photosynthetic impacts of light stress such as fluctuations in cytosolic sugar content, O 2
depletion and ROS production, are of key interest.
1.8
HYPOTHESES
The main hypotheses that are investigated in this thesis are:
1) Mitochondria in plant cells will exhibit fragmentation in response to high cytosolic sugar
content, just as in mammalian cells;
2) Mitochondria in dark grown plants will be elongated, morphologically resembling those of
animal cells. These elongated mitochondria will fragment in response to light;
3) The endoplasmic reticulum will mediate mitochondrial fragmentation and pleomorphy in
plant cells;
4) The interactivity between fragmented mitochondria and peroxisomes will increase in
response to high light and ROS stress; and
5) Peroxules will facilitate mitochondria-peroxisome interactions.
17
CHAPTER 2
MATERIALS AND METHODS
2.1
CLONING OF FLUORESCENT PROTEIN-FUSION CONSTRUCTS
RNA was extracted from Col-0 leaf tissue or whole seedlings with the EZ-10 Spin
Column Plant RNA Mini-Prep Kit (BioBasic Inc.) and used to prepare complementary DNA
(cDNA) using the Thermo Scientific Revertaid H Minus First Strand cDNA Synthesis Kit
(Fermentas). This was then used to PCR amplify the coding sequences (CDS) for each gene of
interest. Promoters were PCR amplified from Col-0 genomic DNA (gDNA). Primers flanked the
sequence upstream of the CDS of the gene of interest, including the 5’-UTR, up to 1kb without
including the sequence of another gene. Primer sequences and restriction enzyme sites
18
introduced for subsequent clonings are available in Appendix III (Table III-1).
Once purified [EZ-10 Spin Column Plasmid DNA Kit (BioBasic Inc.)], all amplified
PCR products were ligated into a base vector, pGEM®-T Easy vector (Promega), and
transformed into Escherichia coli DH5α competent cells. Blue-white colony screening for
successfully ligated products was carried out by plating transformants on Luria-Bertani (LB)
medium (Bertani 1951) with ampicillin and X-galactosidase (Promega). White colonies which
contained the recombinant DNA were selected and used to inoculate overnight LB + ampicillin
cultures. DNA was purified and screened by digestion using the restriction enzymes introduced
by the forward and reverse primers during PCR amplification. The CDS and promoters were
then digested from the pGEM®-T Easy vectors and placed into a pCAMBIA 1300 binary vector
already containing a fluorescent protein (FP) of interest (Fig. 2.1). The cloning strategies for
most FP-fusion constructs were similar, but an example is described here.
HindIII
HindIII
HindIII
p35S
p35S
XbaI
mEosFP
Vector 648
cx
Vector 620
NaeI
backbone)
PMP22
KanR/HygR
Tnos SpeI
EcoRI
XbaI
BamHI
mEosFP
Tnos SacI
EcoRI
cx
Vector 943
(pCAMBIA 1300
(pCAMBIA 1300
(pS0022 backbone)
AmpR/ –
p35S
XbaI
BamHI
backbone)
KanR/HygR
RFP
Tnos
SacI
EcoRI
Figure 2.1 Core vectors used for clonings. The CDS (blue arrows) were replaced with the CDS
of a gene of interest using the restriction enzymes flanking the C- and N-terminus. The p35S
(CaMV35S) was replaced with the native promoter sequence (~1kb upstream of the respective
CDS including the 5’UTR). Vector 943 was created by inserting the RFP sequence (Ching et al.
2012) into the previously available vector 620 in place of the mEosFP sequence in the
BamHI/SacI position. The antibiotic resistance for bacterial/plant selection are indicated. Vector
648 contained a pS0022 backbone which lacks a plant selection marker required for subsequent
transgenic plant screening and was therefore used only as an intermediate vector. Vectors 620
and 943 have a pCAMBIA 1300 backbone which has a hygromycin resistant gene marker. This
was therefore used as a binary vector.
19
pfis1a-YFP-FIS1A (Ruberti 2014) was used to investigate the localization of FIS1A to
peroxisomes, mitochondria and chloroplasts. Also created was a mEosFP targeted FIS1A probe
driven by the constitutive Cauliflower Mosaic Virus 35S (CaMV35S) promoter (referred to as
p35S from here on). This was generated because the green-to-red photo-convertibility of
mEosFP may give further insight into the distribution and possible translocation of FIS1A
between the membranes of the three organelles.
The CDS of FIS1A (AT3G57090.1) was PCR amplified to introduce 5’-NaeI and 3’-SpeI
restriction
enzyme
sites
using
the
ATATGCCGGCATGGATGCTAAGATCGGACA;
following
and
primers:
forward
reverse
5’3’-
GCGCACTAGTTCATTTCTTGCGAGACATCG. This was then ligated into a pGEM®-T Easy
vector and screened as described above. The fis1a gene was then ligated into an existing pS0022
vector in which the green-to-red photo-convertible mEosFP was present in the XhoI and NaeI
position. The mEosFP-FIS1A fusion was excised as XbaI (upstream of the XhoI site in pS0022)
and SpeI and then ligated into an existing pCAMBIA 1300 binary vector. In the binary vector,
p35S was placed into the HindIII and XbaI position of pCAMBIA 1300 and a nos terminator
(Tnos) in the SacI and EcoRI position. The p35S-mEosFP-FIS1A construct was used to
transform electro-competent Agrobacterium (GV301) cells according to Weigel and Glazebrook
(2006), which were plated on Yeast Extract Medium (YEB) medium with rifampicin,
gentamycin and kanamycin. Plates were grown to confluence for 2 days at 28°C. mEosFP-FIS1A
driven by the native promoter was also created by replacing the p35S with pfis1a.
All binary constructs were tested for FP expression using a Nicotiana bentamiana
(tobacco) co-infiltration method. Small streaks the size of a pipette tip from the Agrobacterium
culture plates were resuspended in Agrobacterium infiltration media (AIM) [50mL of ddH2O,
7.5μL of acetosyringone, 0.5mL of 0.5M MgCl and 0.5mL of 2-(N-morpholino)ethanesulfonic
acid (MES)] for 2h at room temperature. The suspension was then diluted if necessary with AIM
to an optical density of 0.8. This was done for the construct of interest as well as a control
construct for which the localization was known, preferably one labeling an organelle in which
the protein of interest was thought to localize [ex. p35S-mEosFP-FIS1A was co-infiltrated with
mito-GFP [GFP-fused to the N-terminal pre-sequence of the mitochondrial β-ATPase subunit
(Logan and Leaver 2000)]. Using a 2mL syringe, equal amounts of each suspension were
20
injected through the abaxial side of ~1month old tobacco leaves. Leaves were checked for
fluorescence 2-3 days later using a Lecia epi-fluorescent microscope or confocal laser scanning
microscopy (CLSM).
2.2
CREATING TRANSGENIC ARABIDOPSIS LINES
A. tumefaciens GV3101 was used to introduce the FP-fusion constructs into Arabidopsis
(Columbia). This strain has a rifampicin resistance gene in its genome and a tumor inducing (Ti)
plasmid that carries a gentamycin resistance gene and virulence (vir) genes that are flanked by a
T-DNA border that incorporates into the plant genome during infection. One reason the
pCAMBIA 1300 backbone was used is that it also has this T-DNA border. 5mL YEB cultures of
the Agrobacterium containing a binary FP-fusion construct incubated at 28°C overnight. The
YEB contained rifampicin, gentamycin and kanamycin for selection. This was then used to
create a 500mL overnight culture. The culture was centrifuged at 3,000rpm for 20min at 4°C and
the pellets were re-suspended in a solution of 5% sucrose and 0.5% Silwet (~100mL). This was
used to transform Arabidopsis plants that were just beginning to flower using the A. tumfaciens
floral dip method (Clough and Bent 1998).
Arabidopsis ecotype Col-0 was transformed with all constructs created (Appendix II and
III). Double and triple transgenics were created in the same manner. For example, mito-GFP
(Logan and Leaver 2000) plants were transformed with pfis1a-mEosFP-FIS1A (double
transgenic) and mito-GFP Yperoxi (YFP-PST1; Mathur et al. 2002) was transformed with RFPER (RER; triple transgenic; Sinclair et al. 2009).
2.2.1 SELECTION OF STABLE LINES
All lines created using the pCAMBIA 1300 backbone were selected by growing sterilized
seeds on Murashige and Skoog (MS) medium (Murashige and Skoog 1962) with hygromycin
(12.5μg/mL). All plants that developed proper root systems that extended well into the medium
and were able to produce their first leaves were screened for fluorescence using an epi- or
confocal microscope. If positive for fluorescence, plants were planted in soil and grown for
second generation seeds. In the case of double and triple transgenics which already had a
transgene(s) and therefore already had the hygromycin resistance gene, seeds were plated on MS
21
without antibiotics. Lines were selected by screening all seedlings for all fluorescent proteins
visually, rather than through genotyping. Doubly or triply positive plants were harvested for
seeds. The second generation seeds were always plated on MS without antibiotic and selected for
fluorescence again. 10-16 plants were harvested for seeds. These were considered stable and
were used for subsequent experimentation. Double and triple transgenics were also created by
crossing and were visually selected during the second and third generations.
2.3
GROWTH CONDITIONS
Sterilized seeds were plated on MS with Gamborg vitamins and 3% sucrose, Parafilm-
sealed, stratified for 2-3 days, and grown for 7 days, unless stated otherwise. When light and
dark comparisons were being made, plants were either grown in light (16h/8h light/dark cycle) or
plates were wrapped with tinfoil immediately after vernalization for growth in the dark. Plates
for both conditions were always kept near one another to exclude temperature differences. When
sugar comparisons were being made, plants were either plated on MS with 3% or with no added
sucrose. The MS medium in all cases contained 3g/L of Phytagel (Sigma-Aldrich).
2.4
COLORIMETRIC SUGAR QUANTIFICATION
The phenol-sulfuric acid colorimetric method for quantifying the total soluble sugar of
plant tissue described by Buysse and Merckx (1993) was implemented to determine the relative
levels of sugar of plants grown in the light and dark. Plants were grown in the light (155μmolm2 -1
s ) or dark for 7 days on MS medium with or without 3% sucrose. 50 plants were quickly and
carefully harvested from each treatment either in light 1.5h into the light cycle or in a room in
near darkness. The plants were immediately placed into pre-weighed 2mL microfuge tubes
during harvest. The tissue was immediately frozen in liquid nitrogen and the tubes were weighed
after. The tissue fresh-weight was calculated by subtracting the post-harvest weight from the preweighed weight of the tube.
22
2.4.1 Sample preparation
The tissue was ground using magnetic beads and a bead beater (Retsch MM301) for 2min
at a frequency of 30.0 1/s and placed on ice immediately. After removing the magnetic beads,
500μl of 80% ethanol was added to the tissue and vortexed to homogenize the tissue in solution.
The tubes were centrifuged at 14,000rpm for 5min at 4°C. The supernatant was transferred to a
new tube on ice. This elution in ethanol was repeated two more times. After the final elution,
tubes were centrifuged one more time to remove residual tissue that could hamper quantification.
2.4.2 Quantification
A stock of 100μg/mL of glucose in 80% ethanol was diluted to 10 to 90μg/mL. These were
used to create a standard line. 500μL of the standard or sugar elution was added to a cuvette. To
each cuvette, 500μL of 28% phenol (w/w) was added. 2.5mL of concentrated sulphuric acid was
added directly to the liquid surface in a steady stream which helped with mixing. After 15min,
the absorbances were read at 490nm. Note that the blank was made the same way, except 80%
ethanol was used as the sample.
2.5
ASSESSING LIGHT-INDUCED STRESS
2.5.1 Induction of High Light Stress
Plants were all grown under intermediate light (50-164μmolm-2s-1). Observations were
made on the seventh day of growth, at least 1.5h into the light cycle for the day of
experimentation. High light (HL) (850 ± 50µmolm-2s-1) was only used for shorter treatments (15min) to observe the more rapid responses to light stress. Observations were taken immediately
after the light treatments. This was possible because the microscope used had a bright field light
which allowed plants to be irradiated directly on the microscope stage so that imaging could
commence immediately after irradiation. Time-lapses were at least 2min long. The time taken to
scan the entire field of view for each frame of the time-lapse was 3.93s.
23
2.5.2 3,3-diaminobenzidine Staining
To compare light-induced production of H2O2 in Col-0 and the anisotrophy1 (any1)
mutant (Fujita et al. 2013), plants were grown in the light (164μmolm-2s-1) for 8 days, transferred
to the dark for 24h and then exposed to light (164μmolm-2 s-1) for 30min, 1h or 2h. To look at the
effects of light intensity, seedlings were grown in the dark for 6 days, transferred to low light
(30μmolm-2s-1) for 2 days and then subjected to 0, 100, 200 or 300μmolm-2 s-1 of light for 24h.
Cotyledon and hypocotyl tissue were submerged in a solution of 3,3-diaminbenzidine (DAB)
(SIGMAFASTTM DAB with Metal Enhancer, Sigma-Aldrich) or distilled water and left under
vacuum (-50KPa) for 4h. The tissue was cleared with ethanol by washing the samples with 100%
ethanol, incubating them in 85% ethanol + 15% methanol overnight, and rinsing them in 70%
ethanol and then distilled water. The samples were mounted in 50% glycerol and sealed with nail
polish. All images were acquired at the same light intensity and microscope settings to permit
direct comparisons between treatments. The DAB stain intensity was measured as the average
inverse grey value using ImageJ (http://imagej.nih.gov/ij/) which was subtracted from the
background (the average inverse grey values of distilled water treated seedlings). The staining
intensity was representative of the amount of H2O2 produced during the relative light intensity
treatments.
2.5.3 H2O2 Responsive Fluorescent Probe
Arabidopsis transgenics expressing cytosolic HyPer-GFP11, hydrogen peroxide
responsive probe (http://www.evrogen.com/products/HyPer/HyPer.shtml, Evrogen, Russia;
Belousov et al. 2006; Costa et al. 2010) were used to look at the real-time production and
dissemination of H2O2 in response to a short light stimulus. Seedlings were given 1 to 5min of
HL and the change in GFP fluorescence was imaged right away. The ImageJ RGB Profile Plot
plugin was used to determine the changes in fluorescence intensity before and after HL
treatment.
2.6
ASSESSING anisotropy1 MUTANT SUSCEPTIBILITY TO STRESS
Scanning electron microscopy (SEM) and toluidine blue (TBO) staining was used to
24
assess the alteration of the cell wall in the any1 mutant. Leaf cross sections of 12 day old
seedlings grown in low light (70µmolm-2 s-1) were stained with TBO to analyse the alterations in
cell isotropy and cell-to-cell connectivity. SEM of whole leaf samples allowed the intercellular
spaces to be visualized with a higher resolution or clarity.
The any1 mutant was used to investigate the effects of altered light penetration, given its
reduced cell wall crystallinity (Fujita et al. 2013) and increased inter-cellular spaces. any1
Yperoxi mito-GFP double transgenic lines were also created to evaluate the mutant’s
susceptibility to HL stress at the subcellular level relative to wild type plants to aid in
understanding organelle interactions. This was done by elucidating if any1 was more susceptible
to HL stress. The production of H2O2 and the duration of interactions between the organelles
were compared in any1 and wild type plants.
2.6.1 Criteria for estimating interactions
mito-GFP Yperoxi and any1 Yperoxi mito-GFP double transgenics were used to visualize
peroxisomes and mitochondria simultaneously. An encounter between the mitochondria and
peroxisomes was considered an interaction if it lasted for ≥4 frames (15.72s) during a given
time-lapse sequence. An encounter was considered coincidental if the organelles moved apart in
Encounter
Not an Encounter
Sustained Interaction
Frame 1
2
3
4
Not a Sustained Interaction
Frame 1
Organelles remain stationary or if
there is motility, they move as a
group for ≥4frames
2
3
4
Organelles do not remain grouped
for ≥4frames; they disperse
Figure 2.2 Criteria for classifying a sustained interaction.
25
less than 4 frames. The diffusion distance of H2 O2 and O·– is ~1μm and 30nm, respectively
(Table 1.2), so an encounter was declared if the fluorescence of the organelles were overlapping
(Figure 2.2). A cut off of 4 frames was used because it was important to distinguish between
coincidence (i.e. the organelles were merely moving in the same direction) and an actual
sustained interaction (the organelles remained in contact long enough for the transfer of ions,
metabolites, membrane constituents etc.).
2.7
MICROSCOPIC VISUALIZATION
2.7.1 Epi-fluorescent microscopy
The Nikon Eclipse 80i epi-fluorescent microscope was used for selecting transgenic lines.
The filters available were Endow EGFP Longpass filter (41018; exciter HQ470/40X, dichroic
Q495LP, emitter HQ500LP), TRITC (41002c; exciter HQ545/30x, dichroic Q570LP, emitter
HQ620/60m) and DAPI (31000v2; exciter AT350/50x, dichroic 400LP, emitter 460/50m). There
were two major limitations with this microscope: it could not be used to visualize organelle
behaviour and punctate localization of FIS1 with high enough resolution; and only one filter
could be used at a time, making simultaneous visualization of organelles near impossible. CLSM
was therefore used for experimental observations.
2.7.2 Confocal Laser Scanning Microscopy
Fluorescent CLSM was used to conduct simultaneous, live imaging of organelle
behaviour and interactions. A Leica DM6000 microscope with a Leica TCS-SP5 scanning head
was used. A 488nm argon and 543nm helium-neon laser was used to excite GFP and RFP based
probes, respectively. Settings used for visualizing multiple FPs at once are shown in Table 2.1.
Time-lapse and 3D imaging was conducted using the Leica microscope software, Confocal LAS
AF. The bright field of this microscope was use for HL treatments. The 40X water immersion
lens (numerical aperture 0.80) was used for all imaging. Further zoom was achieved with the
manual zoom of the Leica software. The format used was 1024 x 512 pixels meaning that the
field of view using the 40X immersion lens with a zoom of 1.00 was 387.5 x 193.56μm. The line
average was set to 3, which meant that it took 3.945s to scan the entire field of view; each frame
26
in a time-lapse series was therefore separated by 3.945s.
Table 2.1 Emissions used to simultaneously visualize multiple FP
FPs Visualized
Green
Red
Chlorophyll
GFP, RFP, Chlorophyll
503-524nm
571-643nm
664-750nm
GFP, YFP, Chlorophyll
503-528nm
550-643nm
643-750nm
EosFP, Chlorophyll
500-536nm
580-653nm
653-754nm
GFP, MitoTracker Orange
500-535nm
555-622nm
634-781nm
2.8
STATISTICAL ANALYSIS
Unless stated otherwise, data was compiled from 4 cells per plant, 5 plants per treatment.
For mitochondrial length analysis, 10 mitochondria per cell were randomly measured. For
mitochondria-peroxisome morphological comparisons, a minimum of 6 of each organelle was
analyzed, and all organelles in the field of view were measured for each image. All experiments
consisted of at least 4 biological and 2 technical replications. When there was an unequal sample
size per treatment, a two-tailed t-test was used to determine the significance of results. For equal
sample sizes, a Tukey’s test of significance was used (Tukey 1949). Significance was
predetermined as having a p-value < 0.05 (95% confidence interval).
27
CHAPTER 3
RESULTS
3.1
IDENTIFYING CONDITIONS TO INDUCE TRANSITIONS BETWEEN
MITOCHONDRIAL MORPHOLOGIES
3.1.1 Mitochondria are predominantly small when grown with exogenous sugar
In hyperglycemic animal cells, elongated mitochondria fragment into small, spherical or
ovate forms (Yu et al. 2006; 2008; Jhun et al. 2013). As mitochondria in plant cells are typically
described as small, spherical to ovate in shape but can also be tubular (Meves 1904; Cavers
1914; Frey-Wyssling and Mühlethaler 1965; Logan and Leaver 2000), it was investigated
whether higher levels of cytosolic sugar can lead to the fragmentation of mitochondria in plant
cells as well. To do this, comparisons of mitochondria length in plants grown on MS medium
with and without sucrose were made.
First however, to be certain that the differences in exogenous sugar between the
treatments were reflected in the cytosolic sugar levels, the total soluble sugar levels were
estimated using a phenol-sulphuric acid colorimetric method (Buysse and Merckx 1993). Indeed,
plants grown on medium with sucrose had a higher level of total soluble sugar than plants grown
without sucrose (Fig. 3.1H). Confocal imaging showed that plants grown with sucrose had a
28
Light Grown
Light Grown
B
Dark Grown
G
3% Sucrose
3% Sucrose
A
Dark Grown
27%
26%
74%
73%
≤0.85μm
>0.85μm
Dark to Light Transfers
1h
F
12h
Total Soluble Sugar (µg/mg f.w.)
H
E
14%
No Sucrose
D
No Sucrose
C
42%
58%
86%
12
10
Light
Dark
8
6
4
2
0
3% Suc
0% Suc
Figure 3.1 Effects of light, dark and sugar on mitochondria length. mito-GFP Arabidopsis
transgenics were grown for 7 days in light (70µmol m-2 s-1) on MS medium with (A) 3% or (C)
no sucrose. Plants were also grown in the dark with (B) 3% or (D) no sucrose. Dark grown
plants grown without sucrose were transferred to light (70µmol m-2 s-1) for (E) 1h and (F) 12h
without sucrose. Sites of fission of tubular mitochondria are indicated (arrows). (G) Graphical
representation of mitochondria length under light and dark conditions when grown with 3% or
no sucrose. The percentage of mitochondria >0.85μm or ≤0.85μm for each treatment is given.
(H) Total soluble sugar [μg/mg of fresh weight (f.w.)] of seedlings grown in the light and dark
with or without sucrose. Standard deviations are indicated (i=4 treatments, j=3 technical
replicates and n=50 plants per treatment). Scale bars = 2µm.
greater proportion of mitochondria ≤0.85μm in length than plants grown without exogenous
sugar (73% and 42%, respectively; Fig. 3.1A-D, G). Mitochondria were indeed small in response
to exogenous sugar uptake. As the production of carbohydrates increases during the process of
photosynthesis (Azcón-Bieto et al. 1983; Azcón-Bieto and Osmond 1983), the length of
mitochondria of plants grown in the light and dark without exogenous sucrose was compared
next.
29
3.1.2 Light induces mitochondrial fission
When plants were grown in the dark, a greater proportion of mitochondria were elongated
and tubular (Fig. 3.1D and G), but mitochondria were primarily small and fragmented when
plants were grown in the light (Fig. 3.1C and G). This trend held true when plants were grown
with sucrose, however it was more apparent in plants grown without. To determine if light could
induce the fragmentation of elongated mitochondria, dark grown plants grown without
exogenous sugar were exposed to light for 1 and 12h. Plants grown in the dark without sucrose
were used here because this treatment yielded the highest proportion of elongated and tubular
mitochondria. During the light treatment, mitochondria started to undergo fission (Fig. 3.1E).
The elongated forms first started to exhibit a beads-on-a-string appearance. As fission occurred,
the mitochondria were aligned at first but later dispersed throughout the cell. This supported that
light induces the fission of mitochondria.
3.1.3 Mitochondria elongate and become giant under O2 limited conditions
While studying mitochondrial pleomorphy by live-imaging, it became apparent that the
longer a plant remained mounted in water between a slide and coverslip for observations, the
more elongated and tubular the mitochondria became (Movie 3.1). After 30min, round
mitochondria began to elongate and by 45min-1h they formed expanded ring-shaped structures
(Fig. 3.2). After 2-3h, extensive giant mitochondria were formed. Using mitoEos transgenics, it
was also observed that the giant mitochondria could fuse with one another, permitting a mixing
of green and green-to-red photo-converted fluorescent protein in the matrix of two of
mitochondria (Fig. 3.3 and Movie 3.2). These mitochondria were similar in appearance to giant
mitochondria formed under low oxygen pressure (Van Gestel and Verbelen 2002). The prospect
that O2 depletion during observations was inducing fusion was therefore investigated next.
Janus green B is used as a supravital stain to differentiate mitochondria from other
organelles (Brenner 1953). The stain is reduced to a colourless leuco-form under anaerobic
conditions. This discolouration takes place over time when observing mitochondria under a
coverslip using Janus green B (Frey-Wyssling and Mühlethaler 1965). To further validate that
giant mitochondria were forming due to oxygen depletion, a mineral oil overlay of plants was
employed.
30
A
B
E
t = 3.93s
F
t = 39.3s
G
t = 78.6s
C
D
H
Figure 3.2 Morphological transition of small to giant mitochondria in response to oxygen
depletion during lengthy imaging. (A) At the beginning of imaging, mitochondria are round
and/or ovate in shape. (B-D) After about 45min-1h, they begin to elongate or form ring shaped
structures. At this point, giant mitochondria also start forming as the mitochondria keep fusing
(D). (E-G) During the early stages of O2 depletion, the giant mitochondria are relatively
stationary, but exhibit some degree of rearrangement, taking on various irregular shapes. (H)
Eventually, after ~2-3h of submergence in water, they form extensive giant and very elongated
mitochondria. Scale = 2μm.
31
G
A
*
R
B
G
R
Figure 3.3 Fusion of giant mitochondria. Transgenics
of mitoEos, where mitochondria were fluorescently
labelled with a green-to-red photo-convertible EosFP,
were submerged in water until giant mitochondria
formed (~2h). (A) One giant mitochondrion here was
UV irradiated, photo-converting it red (*). The others
were not photo-converted, and remained green (yellow
here only because of the settings used). There were two
distinct colours. (B) During the course of the time-lapse
series (Movie 3.2), two giant mitochondria fused and the
contents of the matrix mixed, giving one distinct colour.
The colour threshold maps were created with the ImageJ
Color Inspector 3D plugin.
An overlay of mineral oil over medium is commonly used to culture anaerobic bacteria.
A layer of oil 1cm thick is usually sufficient to limit the diffusion of O2 into and out of the
underlying media. This results in the death of aerobes as O2 becomes depleted (Rahn and
Richardson 1940; Jacobson et al. 2004). Edwards et al. (1947), showed that after 1h of
immersion in mineral oil the average oxygen consumption rate of a fungal culture decreased. As
mineral oil minimizes the diffusion of oxygen and decreases respiration, plants were mounted in
mineral oil to investigate if the elongation of mitochondria in plants mounted in water was due to
the depletion of oxygen over time. Mitochondria were elongated after 1h under water. After 2h,
elongated and giant mitochondria were seen for both treatments (Fig. 3.4). Although it took
longer for small mitochondria to elongate under mineral oil than water, the elongated to giant
mitochondria transition was most rapid under the mineral oil. Thus, O2 limiting conditions
induce fusion, elongation and the subsequent formation of giant mitochondria.
32
Water
Mineral Oil
A
E
B
F
C
G
D
H
0min
30min
1h
2h
Figure 3.4 Giant mitochondria formed by plant submergence in water or mineral oil. Whole
7 day old transgenics expressing mito-GFP were submerged in water (A-D) or mineral oil (E-H)
between a slide and a coverslip for 0min, 30min, 1h or 2h. Mitochondria began elongating
between 30min-1h for both treatments, but elongation was more rapid under water. Between 12h, giant mitochondria formed for both treatments. The transition from ovate to giant
mitochondria occurred quicker in mineral oil than water. Scale bars = 5μm.
3.2
THE ENDOPLASMIC RETICULUM MEDIATES MITOCHONDRIAL
PLEOMORPHY
The ER has been shown to aid in mitochondrial fission in yeast and animal cells
(Friedman et al. 2011). This has not been investigated in plant cells. The notion that the ER could
be a mediator of the various morphological transitions reported here in response to light, sugar
33
and hypoxia was therefore investigated next.
3.2.1 The endoplasmic reticulum is compact in light grown plants
The ER is a meshwork of tubular membrane that forms polygons (Fig. 3.5A and B). It
can also flatten out into a sheet of membrane, forming what is termed cisternae (Fig. 3.5A (*);
Friedman and Voeltz 2011). As mitochondria were predominantly elongated or tubular in dark
grown plants but small and round under light grown conditions, the effect of light on ER polygon
size or morphology in general was investigated first. The average area and perimeter of ER
polygons were significantly greater in dark grown plants (area = 55.07 ± 39.44; perimeter =
30.06 ± 11.06) than light grown plants (area = 3.27 ± 2.05; perimeter = 7.07 ± 2.26; p-value <
0.01, n = 240; Fig. 3.5). It was also noted in dark grown plants that although the ER polygons
were large on average, there were smaller ones within the meshwork (Fig. 3.5B). There was a
greater occurrence of aggregated ER tubules or cisternae in light grown plants than those grown
in the dark (Fig. 3.5A).
*
B
D
70
60
*
50
40
30
20
10
0
Planar Polygon Perimeter (µm)
C
Planar Polygon Area (µm²)
A
35
*
30
25
20
15
10
5
0
Light
Dark
Light
Dark
Figure 3.5 Average planar ER polygon size comparison between light and dark growth
conditions. 8 day old plants of mito-GFP RER Arabidopsis transgenics were grown in the light
(164μmolm-2 s-1, 16h/8h light/dark) (A) and complete darkness (B) were used to determine the
average ER polygon area (C) and perimeter (D) to compare ER arrangement between the two
treatments. Area and perimeter were significantly different between light and dark grown plants
(p-value < 0.01, n = 240). ER cisternae (*) and smaller depressions or polygons within the larger
polygon meshwork of dark grown plants (arrows) are indicated. Standard error bars are shown.
Scale bars = 10μm.
34
3.2.2 The endoplasmic reticulum acts as a mould for mitochondrial shape
Live imaging of mito-GFP RER double transgenics revealed that the various shapes of
mitochondria discussed above were reflected in the arrangement of the surrounding ER. When
mitochondria were round and spherical, as seen in light grown plants, they were found within the
small depressions in the ER polygon meshwork (Fig. 3.6A). When mitochondria were elongated
and tubular, they were outlined by ER tubules (Fig. 3.6C and D). As these tubular forms became
twisted or contorted, they remained aligned with the ER (Movie 3.3 and 3.4). Furthermore, as the
ER flattened out into sheets of cisternae, the mitochondria were also flattened out (Fig. 3.6D;
Movie 3.3). This was especially evident under hypoxic conditions.
Giant mitochondria displayed the most pleomorphy, taking on a wide range of
appearances. Visualization of these shapes made it apparent that as the ER rearranged,
mitochondria followed suit. The mitochondria appeared to flow into gaps in the ER (Movie 3.3).
This phenomenon resembled a fluid (mitochondria) being poured into a mould (the ER
meshwork).
A
Di
mito-GFP
RER
ii
B
Overlay
iii
iv
C
v
Figure 3.6 Mitochondria are embedded in the ER. (A) When mitochondria are small
and spherical, they are embedded within small ER polygons. (C) When mitochondria are
elongated and tubular they are closely aligned by the ER. ER tubules also constrict the
mitochondria at potential sites of fission (arrow). (D) As the ER flattens out into sheets of
cisternae, mitochondria expand and also flatten. As the ER rearranges, the mitochondria
follow suit, acting in a fluid-like motion and flowing into gaps created by the ER.
RFP=ER; GFP=mitochondria. Scale bars = 2μm.
35
3.2.3 The endoplasmic reticulum aids in mitochondrial fragmentation
The ER has been shown in yeast and animal cells to aid in the fission of mitochondria by
wrapping around the mitochondrial fission sites (Friedman et al. 2011). This has not been shown
in plant cells. This aspect of the ER-mitochondrial relationship was investigated next.
Mitochondrial elongation was induced by either growing plants in the dark or by inducing
hypoxia by submerging the plants in water for ≥1h. Plants were then exposed to HL to induce
mitochondrial fission. The elongated mitochondria were closely surrounded by ER tubules which
often wrapped around the mitochondria, constricting them at sites of fission (Fig. 3.7; Movie
3.4). As the ER was rearranged, the mitochondria also appeared to be tugged accordingly,
suggesting that the ER also applies a pulling force that allows mitochondria to be pulled apart
during fission. This separation also aids in mitochondrial dispersal as the fragmented portions are
pulled in different directions.
3.2.4 Mitochondrial beading and the endoplasmic reticulum
Mutants where mitochondrial fission is impaired were used to further validate the ER
involvement in this process. The drp3a/apm1-13 mutant displays an aberrant mitochondrial
phenotype (Fig. 3.7B, C; Mano et al. 2004). Plants of this mutant were grown in the dark for 7
days and stained with MitoTracker Orange for 10min prior to live imaging. When treated with
HL for 2min, the elongated, tubular mitochondria typical of this mutant displayed a beads-on-astring appearance as they were stretched throughout the cell (Fig. 3.7D). This beading
phenomenon was also seen in plants of elm1-1. As the beading is also seen in wild type plants
when tubular mitochondria are undergoing fission, it was investigated whether the ER
constriction was actually the cause of the beading.
e1m-1 Mt-GFP (mitochondrial targeted GFP; Feng et al. 2004; Arimura et al. 2008) were
transformed with RER to gain insight into the stretching and beading phenomenon. Elongated
mitochondria appeared to thread through the ER polygons. When the ER was rearranged, the
mitochondria often became constricted at points where the ER became constricted. This resulted
in the beading appearance. Furthermore, as tubular mitochondria moved through a ‘tunnel’ or
between ER tubules, they remained thin. Once it reached an area of this ER tunnel that was
36
A
WT
B
apm1-13 MitoTracker Orange
C
E
elm1-1 RER
F
G
D
Before Light
H
I
37
After 2min HL
Figure 3.7 ER-mediated fission and beading of elongated mitochondria. (A) Sequence of mitochondrial fission from Movie 3.4.
The solid arrows indicate sites where the ER constricts the mitochondrion. Dotted arrows indicate the movement of the ER a nd
subsequent movement of the fragmented mitochondrion. The white occurred before the yellow which occurred before the blue. The
sequences show that the ER constricts the mitochondrion and as the ER is rearranged, the mitochondrion fragments at this site. The
fragmented portion of the mitochondrion disperses in the direction of the ER rearrangement. (B-D) Plants of drp3a/apm1-13 stably
expressing GFP-labelled peroxisomes were stained with MitoTracker Orange (Molecular Probes). Both organelles mostly displayed
an aberrant phenotype whereby they were very elongated. As the organelles stretched, they beaded (C, arrows). (D) When given a
2min HL treatment, the mitochondria rapidly stretched and exhibited beading. (E-I) Mutants of elm1-1 stably expressing GFPlabelled mitochondria were transformed with RER. (E) Mitochondria were typically elongated, but were capable of beading as well
in this mutant (F-I). Where the tubular mitochondria threaded through the ER, beading was often seen (solid arrows). This was
especially the case when the mitochondrion encountered aggregated ER (dotted arrows). H is a magnification of the boxed-in region
of G. apm1-13 and elm1-1 seeds were kindly provided by Shoji Mano and Shin-ichi Arimura, respectively.
55
37
dilated, the mitochondria followed suit and also dilated (Fig. 3.7F-I). A similar beading
phenomenon was also witnessed with elongated peroxisomes (Fig. 3.7B; Sinclair et al. 2009;
Delille et al. 2010) and stromules (Pkye and Howells 2002; Gunning 2005; Hanson and
Sattarzadeh 2008; Schattat et al. 2011).
3.3
THE ER CAGES MITOCHONDRIA, PLASTIDS AND PEROXISOMES
The ER clearly plays a major role in the pleomorphic nature of mitochondria, peroxisomes
and plastids. To gain a better understanding of how all four organelles interact in a live system,
triple Arabidopsis transgenics, mito-GFP Yperoxi RER, were created. This was done by crossing
mito-GFP (Logan and Leaver 2000) plants with Yperoxi (Mathur et al. 2002) and transforming
the stable transgenic plants with RER (Sinclair et al. 2009). The chlorophyll auto-fluorescence
could be used to visualize chloroplasts as well, therefore simultaneous visualization of all four
organelles was possible.
It was previously shown that the ER forms a cage that can enclose plastids (Schattat et al.
2011). As the cage moved, the plastids moved with it. Live-imaging of the triple transgenics here
showed that mitochondria and peroxisomes also become stuck in this cage (Fig. 3.8; Movie 3.5).
Mitochondria and peroxisomes were also seen moving along ER bundles in the cytoplasmic
stream, but where this may be co-incidental, the ER cage which has minimal movement in
comparison to cytoplasmic stream may serve a higher purpose. Containment of mitochondria,
peroxisomes and chloroplasts within the ER network may consequently permit sustained
interactions to increase the efficiency of metabolite or ROS transfers between all four organelles.
Figure 3.8 The ER cages mitochondria, peroxisomes and chloroplasts. A cluster
of organelles maintaining a sustained interaction is shown. GFP=mitochondria;
YFP=peroxisome; RFP=ER; auto-fluorescence false coloured blue=chloroplasts.
38
Since the ER plays a role in regulating peroxisome (Sinclair et al. 2009; Barton et al. 2014)
and mitochondrial pleomorphy, and both organelles were found to exist in close association with
one another in ER cages, the mitochondria-peroxisome interactions were investigated in more
depth next. The interactive behaviours in response to light, sugar and ROS stress were of
particular interest because these are all factors that link the two organelles and suggest a cause
for their close interactions.
3.4
INVESTIGATION OF MITOCHONDRIA AND PEROXISOME INTERACTIONS
3.4.1 Mitochondria and peroxisome morphology in response to H2O2
mito-GFP Yperoxi plants were grown in the dark for 7 days and then placed in water, 0.3%
or 3% H2O2 for 30s. The plants were quickly rinsed with and mounted in water. Plants grown
continuously in the dark without sucrose were used because mitochondria in these plants were
most elongated. Since ROS has been shown to fragment mitochondria, responses may be most
apparent in these plants. Mitochondria were significantly longer when plants were treated with
0.3% and 3% H2O2 (Fig. 3.9; p-value < 0.01, n = 204). There was no significant difference in
length between the two 0.3% and 3% treatments, however. Peroxisomes were elongated in all
three treatments, but H2O2 did not result in a significant increase in the average length (p-value >
0.05, n = 201). Peroxules did form occasionally, and some beading of elongated mitochondria
and peroxisomes were seen (Fig. 3.9). No clear interaction between mitochondria and
peroxisomes were seen.
3.4.2 Peroxisome morphology in response to light, sugar and low O2 conditions
mito-GFP Yperoxi double transgenics were grown either in the light (16h/8h light/dark
cycle) or continuously in the dark on MS medium with or without sucrose. Mitochondria were
predominantly longer (>1.3μm in length) when plants were grown in the dark without sucrose
but were predominantly shorter (≤1.3μm in length) when grown in the light with sucrose (pvalue < 0.05; Fig. 3.10A and C-F). This was consistent with the above results. The trends
reflected in peroxisomal morphology were nearly opposite. There was a significantly greater
proportion of large (>1.3μm in length) peroxisomes than small (≤1.3μm in length) ones when
39
B
G
H
D
E
F
Average Length of
Peroxisomes (μm)
3% H2O2
0.3% H2O2
C
Average Length of
Mitochondria (μm)
0% H2O2
A
4.0
3.0
2.0
*
1.0
0.0
0%
0.3%
3%
0%
0.3%
3%
2.5
2.0
1.5
1.0
0.5
0.0
% H2O2
Figure 3.9 Effects of H2O2 on mitochondria and peroxisome morphology. mito-GFP
Yperoxi plants grown in the dark without sucrose for 7 days were placed in a solution of
0.3% or 3% H2O2, or in water (0%) for 30s. Mitochondria were significantly longer when
treated with H2O2. Although peroxisomes were elongated, there was no significant
increase in length. Mitochondria and peroxisomes did exhibit some beading during
fission (arrows). GFP=mitochondria; YFP=peroxisomes. Scale bars = 2μm.
plants were grown in the light with and without sugar (p-value < 0.05; Fig. 3.10B, C-F). There
was also a significant decrease in the proportion of large or elongated peroxisomes when plants
were grown in the dark without sucrose (p-value < 0.05; Fig. 3.10B-F).
Peroxules and actively dividing peroxisomes were rarely seen even in the light grown
plants. Since the plants were grown under the respective treatments for 7 days prior to analysis,
they may have adapted and achieved homeostasis. It has been suggested that peroxisomes are
constantly sensing or monitoring cellular ROS levels given their role in ROS scavenging
(Sinclair et al. 2009; Mathur et al. 2012). As such, the effects of light on peroxisomal
pleomorphy may be more rapid and apparent under shorter treatments where the cell is quickly
trying to attain homeostasis.
Arabidopsis transgenics expressing HyPer-GFP were given a short HL treatment to look
at the rapidity of H2O2 production in response to light. Prior to HL treatment, the intensity of the
40
C
a
*
*
60%
>1.3
40%
≤1.3
20%
0%
- Suc
+ Suc
- Suc
+ Suc
Dark, No Dark, Suc Light, No Light, Suc
Dark
Light
Suc
Suc
a
NS
80%
*
a
*
60%
E
F
G
Hypoxia
H
40%
20%
0%
Dark Grown
D
3% Sucrose
*
80%
B
Proportion of short and
long peroxisomes
Light Grown
a
100%
No Sucrose
Proportion of short and
long mitochondria
A
- Suc
+ Suc
- Suc
+ Suc
Dark, No Dark, Suc Light, No Light, Suc
Suc Dark
Suc Light
≤1.3μm
>1.3μm
Figure 3.10 Morphological comparisons of mitochondria and peroxisomes in response to
light, sugar and hypoxia. The average lengths of mitochondria and peroxisomes were
measured using 7 day old mito-GFP Yperoxi plants grown in the light or dark, with or without
sucrose in the medium. An organelle was considered large if the length was >1.3μm and small
if it was ≤1.3μm. (A) Mitochondria were more elongated or larger when plants were grown in
the dark (D, F), and shorter when grown in the light (C, E) (p-value < 0.05). (B) Peroxisomes
were predominantly larger when plants were grown in the light, with and without sucrose than
dark grown plants without sucrose (p-value < 0.05). Peroxules and elongated peroxisomes were
rarely seen in all treatments. Standard deviation bars are shown. * = significant difference in
the proportion of long and short organelles within each treatment; a = significant difference in
the proportions between treatments; NS = no significant difference; α=0.05. (G-H) During
hypoxia, one or more spherical peroxisomes were seen often clustered to ring-shaped or giant
mitochondria. Ends of peroxules were never witnessed interacting with mitochondria exhibiting
hypoxic morphologies. Scale bars = 2μm.
basal GFP fluorescence was low (Fig. 3.11A and C). After only 5min of HL, the intensity
increased (Fig. 3.11B and D). This showed that the H2O2 levels can indeed increase very rapidly
in response to HL. HL irradiation of dark grown plants for short intervals – 30s, 1, 2 or 4min –
were therefore used to investigate rapid morphological responses of mitochondria and
41
B
C
200
Intensity
A
100
0.24
0.26
0.28
0.26
0.28
0.22
0.24
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
Distance Measured (Inches)
D
0.22
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0
0.02
100
0.00
Intensity
200
Distance Measured (Inches)
Figure 3.11 H2O2 production in response to short HL irradiation. Plants expressing a
cytosolic hydrogen peroxide responsive probe, HyPer-GFP, were given a high light (HL)
treatment for 1-5min. The intensity of the GFP fluorescence increased after the light treatment
(B), suggesting an increase in H2O2 from the basal level (A). A Red-Green plot (ImageJ) shows
this graphically. The fluorescence intensity of GFP increased after treatment, while the
chloroplast auto-fluorescence (red) remained constant. The distance measured corresponds with
the boxed area in (A) and (B), left to right.
peroxisomes in response to HL-induced ROS stress. But again, no clear morphological responses
of peroxisomes to the shorter HL treatments were observed. What was witnessed however was
an interactive behaviour of peroxisomes around mitochondria.
This interactivity was also seen during hypoxia. When hypoxia was induced by
submerging mito-GFP Yperoxi plants in water for 45min onwards, ring-shaped and giant
mitochondria were formed reproducibly as before. These mitochondria often had one or more
spherical peroxisomes clustered around them (Fig. 3.10G and H). The interactivity of these two
organelles was investigated in more depth.
3.4.3 Mitochondria and peroxisomes have sustained interactions
Mitochondria and peroxisomes are linked metabolically and biochemically, but their
physical interactions have not been fully investigated in plants. This was looked at next.
Simultaneous visualization of mitochondria and peroxisomes using mito-GFP Yperoxi double
transgenics showed that although the two organelles would often move along similar paths, for
example along the cytoplasmic stream (Movie 3.6) and sometimes crossed paths, there was no
42
impression of an interaction. However, often mitochondria would cluster around peroxisome(s)
for long periods of time (Fig. 3.12; Movie 3.6, 3.7). The number of these interactions between
the two organelles peaked after 1min of HL irradiation, but no clear trend was observed (Fig.
3.12E). However, the duration of the interactions did increase between 0-2min of HL, but
decreased after 4min. Furthermore, the motility of these clusters was minimal in comparison to
the rapid movement of the surrounding organelles. The clusters often consisted of small,
spherical mitochondria around spherical peroxisomes with and without peroxules (Fig. 3.12A
and B). Since the fluctuation of light is a major contributor to ROS production, a mutant where
the effects of light were implicated was of interest.
3.5
THE anisotropy1 MUTANT RESPONSE TO LIGHT
Plant epidermal cells act as lenses (Haberlandt 1914; Vogelmann 1993; Vogelmann et al.
1996). When collimated light strikes these cells, it is refracted and passes through the epidermis
to the underlying mesophyll layer where it is absorbed by plastids. The way the light is refracted
and how it is focused in the cells below the epidermal cell wall is determined by such
characteristics as the cell surface area, cell wall composition, waxes, and the degree and
uniformity of curvature (Vogelmann et al. 1996). If the radius of curvature (r) is small (i.e. the
cell is very round), the light is focused with minimal scattering immediately below the epidermis
at the top of the mesophyll layer. Conversely, if r is large (i.e. the cell is flattened), the light is
focussed farther below the epidermis. But the deeper the focal point of light, the greater the
scattering and hindrance of absorption there is (Vogelmann et al. 1996). It would therefore be
expected that the chloroplasts of the mesophyll layer would absorb the light better if the cells in
the outermost epidermal layer are round – the light would be better focused. This would also
mean that the more light absorbed by the chloroplasts, the higher the rate of photosynthesis and
therefore the higher the levels of ROS. The expectation would be that the interactivity between
the mitochondria and peroxisomes in response to light-induced ROS would be heightened in a
mutant exhibiting such a phenomenon.
43
B
C
Number of
E
20
18
16
14
12
10
8
6
4
2
0
interaction
A
0s
30s
1min
2min
4min
F
D
Duration of Interaction (s)
Length of high light exposure
65
60
55
50
45
40
35
0s
30s
1min
2min
4min
Length of high light exposure
Figure 3.12 Sustained interactions between mitochondria and peroxisomes. (A) Successive
frames showing a cluster of mitochondria, peroxisomes and a chloroplast. This cluster remains
intact and relatively stationary as mitochondria and peroxisomes outside of the cluster move in
and out of frame. (B) Successive frames showing a peroxule retract with a cluster of small
mitochondria around it. The peroxisome body remains in contact with chloroplasts as well. A
second peroxisome maintains contact with a mitochondrion, this time on the peroxisome body
rather than the peroxule. (D) An interactive 3D plot (ImageJ) of (C) illustrating a peroxisome
and peroxule closely wrapped around a chloroplast. Small mitochondria are also closely
associated with the peroxisome body and peroxule. (E) 7 day old mito-GFP Yperoxi plants
grown in the dark with no sucrose were irradiated with HL for 30s, 1, 2 and 4min. A 2min
time-lapse was taken immediately after the light treatment. The average number (E) and
duration (F) of interactions captured within those time-lapses are shown. Scale bars = 5μm.
3.5.1 Production of light-induced H2O2 is more rapid in any1 than wild type
One of the mutants available was anisotropy1 (any1). any1 has a mutation in a cellulose
synthase (CesA) gene that causes a reduction in cell wall crystallinity (Fujita et al. 2013). As the
44
Figure 3.13 Comparisons of wild type and any1 cell shape and arrangement. Trichomes
(tr) of wild type are branched (A), but in any1 they are bulbous and swollen (B, b). Epidermal
cells in any1 are also anisotropic and swollen (D, F) compared to wild type (C, E). Toluidine
blue staining of (G) wild type and (H) any1 shows that there are more intercellular spaces or
pockets (*) in this mutant. Scale bars = A, B = 250 µm; C-F = 100µm; G, H = 50µm.
cell wall crystallinity was affected, the refraction of light would be expected to be altered as well.
Comparisons of wild type and any1 revealed that the epidermal cells in any1 are nacreous and
often swollen or bulged (Fig. 3.13). The reduction in growth anisotropy of the cells also created
larger intercellular spaces. As the arrangement and the curvature of the cells were altered, this
mutant was a good contender for investigating the effects of light-induced ROS production.
Columbia and any1 seedlings were grown in the dark for 6 days and then transferred to
low light (30μmolm-2s-1) for 2 days prior to subjecting them to 0, 100, 200 or 300μmolm-2s-1 of
light for 24h. At this stage the seedlings did not have their first true leaves fully emerged yet. The
plants were submerged in a solution of DAB with a metal enhancer (Sigma-Aldrich) under
vacuum (-50KPa) for 4h. The tissue was cleared with ethanol and mounted in 50% glycerol.
Images were acquired at the same light intensity and microscope settings for direct comparisons.
The staining was representative of the amount of H2O2 produced during the relative light
intensity treatments. DAB staining illustrated that any1 produces more H2O2 in response to light
than wild type (Fig. 3.14B). When seedlings were transferred to the dark for 24h and then
exposed to light (164μmolm-2 s-1) for 30min, 1h or 2h, H2O2 levels were also greater in any1
seedlings in each case (Fig. 3.14A). This also showed that response to light occurs well before
24h, as staining intensified over the span of 2h.
45
30min
1h
2h
B
Average Inverse Grey Value
Dark
any1
WT
A
any1 Cotyledon
any1 Hypocotyl
WT Cotyledon
WT Hypocotyl
12
10
46
WT
8
any1
any1
6
WT
4
D
2
C
Average Mitochondria Length (μm)
Light intensity (μmol m-2 s-1)
1
Dark
2
3
Light
4
Figure 3.14 Comparisons of wild type (WT) and any1 responses to light. (A) Seedlings of any1 and WT were transferred to the
dark for 24h prior to their exposure to light (164μmolm-2 s-1) for 0, 0.5, 1 or 2h prior to DAB staining. (B) Dark grown seedlings were
also transferred to low light (30μmolm-2s-1) for 2 days and then transferred to 0, 100, 200 or 300μmolm-2 s-1 of light for 24h prior to
DAB staining. The average inverse gray values of DAB staining for H2O2 were greater for any1 in the cotyledon and hypocotyl. (D)
A boxplot (R, http://www.r-project.org/) showing that mitochondria were smaller on average in any1. (C) The interactions between
mitochondria and peroxisomes and peroxules were more apparent and frequent in the mutant. Scale bars = 0.5mm and 2μm, A and C,
respectively.
55
46
3.5.2 Mitochondria-peroxisome interactions in any1
Using mitochondrial size as an indicator for light stress, 7 day old any1 mutants were
grown in the light or dark, with or without sucrose. The average length of mitochondria was
smaller in any1 than wild type for all treatments (Fig. 3.14D). The mitochondria-peroxisome
interaction was more apparent in these plants than wild type as well (Fig. 3.14C; Movie 3.8).
Peroxules were also observed more frequently. These extensions were seen interacting with
small, fragmented mitochondria and within clusters of chloroplasts. These observations were
very apparent in cells that exhibited the most isotropy and were bulbous or nacreous. Overall, the
morphological transitions and interactive behaviour of mitochondria and peroxisomes in
response to light stress was most apparent in any1 plants than wild type.
3.6
INVESTIGATION OF MITOCHONDRIA FISSION MACHINERY
Investigations of mitochondria and peroxisome morphology brought to light overlaps in
the appearances of each organelle, in particular during their fission (Fig. 1.5). When undergoing
fission, both organelles elongate. They then become constricted sometimes exhibiting a beadson-a-string appearance. Fission then takes place at the site(s) of constriction and the new entities
disperse throughout the cell. As mitochondria and peroxisomes share this morphological
procession during division, the mechanism behind fission was looked at closer. The possibility
that FIS1A, a tail-anchored membrane protein, in particular could be exchanged between
mitochondria and peroxisomes through mitochondrial derived vesicles (MDVs) was explored in
particular.
3.6.1 DAL as possible mechanism of hierarchical fission between mitochondria and
peroxisomes
Neuspiel et al. (2008) described a novel interaction between mitochondria and
peroxisomes in mammalian cells by demonstrating that mitochondria derived vesicles (MDVs)
carrying an outer membrane Mitochondria-Anchored Protein Ligase (MAPL) were transported
from mitochondria to peroxisomes. They demonstrated that MAPL becomes enriched along
tubular mitochondria and that MAPL over-expression resulted in fragmentation of mitochondria,
47
a phenomenon that was deemed dependent on the MAPL-RING domain and Drp1.
Since 1) FIS1 is anchored to the peroxisomal membrane and the OMM, 2) MAPL is
localized to the OMM and 3) MDVs bud off of the OMM, it was questioned if the MDVs
translocate MAPL and FIS1 to peroxisomes, which could be the result of close interactions seen
with small mitochondria and peroxisomes and/or peroxules. This could result in the subsequent
fragmentation of peroxisomes as well.
FIS1 is C-terminally anchored to the peroxisome membrane and to the OMM, thus
mEosFP was N-terminally fused to FIS1A and to FIS1B. The expressions of both fusions were
driven by the p35S. A construct in which Eos-FIS1A was driven by the native promoter (pfis1aEos-FIS1A) was also created. When N. benthamiana was co-infiltrated with Eos-FIS1B, mitoGFP and Yperoxi, FIS1B was localized to mitochondria, peroxisomes, plastids and stromules
(Fig. 3.15; Ruberti 2014). This was also the case for Eos-FIS1A under the p35S and pfis1a. As
this could have been an artifact of transient expression, stable lines of YFP-FIS1A (Ruberti
2014) crossed with mito-GFP and with FNR-GFP were created. Again, FIS1A was localized to
the entire OMM, plastid bodies and stromules (Fig. 3.15). Dal1-Eos and Dal2-Eos lines were
also created, where DAL1 and 2 are the plant homologues of MAPL. A major issue in testing the
hypothesis that FIS1A or FIS1B and DAL1 and/or DAL2 were translocated from fragmented
mitochondria to peroxisomes during their interaction for subsequent peroxisome fission was the
experimental approach. mEosFP was used in particular with the hopes of photo-converting a
small region or subset of FIS1/DAL1/DAL2 on mitochondria from green to red. The red would
then be tracked to see if they are indeed translocated to peroxisomes during fission. Given the
motility and dispersal of these organelles during fission, it was not possible to do that. The lines
are available however, if approaches are available in the future.
48
Chlorophyll
m
mito-GFP x YFP-FIS1A
YFP-FIS1A
mito-GFP
Overlay
c
p
G
p35S-Eos-FIS1B mito-GFP Yperoxi
H
I
p35S-Eos-FIS1A mito-GFP Yperoxi
J
K
pfis1a-Eos-FIS1A mito-GFP Yperoxi
L
m
A
B
YFP-FIS1A
mito-GFP
Overlay
D
E
C
49
str
Chlorophyll
YFP-FIS1A x FNRGFP
YFP-FIS1A
FNRGFP
Overlay
F
Figure 3.15 Localization of FIS1A and FIS1B to mitochondria, peroxisomes and plastids. (A-E) mito-GFP plants crossed with
YFP-FIS1A. FIS1A was localized to mitochondria, as shown by the green-red overlay making yellow. It was also localized to
peroxisomes and peroxules, which are indicated (p and px, respectively) by just the yellow in B and C. It formed rings around
chloroplasts and also localized to stromules (str). (F) YFP-FIS1A was crossed to FNRGFP (GFP labelling plastid stroma) and
further validated that FIS1A localized to the plastids and stromules. (H-L) The Eos-FIS1A and Eos-FIS1B constructs co-infiltrated
with mito-GFP and Yperoxi showed that FIS1A and FIS1B (seen in red in all images) were localized to plastids as well. Note that
it was difficult to photo-convert a single or very small portion of the mitochondria and peroxisome population.
55
49
CHAPTER 4
DISCUSSION
4.1
MITOCHONDRIA MORPHOLOGY IN RESPONSE TO LIGHT, SUGAR AND O2
LIMITATED CONDITIONS
4.1.1 Morphological differences between mitochondria in light and dark grown plants
Fluctuating cytosolic sugar levels is a phenomenon plant cells must constantly overcome.
This is because of fluctuations in light and photosynthetic activity (Azcón-Bieto et al. 1983;
Azcón-Bieto and Osmond 1983). Because of their photosynthetic capabilities, plants are able to
synthesize carbohydrates when they are exposed to light. Any excess sugar produced is
converted to and stored as starch which is broken down when cytosolic sugar levels are too low,
such as at night (Fig. 4.1). The carbohydrates produced are metabolized by glycolysis and the
TCA cycle during cellular respiration to produce ATP, NADH and FADH2. The latter two are
reduced by complexes I and II of the ETC in mitochondria to supply electrons. This is necessary
to create an electrochemical gradient that is needed to drive ATP synthesis (Fig. 1.1).
One of the major causes of damaged mitochondrial constituents is an overwhelmed ETC,
where there is an excessive amount of electrons that are not reduced. These electrons then react
with O2, producing ROS. Such a situation is seen in animal cells when hyperglycemic conditions
50
provide an overwhelming metabolic input that causes the ETC to overwork. There is a
consequential rise in ROS that leads to cell death (Yu et al. 2006; 2008). Overcoming
detrimental levels of ROS is a challenge photosynthetic plants are constantly faced with because
excessive amounts of light can be quite damaging by producing too much ROS (Bhattacharjee
2011). Mitochondria of animal cells fragment under hyperglycemic conditions (Yu et al. 2006;
2008; Jhun et al. 2013), but mitochondrial pleomorphy in response to fluctuations in cytosolic
sugar in plant cells had not been investigated. As mitochondria are essential for cellular
respiration in eukaryotes, and mitochondrial fission and fusion play an important role in
regulating this activity, an investigation on the pleomorphy of mitochondria in response to light
and sugar was carried out.
When plants are grown without exogenous sugar, mitochondria are predominantly
elongated. When plants are grown with sucrose however, mitochondria are primarily small and
round to ovate in shape. Thus, sugar is an inducer of mitochondrial fragmentation in plant cells
as well. Most striking however, is the effect light has on the appearance of mitochondria. When
plants are grown in the light, the mitochondrial population consists of fragmented, small and
spherical entities but more tubular and elongated mitochondria are seen in cells of dark grown
plants. This trend holds true whether plants are grown with or without exogenous sugar. When
dark grown plants are exposed to light, the mitochondria are quick to undergo fission, rapidly
fragmenting from a tubular, elongated population to a small and spherical one. Light therefore
has a greater influence on what the more dominant shape and size of the mitochondria is than
exogenous sugar levels.
Dark grown plants are unable to photosynthesize and they quickly deplete the lipids, starch
and carbohydrates stored in their cotyledons (Fig. 4.2). As the cell is not respiring, NADH and
FADH2 are low; electrons are not provided to the ETC to create the electrochemical gradient
required to produce ATP. Mitochondria undergo fusion when the ΔΨm is depolarized which
explains why mitochondria are predominantly longer in plants grown in the dark without
sucrose; fusing of the membranes allows the repolarization of the ΔΨm so that oxidative
respiration can still occur to synthesize ATP.
51
↑ADP
Fatty Acids
↓ATP
Dispersal
of Energy
Light
β-oxidation
Depolarization
of ΔΨm
Starch
Synthesis
Fission
Sugar
CO2
Glycolysis
Pyruvate
Exogenous
Sugar
Orthodox
State
CO2
ATP
52
NADH
ATP
TCA
cycle
Hyperpolarization
of ΔΨm
ETC
Oxidative
Phosphorylation
GTP
NADH
FADH2
Figure 4.1 Influence of light, sugar and energy status on mitochondrial shape. When there is light, plants are able to
photosynthesize and produce carbohydrates. Excess sugar is converted to starch which is broken down when cytosolic sugar levels
are too low, such as at night. Under controlled conditions discussed in this thesis, plants are also able to uptake exogenous sugar
supplied in the growth medium (see Section 3.1.1). Glycolysis breaks down glucose, yielding ATP, NADH and pyruvate. The TCA
cycle further metabolizes pyruvate, producing GTP, NADH and FADH 2. NADH is reduced by NADH dehydrogenase (complex I)
and FADH2 by succinate dehydrogenase (complex II) of the ETC during oxidative phosphorylation. This occurs on the IMM. The
electrons released are transported down the ETC pathway as H + are pumped out of the matrix into the IMS creating an
electrochemical gradient. The electrons are accepted by O 2 (Fig. 1.1). The gradient is used by ATP synthase to produce ATP. The
mitochondria would then be in the orthodox state, which occurs when ATP levels are high. Mitochondria then undergo fission,
increasing their numbers. The cell is in a high energy state now, and the mitochondria are constantly dispersing to other areas of the
cell. They will interact with one another, transferring the energy to each other and parts of the cell where energy is required. ATP
levels are decreasing at this point, and ADP levels are increasing. As the membrane is therefore depolarizing, mitochondria must keep
keep respiring to replenish ATP.
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52
Hypoxia
β-oxidation
GTP
Starch
Depleted
No
Light
Oxidative
Phosphorylation
TCA
cycle
NADH
FADH2
Fatty Acids
CO2
Starch
Degradation
Sugar
ATP
Depolarization
of ΔΨm
Condensed
State
Fusion
↑ΔΨm
No
Exogenous
Sugar
ATP
53
Starvation
No Energy
Dispersal
Immobilization
of mitochondria
ATP
Depleted
Figure 4.2 The effects of dark, hypoxia and starvation on respiration and mitochondria morphology. Plants were grown in the
dark for seven days post germination. As no light was available, photosynthesis was not occurring and carbohydrates were not being
produced. Seedlings were then entirely dependent on lipids, carbohydrates and other nutrients stored in the cotyledons and also on
the nutrients other than sugar in MS medium. Once the starch, lipid and carbohydrate stores were depleted, ATP levels became low.
With no NADH and FADH2 feeding the ETC due to the lack of glycolysis, β-oxidation and TCA cycling, oxidative phosphorylation
is also not taking place. The IMM becomes depolarized. Mitochondria attempt to fuse, which helps to increase the ΔΨm, however any
ATP produced becomes depleted as well. Mitochondria also have minimal motility and very little energy is being dispersed. The cell
continues to starve. A similar situation occurs when a plant is submerged in nutrient-free water, except O2 is also being depleted and
eventually becomes unavailable for oxidative phosphorylation.
55
53
After the plants are given a HL treatment, photosynthesis can occur, thereby increasing
cytosolic sugar levels (Fig. 4.3). Once carbohydrates become available, respiration may
commence. At this point, the IMM becomes progressively hyperpolarized and ATP levels rises.
The mitochondria then undergo fission to disperse the energy throughout the cell (Amchenkova
et al. 1988; Bereiter-Hahn and Vöth 1994). High energy mitochondria might also fuse with those
of lower ΔΨm to achieve an intermediate state of polarization. This fission and fusion activity
continues until a proper balance is achieved. Since plants grown in the light have higher levels of
total soluble sugar than those grown in the dark, it makes sense that the mitochondria are
fragmented in the light grown plants; there is a greater metabolic supply for respiration in the
presence of light which essentially leads to more hyperpolarized, energy-filled mitochondria that
must constantly disperse the energy throughout the cell. A similar situation occurs when a plant
is submerged in water during microscopic observations for too long.
4.1.2 Mitochondria morphology under O2 deplete conditions
Whole plants are mounted in water on a slide and coverslip during observations. When
they are left submerged for long periods of time, the carbohydrates, starch, lipid stores etc. get
depleted. As cells respire, O2 is also consumed (Frey-Wyssling and Mühlethaler 1965;
Hackenbrock et al. 1971) and eventually becomes unavailable for oxidative phosphorylation
(Fig. 4.2). If O2 is too low, electrons start to accumulate. This accumulation results in an increase
in ROS which triggers mitochondrial fragmentation and mitophagy (Yu et al. 2008). However,
after being submerged in water ≥1h, nutrients (carbohydrate, starch and lipid stores etc.) become
depleted as well. Just as in the dark grown plants, very little electrons are therefore fed into the
ETC regardless. This again causes a depolarization of the ΔΨm. Mitochondria are forced to fuse
in an attempt to repolarize the IMM. As both the metabolic input and O 2 availability for
oxidative phosphorylation are low, the fusion has to be more extensive. Giant mitochondria
form.
54
HL
Short HL or
short time after HL
55
Fusion
1.
Before HL: The mitochondrion is elongated. ATP levels are low or basal, but there is no
metabolic input; the cell is not respiring. Intracristal spaces are narrow; the orthodox state is
assumed.
2.
After HL: Photosynthesis yields an increase in cytosolic sugar levels. Respiration commences.
The intracristal spaces have expanded; the condensed state is acquired. The mitochondrion is
still elongated.
3.
The ATP levels are high and the IMM is hyperpolarized in this condensed state. The
mitochondrion undergoes fission and disperses throughout the cell, usually to an area of high
energy demand.
4.
A high energy mitochondrion fuses with one of lower ATP and lower ΔΨm which may be in the
orthodox state. The longer the HL treatment, the greater the energy input and the higher the
rate of fission and motility. The population may become predominantly small and spherical.
5.
After fusion, the mitochondrion is in an intermediate state of ΔΨm and ATP. If the membrane is
still too hyperpolarized, steps 3 and 4 may be repeated until homeostasis is achieved or the
cell has sufficient energy to function.
Long HL or
long time after HL
Fusion
Figure 4.3 Proposed model for orthodox to condensed state transition to explain fusion-fission activity in response to HL. When
plants with a predominantly elongated population receive a HL treatment, the mitochondria may undergo an ultrastructural and then
energetic state transition. (1) An elongated mitochondrion in the orthodox state (i.e. the intracristal spaces are narrow, the matrix is
expanded and ATP levels are low) transitions to (2) the condensed state upon HL irradiation. With the HL stimulus, the plant is
actively respiring and the mitochondria undergo oxidative phosphorylation. (3) The IMM becomes hyperpolarized and the
mitochondrion must undergo fission to disperse the energy it now contains. (4) After they disperse throughout the cell, the
hyperpolarized, energy-filled mitochondria may fuse to a mitochondrion of lower ΔΨm or lower energy. This transferral of
electrochemical potential and energy results in a mitochondrion of intermediate ΔΨm and an ultrastructure somewhere in between the
orthodox and condensed state. Red = orthodox state, low ATP, high ADP; green = condensed state, high ATP, low ADP; yellow =
intermediate orthodox-condensed state and ATP/ADP levels; the darker shaded regions are the intracristal spaces and the light regions
are the matrix to better distinguish between the orthodox (more lighter regions/expanded matrix) and condensed state (more darker
regions/expanded intracristal spaces).
55
55
4.2
MITOCHONDRIA AND ENDOPLASMIC RETICULUM INTERACTIONS
The ER has been shown to be a key player in peroxisome (Sinclair et al. 2009; Barton et al.
2014) and plastid morphology (Pyke and Howells 2002; Gunning 2005; Hanson and Sattarzadeh
2008; Schattat et al. 2011). The extent of the ER-mitochondria interactions in plants cells had not
been elucidated yet. Mitochondria exhibit quite an array of morphological appearances
(Appendix I). Not only can they exist as ‘fila’ or ‘plastosomes’, but when they form giant
mitochondria, they display very irregular shapes, which were shown here to be mediated by the
ER. As the ER rearranges, the mitochondria follow suit: flattened ER result in flattened
mitochondria; small mitochondria are embedded within small ER polygons; elongated, beaded
mitochondria are constricted by ER tubules; and ER rearrangement pulls or tugs constricted
mitochondria, and as they undergo fission, they disperse with the ER.
Membrane contact sites (MCS) have been suggested to exist between mitochondria and the
ER. These MCS are thought to have several purposes, such as tethering the ER and
mitochondria, lipid transfer, Ca2+ transport and apoptosis signalling (Rizzuto et al. 1998;
Achleitner et al. 1999; De Brito and Scorrano 2008; Osman et al. 2011; Prinz 2014; Vance
2014). Interestingly, one of the proposed tethers between these two organelles is Fis1 in the
OMM which is thought to tether to Bap31 in the ER membrane (Prinz 2014). This may explain
why the mitochondria can be tugged by the ER during fission and why DRP3, which binds to
Fis1, is found at sites of ER-constricted mitochondria (Fig. 4.4). If Fis1 tethers the ER around a
tubular mitochondrion, it would allow the ER to constrict the mitochondrion tight enough and
long enough for DRP3 to bind to FIS1 and form helices around the constricted site. DRP3 could
then mechanochemically cause fission and refuse the ends of the IMM and the OMM. If the
newly divided mitochondria are still tethered to the ER, either through FIS1 or other tethering
proteins (i.e. VDAC for Ca2+ transport), when the ER rearranges, it would pull the mitochondria
along with it. The mitochondria would disperse throughout the cell (Fig. 4.4E). Simultaneous
visualization of mito-GFP RER double transgenics here supports this.
It was also shown here that when the ER is flattened out (i.e. sheets of cisternae),
mitochondria often are as well. As the ER is stretched out, a meshwork of distinct tubules is
created. As this occurs, mitochondria too, are stretched out, and become tubular. It is proposed
56
that this stretching out and tubulation of these two organelles are correlated and may be due to
membrane contact sites (MCS). If mitochondria are attached to the ER through MCS, as the ER
membrane is stretched, the MCS are pulled along with it causing the mitochondrial membrane to
stretch as well (Fig. 4.4D). In some situations, it was noticed that a piece of the mitochondrion
that fragmented from a tubular mitochondrion appeared small and round. This may be because
there are now less MCS with the ER to allow the mitochondria membranes to be stretched out or
elongate; the mitochondria by default would ball up. This may explain why mitochondria are
described as spherical structures in mitochondria isolations; there is no ER to stretch the outer
membrane out.
It is interesting that FIS1 is suggested to be involved in tethering mitochondria. The ER
has also been shown to play a role in peroxisome and plastid morphology and FIS1 was shown
here and by Ruberti (2014) to localize to mitochondria, peroxisomes and plastids. It was also
shown here through simultaneous visualization of triple transgenics that the ER cages these three
organelles. Other tethering proteins have been suggested between the ER and the other
organelles, so perhaps MCS are involved in bringing these organelles together during interorganelle transfers. The metabolic and biochemical basis for close ER-plastid, ER-peroxisome
and mitochondria-plastid interactions have been discussed previously, but analysis of
mitochondria-peroxisome physical interactions in plant cells was lacking. The clustering of
mitochondria and peroxisomes was therefore investigated further.
4.3
MITOCHONDRIA AND PEROXISOME INTERACTIONS
Both mitochondria and peroxisomes can exist in tubular and spherical forms. During
fission, both organelles constrict and divide to increase their population and dispersal during
various stresses. Since mitochondria fragment in response to light and increased cytosolic sugar,
the effects these factors may have on peroxisome morphology was investigated.
57
A
C
Ca2+
DRP3
ER
FIS1
Cyotplasm
BAP31
OMM
IP3R
IMM
VDAC
MCU
B
D
Pulling/pushing force by ER
Fusion
Constriction (ER, FIS1, DRP3)
Pulling force by ER
Fission
E
MCS
Flattened ER
Y
As the ER is
stretched in the
X direction, it
‘shrinks’ in the Z
and Y directions
too
Pulling Force
on the ER
Weakened Point
in Mitochondria
Membrane
Minimal MCS
Mitochondria
Membrane
can ball up
Z
X
Figure 4.4 Proposed models to explain ER-mediated mitochondrial pleomorphy. (A, B)
FIS1 acts as a tether for mitochondria to the ER during mitochondrial fission. Tethered to the
mitochondrion, the ER constricts it. DRP3 may bind to FIS1, but as the mitochondrion is
constricted, DRP3 can form a helix around the mitochondria and mechanochemically cause
fission. (C) VDAC can also act as an ER-mitochondrial tether during Ca2+ transport. (D)
Mitochondria are often embedded or surrounded by the ER. During mitochondrial fission, the
ER constricts a mitochondrion. Narrow enough now, DRP3 wraps around this site. Fission
occurs as the ER also helps to apply a pulling force. (E) as the ER rearranges, the mitochondria,
tethered to the ER by MCS(s), moves along with the ER. Based on observations reported here
and adapted from models proposed by Friedman and Voeltz 2011; Westerman 2011; Prinz 2014
in the yeast and animal systems.
58
Mitochondria and peroxisomes are both essential organelles in eukaryotic cells. In plant
cells, peroxisomes are the only site of β-oxidation, products of which enter the mitochondria
matrix for use in ATP synthesis (Schrader and Yoon 2007). During ATP synthesis, electron
leakage from the ETC often occurs. When these electrons spontaneously react with O 2, ROS are
produced. Although mitochondria house enzymes to reduce these highly reactive species, ROS
such as H2O2 can diffuse out of the mitochondrial membranes into the cytosol. Being notorious
for causing lipid peroxidation, this increase in ROS can be damaging to all membranes in the
cell. Plants are quite vulnerable to lipid peroxidation because, where mitochondria are the major
producers of ROS in animal and yeast cells (Foyer and Noctor 2003; Apel and Hirt 2004; Ježek
and Plecitá-Hlavatá 2009), chloroplasts also produce them in plants during photorespiration. Just
as with mitochondria, ROS produced by chloroplasts may also diffuse through the chloroplast
membranes into the cytosol. Peroxisomes, which are also producers of ROS themselves, are most
famous for their ROS scavenging capabilities. The ROS released into the cytosol can enter the
peroxisomal matrix. Having enzymes such as catalase and peroxidase, the ROS can then be
reduced to more stable forms, i.e. catalase reduction of H2O2 to H2O (Foyer and Noctor 2003;
Apel and Hirt 2004; Schrader and Yoon 2007; Bhattacharjee 2011).
It is quite clear that these organelles are linked metabolically and biochemically in the cell.
What has also become apparent, but not investigated in depth, is their physical interactivity. The
half-life of O2˙ˉ is seconds and that of 1O2 is a matter of microseconds. This means that they
have short diffusion distances (Halliwell and Gutteridge 1999). For this reason, it would be
expected that the scavenger, peroxisomes, would be found within very short distances of the
ROS producers, chloroplasts and mitochondria. Fredrick and Newcomb (1969) published a series
of TEM images which supported this. They showed peroxisomes in close, physical proximity
with mitochondria and chloroplasts. When transgenics expressing a cytosolic hydrogen peroxide
responsive probe, HyPer-GFP (Costa et al. 2010), were given a short 5min HL irradiation, the
GFP fluorescence in the cytosol increased. This demonstrates the sheer rapidity of H2O2
production and release into the cytosol during HL irradiation. Simultaneous visualization of
mitochondria and peroxisomes revealed that the length of interaction between these two
organelles in dark grown plants increased within 30s to 2min of HL. This further supports that
ROS production-scavenging in response to light stress is a contributor to mitochondria-
59
peroxisome interactions, which may be one of the necessary efficient mechanisms for
maintaining ROS homeostasis.
The duration of interactions between the two organelles did however decrease after 4min
of HL irradiation. Dark grown plants have etioplasts which contain protochlorophyllide. When
etioplasts are photoactivated, they become chloroplasts because protochlorophyllide is converted
to chlorophyll. Protochlorophyllide accumulates in the dark and when the dark-to-light shift
occurs, it becomes chlorophyll. But 1O2 is produced in the process. If the photoperiod and light
intensity are too high, 1O2 levels become cytotoxic and cell death occurs (Kim et al. 2008;
Przybyla et al. 2008; Gechev et al. 2010). The decrease in interactivity of mitochondria and
peroxisomes may have been because the HL stimulus caused a cytotoxic level of 1O2 to be
produced between 2-4min of irradiation, causing the cell to prepare for death or the cell was in
disarray, attempting a last ditch effort to save itself.
FIS1 and DRP3 are involved in the fission of both peroxisomes and mitochondria (Mano et
al. 2004; Arimura et al. 2008; Zhang and Hu 2008; 2009; Pan and Hu 2011; Logan et al. 2004;
Scott et al. 2006; Schrader 2006; Delille et al. 2009). Neuspiel et al. (2008) showed that MDVs
carrying MAPL was translocated from the OMM to the peroxisome membrane. MDVs have
been suggested to be one way in which intramembranous material and even lipids can be
delivered from mitochondria to peroxisomes (Schumann and Subramani 2008). Another means
of transfer between these two organelles that has been suggested is through the increase of
surface area of one of the organelles to increase the physical contact available for efficient
transfer (Schumann and Subramani 2008). This was of particular interest.
Both peroxisome and mitochondrial extensions called peroxules and matrixules,
respectively, have been reported (peroxules: Scott et al. 2007; Sinclair et al. 2009; Delille et al.
2010; Barton et al. 2014; matrixules: Logan et al. 2004; Logan 2006). Their functions however,
have yet to be elucidated. Extensions are thought to permit increased surface area for interorganelle transport. Sustained interactions between peroxules and small, fragmented
mitochondria were reported here. This interaction is more apparent in any1, a mutant that was
shown to be more vulnerable to light stress, than wild type. Given that 1) this mutant is more
susceptible to light stress, 2) peroxules have been suggested to be stress sensors (Mathur et al.
60
2012) and 3) that peroxules are produced during ROS stress, the interaction may be a means of
transporting ROS and Ca2+, both of which are signals involved in regulating stress responses
(Brookes et al. 2004).
Light
Dark grown or etiolated:
Light
HL stimulus:
- With no light, etioplasts
have protochlorophyllide
and no chlorophyll
- Protochlorophyllide
becomes chlorophyll,
which creates ROS (1O2)
- With no metabolic input
(i.e. no photosynthesis),
there is no active cell
respiration and therefore
no interaction or transport
between organelles
- Photosynthesis may now
commence and
metabolites are produced
Metabolites become
available:
- The organelles start
interacting. The ER (not
shown) becomes more
dynamic
- Metabolites and other
signals are transported
between organelles
- Mitochondria beading may
be seen as really
elongated mitochondria
thread through the ER
meshwork which may
contain some small
polygons
- As mitochondria start
oxidative phosphorylation,
ROS is produced
- Mitochondria start dividing
to disperse energy from
the ETC
Light
ROS levels rise:
- During photosynthesis and
cellular respiration, ROS is
being produced
- If ROS production >
scavenging/disposal, the
cell is in distress (ex. lipid
peroxidation)
- ROS signals apoptosis
- The interactions between
organelles become minimal
as mitochondria and
peroxisomes are
dispersing, trying to save
the cell or working towards
cell death
Figure 4.5 Model to explain effects of light on mitochondria, peroxisomes and plastids. Note
that the ER is not shown for simplicity, but as the cells start photosynthesizing and respiring, the
ER becomes more dynamic as it plays a major role in transporting protein, lipid etc.
Green=mitochondria; yellow=peroxisomes; blue=ROS; pink to dark red gradient=etioplasts
transitioning into chloroplasts.
CONCLUSIONS
Mitochondria are one of the most pleomorphic organelles found in eukaryotic cells
because of their constant fusion and fission. Light and sugar induce the fission of mitochondria
into small, spherical forms typically seen in plant cells. O 2 limited conditions however, induce
mitochondrial fusion which results in elongation and eventually expanded, giant mitochondria.
61
The ER is a mediator of these transitions because it constricts tubular mitochondria and applies a
pulling force to separate the mitochondria after fission. Also, as ER tubules rearrange or flatten
out into cisternae, the mitochondria rearrange accordingly. Mitochondria also interact with
peroxisomes. During HL stress, which results in increased ROS levels, peroxisomes extend thin
membrane projections called peroxules. Peroxules often extend around chloroplasts and have
small mitochondria clustered around them. Simultaneous, live-imaging further reveals that ER
tubules form a cage around mitochondria, peroxisomes and chloroplasts and that all four
organelles are truly interactive.
FUTURE DIRECTIONS
The effects of light, sugar and O2 depletion on mitochondria morphology in plant cells
was of key interest here. These factors were shown to induce morphological transitions that may
ultimately be linked to the metabolic status of the cell, which could be explored further. Future
studies could also apply these conditions to better understand the mechanism behind fusion and
fission. For example, the ER is now known to constrict mitochondria during fission in yeast,
mammalian and plant cells. Since dynamin related proteins have a smaller diameter than that of a
tubular mitochondrion, it has been suggested that the ER must constrict the mitochondrion
enough to allow the dynamin to wrap around the membrane to facilitate fission (Friedman et al.
2011). However, why it is the ER that does this and how the site of fission is decided remains
unknown. Investigations into MCS may give insight into this. One MCS that may be of interest
is the voltage dependent anion channel (VDAC), for which primers and base vectors are
available (Appendix III). This may give insight into one of the major reasons for ERmitochondria interactions: the transport of Ca2+, a key stress signal that has also been shown to
regulate mitochondrial fission (Brookes et al. 2004).
Simultaneous live imaging of the ER, mitochondria, peroxisomes and chloroplasts was
also carried out. This gave a greater appreciation for how dynamic and interactive the living cell
is. These organelles are often found in close, sustained clusters which may permit inter-organelle
transfer of lipids, metabolites, and signalling molecules such as ROS and Ca2+. One means of
62
enabling efficient transfers between organelles is by increasing membrane surface area such as
through extensions. Peroxules were shown here to interact with small, fragmented mitochondria.
ROS is released from mitochondria during their fragmentation. Peroxules may be one way to
effectively transport ROS or other metabolites. MDVs carrying MAPL have been shown
previously to translocate from mitochondria to peroxisomes. Transgenic lines of DAL1-Eos and
DAL2-Eos, Arabidopsis homologues of MAPL have been created and may be of interest to
further investigate the transport aspect of explaining the sustained mitochondria-peroxisome
interactions reported here. How peroxules are extended during these interactions could also be
investigated. Peroxin11 (PEX11) is known to be a peroxisome elongation factor (Koch et al.
2010) and may be of interest in such an investigation.
Sui generis: in a class of its own; unique. This phrase was initially used to describe the
very pleomorphic organelle, mitochondria (Cavers 1914). However, investigations of their
morphological behaviours and interactions with other organelles during stress contradict this. No
organelle is truly in a class of its own; they are all interactive entities that work together to permit
life.
63
REFERENCES
Achleitner G, Gaigg B, Krasser a., Kainersdorfer E, Kohlwein SD, Perktold a., Zellnig G,
Daum G (1999) Association between the endoplasmic reticulum and mitochondria of
yeast facilitates interorganelle transport of phospholipids through membrane contact. Eur
J Biochem 264:545-553
Altmann R (1890) Die elemtaroganismen und ihre bexiehungen zu den dellen. 1 st ed. Veit,
Leipzig
Amchenkova AA, Bakeeva LE, Chentsov YS, Skulachev VP, Zorov DB (1988) Coupling
membranes as energy-transmitting cables. I. Filamentous mitochondria in fibroblasts and
mitochondrial clusters in cardiomyocytes. J Cell Biol 107:481-495
Apel K, Hirt H (2004) Reactive oxygen species: Metabolism, oxidative stress, and signal
transduction. Ann Rev Plant Biol 55:373-399
Arimura S, Aida GP, Fujimoto M, Nakazono M, Tsutsumi N (2004) Arabidopsis dynaminlike protein 2a (ADL2a), like ADL2b, is involved in plant mitochondrial division. Plant
Cell Physiol 45:236-242
Arimura S, Fujimoto M, Doniwa Y, Kadoya N, Nakazono M, Sakamoto W, Tsutsumi N
(2008) Arabidopsis ELONGATED MITOCHONDRIA1 is required for localization of
DYNAMIN-RELATED PROTEIN3A to mitochondrial fission sites. Plant Cell 20:15551566
Arimura S, Tsutsumi N (2002) A dynamin-like protein (ADL2b), rather than FtsZ, is involved
in Arabidopsis mitochondrial division. Proc Natl Acad Sci USA 99:5727-5731
Arimura S, Yamamoto J, Aida GP, Nakazono M, Tsutsumi N (2004) Frequent fusion and
fission of plant mitochondria with unequal nucleoid distribution. Proc Natl Acad Sci USA
101:7805-7808
Asada K (1999) The water-water cycle in chloroplast: scavenging oxygens and siddispartion of
excess protons. Annu Rev Plant Physiol Mol Biol 50:601-639
Asada K (2006) Production and scavenging of reactive oxygen species in chloroplasts and their
functions. Am Soc Plant Biol 141:391-396
Azcón-Bieto J, Lambers H, Day DA (1983) Effects of photosynthesis and carbohydrate status
on respiratory rates and the involvement of the alternative pathway in leaf respiration.
Plant Physiol 71:598-603
Azcón-Bieto J, Osmond CB (1983) Relationship between photosynthesis and respiration. The
effects of carbohydrate status on the rate of CO2 production by respiration in darkened
and illuminated wheat leaves. Plant Physiol 71:574-581
64
Barsoum MJ, Yuan H, Gerencser AA, Liot G, Kushnareva Y, Gräber S, et al. (2006) Nitric
oxide-induced mitochondrial fission is regulated by dynamin-related GTPases in neurons.
EMBO J 25:3900-3911
Barton KA, Jaipargas EA, Griffiths N, Mathur N, Mathur J (2014) Live imaging of
peroxisomes and peroxules in plants. Molecular Machines Involved In Peroxisome
Biogenesis and Maintenance. Springer Publ pp. 233-254
Beevers H (1979) Microbodies in higher plants. Annu Rev Plant Physiol 30:159-193
Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, Terskikh AV,
Lukyanov S (2006) Genetically encoded fluorescent indicator for intracellular hydrogen
peroxide. Nat Methods 3:281-286.
Benard G, Peng G, Karbowski M (2009) Mitochondria fusion and fission. ELS. JohnWiley &
Sons, Ltd pp. 1-11
Benda C (1897) Über die Spermatogenese der Vertebraten und höheren Evertebraten. Verhanl
Physik Ges Berlin pp. 14-17
Bereiter-Hahn J, Vöth M (1994) Dynamics of mitochondria in living cells: shape changes,
dislocations, fusion, and fission of mitochondria. Micro Res Tech 27:198-219
Bertani G (1951) STUDIES ON LYSOGENESIS I.: The mode of phage liberation by lysogenic
Escherichia coli. J Bacteriol 62:293-300
Bhattacharjee (2011) Sites of generation and physicochemical basis of formation of reactive
oxygen species in plant cell. Reactive oxygen species and antioxidants in higher plants.
Science Publ pp. 1-30
Boustany NN, Drezek R, Thakor NV (2002) Calcium-induced alterations in mitochondrial
morphology quantified in situ with optical scattering imaging. Biophys J 83:1691-1700
Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu S (2004) Calcium, ATP, and ROS: a
mitochondrial love-hate triangle. Am J Physiol 287:C817-C833
Buysse J, Merckx R (1993) An improved colorimetric method to quantify sugar content of plant
tissue. J Exp Bot 44:1627-1629
Cavers F (1914) Chondriosomes (mitochondria) and their significance. New Phytol 13:96-109
Chan DC (2006) Mitochondrial dynamic organelles in disease, aging, and development. Cell
125:1241-1252
Chazotte B (2011) Labeling mitochondria with MitoTracker dyes. Cold Springs Harb Protoc
8:990-992
65
Chen H, Chomyn A, Chan DC (2005) Disruption of fusion results in mitochondrial
heterogeneity and dysfunction. J Biol Chem 280:26185-26192
Chen Q, Vazquez EJ, Moghaddas S, Hoppel CL, Lesnefsky EJ (2003) Production of reactive
oxygen species by mitochondria: central role of complex III. J Biol Chem 278:3602736031
Ching SLK, Gidda SK, Rochon A, van Cauwenberghe OR, Shelp BJ, Mullen RT (2012)
Glyoxylate Reductase Isoform 1 is localized in the cytosol and not peroxisomes in plant
cells. Int Plant Biol 54:152-168
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated
transformation of Arabidopsis thaliana. Plant J 16:735-743
Costa A, Drago I, Behera S, Zottini M, Pizzo P, Schroeder JI, Pozzan T, Lo Schiavo, F
(2010) H2O2 in plant peroxisomes: An in vivo analysis uncovers a Ca(2þ)-dependent
scavenging system. Plant J 62:760-772
De Brito OM, Scorrano L (2008) Mitofusin 2 tethers endoplasmic reticulum to mitochondria.
Nature 456:605-610
De Duve C, Baudhuin P (1966) Peroxisomes (microbodies and related particles). Physiol Rev
46:323-357
del Río LA, Sandalio LM, Corpas FJ, Palma JM, Barroso JB (2006) Reactive oxygen species
and reactive nitrogen species in peroxisomes. Production, scavenging, and role in cell
signalling. Am Soc Plant Biol 141:330-335
Delille HK, Agricola B, Guimaraes SC, Borta H, Lüers GH, Fransen M, Schrader M (2010)
Pex11pβ-mediated growth and division of mammalian peroxisomes follows a maturation
pathway. J Cell Sci 123: 2750-2762
Delille HK, Alves R, Schrader M (2009) Biogenesis of peroxisomes and mitochondria: Linked
by division. Histochem Cell Biol 131: 441–446
Eastmond PJ, Quettier AL, Kroon JT, Craddock C, Adams N, Siabas AR (2010)
PHOSPHATIDIC ACID PHOSPHOHYDROLASE1 and 2 regulate phospholipid
synthesis at the endoplasmic reticulum in Arabidopsis. Plant Cell 22:2796-2811
Edwards GA, Buell CB, Weston WH (1947) The influence of mineral oil upon the oxygen
consumption of Sordaria fimicola. Am J Bot 34:551-555
Feng XG, Arimura S, Hirano HY, Sakamoto W, Tsutsumi N (2004). Isolation of mutants
with aberrant mitochondrial morphology from Arabidopsis thaliana. Genes Genet Syst
79:301-305
66
Flemming W (1882) Zellsubstanz, Kern- und Zellteilung. Verlag Vogel, Leipzig
Foyer CH, Noctor G (2003) Redox sensing and signalling associated with reactive oxygen in
chloroplasts, peroxisomes and mitochondria. Physiol Plant 119:355-364
Frank M, Duvezin-Caubet S, Koob S, Occhipinti A, Jagasia R, Petcherki A, Ruonala MO,
Priault M, Salin B, Reichert AS (2012) Mitophagy is triggered by mild oxidative stress
in a mitochondrial fission dependent manner. Biochim Biophys Acta 1823:2297-2310
Frederick SE, Newcomb EH (1969) Cytochemical localization of catalase in leaf microbodies
(peroxisomes). J Cell Biol 43:343-353
Frey-Wyssling A, Mühlethaler K (1965) Ultrastructural plant cytology with an introduction to
molecular biology. Elsevier Publ Co
Friedman JR, Lackner LL, West M, DiBenedetto JR, Nunnari J, Voeltz GK (2011) ER
Tubules Mark Sites of Mitochondrial Division. Science 334:358-362
Friedman JR, Voeltz GK (2011) The ER in 3D: A multifunctional dynamic membrane
network. Trends Cell Biol 21:709-717
Fujita M, Himmelspach R, Ward J, Whittington A, Hasenbein N, Liu C, Truong TT,
Galway ME, Mansdield SD, Hocart CH, Wasteneys GO (2013) The anisotropy1
D604N mutation in the Arabidopsis Cellulose Synthase1 catalytic domain reduces cell
wall crystallinity and the velocity of cellulose synthase complexes. Plant Physiol 162:7485
Gechev T, Petrov V, Minkov I (2010) Reactive oxygen species and antioxidants in higher
plants: Reactive oxygen species and programmed cell death. CCS Press 65-79
Giepmans BN, Adams SR, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for
assessing protein location and function. Science 312:217-224
Gunning BES (2005) Plastid stromules: video microscopy of their outgrowth, retraction,
tensioning, anchoring, branching, bridging, and tip-shedding. Protoplasma 225:33-42
Haberlandt G (1914) Physiological plant anatomy. Mac Millan: London pp. 613-630
Hackenbrock CR (1966) Ultrastructural bases for metabolically linked mechanical activity in
mitochondria. J Cell Biol 30:269-297
Hackenbrock CR (1968) Ultrastructural bases for metabolically linked mechanical activity in
mitochondria. II. Electron transport-linked ultrastructural transformations in
mitochondria. J Cell Biol 37:345-369
Hackenbrock CR, Rehn TG, Weinbach EC, Lemasters JJ (1971) Oxidative phosphorylation
67
and ultrastructural transformation in mitochondria in the intact ascites tumor cell. J Cell
Bio 51:123-137
Halliwell B, Gutteridge LMC (1989) Free radicals in biology and medicine. 2 nd ed., Oxford:
Clarendon
Hanson MR and Sattarzadeh A (2008) Dynamic morphology of plastids and stromules in
angiosperm plants. Plant Cell Enviro 31:646-657
Haseloff J, Siemering KR, Prasher DC, Hodge S (1997) Removal of a cryptic intron and
subcellular localization of green fluorescent protein are required to mark transgenic
Arabidopsis plants brightly. Proc Natl Acad Sci USA 94:2122-2127
Hideg É, Barta C, Kalai T, Vass I, Hideg K, Asada K (2002) Detection of singlet oxygen and
superoxide with fluorescent sensors in leaves under stress by photo inhibition or UV
radiation. Plant Cell Physiol 43:154-64
Hoefnagel MHN, Atkin OK, Wiskich JT (1998) Interdependence between chloroplasts and
mitochondria in the light and the dark. Biochim Biophys Acta - Bioenerg 1366:235-255
Hoffman HP, Avers CJ (1973). Mitochondrion of yeast: ultrastructural evidence for one giant,
branched organelle per cell. Science 181:749-751
Ingerman E, Perkins EM, Marino M, Mears JA, McCaffery JM, Hinshaw JE, Nunnari J
(2005) Dnm1 forms spirals that are structurally tailored to fit mitochondria. J Cell Biol
170:1021-1027
Jacobson AP, Thunberg RL, Johnson ML, Hammack TS, Andrews WH (2004) Alternative
anaerobic enrichments to the Bacteriological Analytical Manual culture method for
isolation of Shigella sonnei from selected types of fresh produce. J AOAC Int 87:11151122
Jendrach M, Mai S, Pohl S, Vöth M, Bereiter-Hahn J (2008) Short- and long-term alterations
of mitochondrial morphology, dynamics and mtDNA after transient oxidative stress.
Mitochondrion 8:293-304
Ježek P, Plecitá-Hlavatá L (2009) Mitochondrial reticulum network dynamics in relation to
oxidative stress, redox regulation, and hypoxia. Int J Biochem Cell Biol 41:1790-1804
Jhun BS, Lee H, Jin ZG, Yoon Y (2013) Glucose stimulation induces dynamic change of
mitochondrial morphology to promote insulin secretion in the insulinoma cell line INS1E. PLoS ONE 8:e60810
Kim C , Meskauskiene R, Apel K, Laloi C (2008) No single way to understand singlet oxygen
signaling in plants. EMBO Rep 9:435-439
68
Klotz L-O (2002) Oxidant-induced signaling: effects of peroxynitrite and singlet oxygen. Biol
Chem 383:443
Kobayashi S, Tanaka A, Fujiki Y (2007) Fis1, DLP1, and Pex11p coordinately regulate
peroxisome morphogenesis. Exp Cell Res 313:1675-1686
Koch J, Pranjic K, Huber A, Ellinger A, Hartig A, Kragler F, Brocard C (2010) PEX11
family members are membrane elongation factors that coordinate peroxisome
proliferation and maintenance. J Cell Sci 123:3389-3400
Legros F, Lombès A, Frachon P, Rojo M (2002) Mitochondrial fusion in human cells is
efficient, requires the inner membrane potential, and is mediated by mitofusins. Mol Biol
Cell 13:4343-4354
Lewis MR, Lewis WH (1914) Mitochondria (and other cytoplasmic structures) in tissue
cultures. Am J Anat 17:339-401
Lingard MJ, Gidda SK, Bingham S, Rothstein SJ, Mullen RT, Trelease RN (2008)
Arabidopsis PEROXIN11c-e, FISSION1b, and DYNAMIN-RELATED PROTEIN3A
cooperate in cell cycle-associated replication of peroxisomes. Plant Cell 20:1567-1585
Logan DC (2006) Plant mitochondrial dynamics. Biochmica et Biophysica Acta 1763:430-441
Logan DC (2010) Chapter 11, essay 11.3. Mitochondrial dynamics: When form meets function.
5th ed., Plant Physiology Online
Logan DC, Leaver CJ (2000) Mitochondria-targeted GFP highlights the heterogeneity of
mitochondrial shape, size and movement within living plant cells. J Exp Bot 51:864-871
Logan DC, Scott I, Tobin AK (2004) ADL2a, like ADL2b, is involved in the control of higher
plant mitochondrial morphology. J Exp Botany 55:783-785
Mano S, Nakamori C, Kondo M, Hayashi M, Nishimura M (2004) An Arabidopsis dynaminrelated protein, DRP3A, controls both peroxisomal and mitochondrial division. Plant J
38:487-498
Mathur J (2007) The illuminated plant cell. Trends Plant Sci 12:506–513
Mathur J, Mammone A, Barton KA (2012) Organelle extensions in plant cells. J Int Plant Biol
54:851-867
Mathur J, Mathur N, Hulskamp M (2002) Simultaneous visualization of peroxisomes and
cytoskeletal elements reveals actin and not microtubule based peroxisome motility in
plants. Plant Physiol 128:1031-1045
69
Mathur J, Radhamony R, Sinclair AM, Donoso A, Dunn N, Roach E, Radford D,
Mohaghegh PSM, Logan DC, Kokolic K, et al (2010) mEosFP-based green-to-red
photoconvertible subcellular probes for plants. Plant Physiol 154:1573-1587
Mehler AH (1951) Studies on reactions of illuminated chloroplasts. II. Stimulation and
inhibition of reaction with oxygen. Arch Biochem Biophys 33:65-77
Meves F (1904) Über das vorkommen von mitochondrion bezw. Chrondromiten der Pflanzen
zellen. Ber deutsch bot Ges 22:264-286
Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene
network of plants. Trends Plant Sci 9:490-498
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco
tissue cultures. Physiol Plant 15:473-797
Nakada K, Inoue K, Ono T, Isobe K, Ogura A, Goto YI, Nonaka I, Hayashi JI (2001) Intermitochondrial complementation: mitochondria-specific system preventing mice from
expression of disease phenotypes by mutant mtDNA. Nat Med 7:934-940
Neuspiel M, Schauss AC, Braschi E, Zunino R, Rippstein P, Rachubinski RA, AndradeNavarro MA, McBride HM (2008) Cargo-selected transport from the mitochondria to
peroxisomes is mediated by vesicular carriers. Curr Biol 18:102-108
Novikoff AB, Holtzman E (1976) Cells and organelles. 2nd ed., Holt, Rinehart and Winston:
USA
Nunnari J, Marshall WF, Straight A, Murray A, Sedat JW, Walter P (1997) Mitochondrial
transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial
fusion and fission and the intramitochondrial segregation of mitochondrial DNA. Mol Biol
Cell 8:1233-1242
Osman C, VoelkerDR, Langer T (2011) Making heads or tails of phospholipids in
mitochondria. J Cell Biol 192:7-16
Packer L (1960) Metabolic and structural states of mitochondria. J Bio Chem 235:242-249.
Pan R, Hu J (2011) The conserved fission complex on peroxisomes and mitochondria. Plant
Signal Behav 6:870-872
Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ (1992)
Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111:229-233
Prinz W (2014) Bridging the gap: Membrane contact sites in signaling, metabolism, and
organelle dynamics. J Cell Biol 205:759-769
70
Przybyla D, Gobel C, Imboden A, Hamberg M, Feussner I, Apel K (2008) Enzymatic, but
not non-enzymatic, 1O2-mediated peroxidation of polyunsaturated fatty acids forms part
of the EXECUTER1-dependent stress response program in the flu mutant of Arabidopsis
thaliana. Plant J 54:236-248
Pyke KA and Howells CA (2002) Plastid and stromule morphogenesis in tomato. Annals of
Botany 90:559-566
Rahn O, Richardson GL (1940) Oxygen demand and supply. J Bact 41:225-249
Rhodin J (1954) Correlation of ultrastructural organization and function in normal
experimentally changed convoluted tubule cells of the mouse kidney, PhD thesis,
Aktiebolaget Godvil, Stockholm
Rintoul GL, Filiano AJ, Brocard JB, Kress GJ, Reynolds IJ (2003) Glutamate decreases
mitochondrial size and movement in primary forebrain neurons. J Neurosci 23:78817888
Rizzuto R, PintonP, Carrington W, Fay FS, Fogarty KE, Lifshitz LM, Tuft RA, Pozzan T
(1998) Close contacts with the endoplasmic reticulum as determinants of mitochondrial
Ca2+ responses. Science 280:1763-1766
Ruberti C, Costa A, Pedrazzini E, Lo Schiavo F, Zottini M (2014) FISSION1A, an
Arabidopsis Tail-Anchored Protein, Is Localized to Three Subcellular Compartments. Mol
Plant 7:1393-1396
Schattat M, Barton K, Baudisch B, Klösgen RB, Mathur J (2011) Plastid stromule branching
coincides with contiguous endoplasmic reticulum dynamics. Plant Physiol 155:16671677
Schrader M (2006) Shared components of mitochondrial and peroxisomal division. Biochim
Biophys Acta - Mol Cell Res 1763:531-541
Schrader M, Wodopia R, Fahimi HD (1999) Induction of tubular peroxisomes by UV
irradiation and reactive oxygen species in HepG2 cells. J Histochem Cytochem 47:11411148
Schrader M, Yoon Y (2007) Mitochondria and peroxisomes: Are the “Big Brother” and the
“Little Sister” closer than assumed? BioEssays 29:1105-1114
Schumann U, Subramani S (2008) Special delivery from mitochondria to peroxisomes. Trends
Cell Biol 18:253-256
Scott I, Logan DC (2007) Mitochondrial morphology transition is an early indicator of
subsequent cell death in Arabidopsis. New Phytol 177:90-101
71
Scott I, Sparkes IA, Logan DC (2007) The missing link: inter-organellar connections in
mitochondria and peroxisomes? Trends Plant Sci 12:380-381
Scott I, Tobin AK, Logan DC (2006) BIGYIN, an orthologue of human and yeast FIS1 genes
functions in the control of mitochondrial size and number in Arabidopsis thaliana. J Exp
Bot 57:1275-1280
Shockey JM, Gidda SK, Chapital DC, Kuan JC, Dhanoa PK, Bland JM, Rothstein SJ,
Mullen RT, Dyer JM (2006) Tung tree DGAT1 and DGAT2 have nonredundant functions
in triacylglycerol biosynthesis and are localized to different subdomains of the endoplasmic
reticulum. Plant Cell 18:2294-2313
Sinclair AM, Trobacher CP, Mathur N, Greenwood JS, Mathur J (2009) Peroxule extension
over ER-defined paths constitutes a rapid subcellular response to hydroxyl stress. Plant J
59:231-242
Skulachev VP (2001) Mitochondrial filaments and clusters as intracellular power-transmitting
cables. Trends Biochem Sci 26:23-29
Stevens B (1981) Mitochondrial structure. The Molecular Biology of the Yeast Saccharomyces,
Life Cycle and Inheritance, vol. 1, Cold Spring Harbor Lab Press: NY pp.471-504
Stolz B, Bereiter-Hahn J (1987) Sequestration of iontophoretically injected calcium by living
endothelial cell. Cell Calcium 8:103-21
Tabak HF, Hoepfner D, Zand A, Geuze HJ, Braakman I, Huynen MA (2006) Formation of
peroxisomes: Present and past. Biochim Biophys Acta 1763:1647-1654
Tandler B, Erlandson RA, Smith AL, Wynder EL (1969) Ribflavin and mouse hepatic cell
structure and function. II. Division of mitochondria during recovery from simple
deficiency. J Cell Biol 41:477-493
Titorenko VI, Mullen RT (2006) Peroxisome biogenesis: The peroxisomal endomembrane
system and the role of the ER. J Cell Biol 174:11-17
Tobin AK, Thorpe JR, Hylton CM, Rawsthorne S (1989) Spatial and temporal influences on
the cell-specific distribution of glycine decarboxylase in leaves of wheat (Triticum
aestivum L.) and pea (Pisum sativum L.). Plant Physiol 91:1219-1225
Tukey JW (1949) Comparing individual means in the analysis of variance. Biometrics 5:99-114
Twig G, Elorza A, Molina AJA, Mohamed H, Wikstrom JD, Walzer G, Stiles L, Haigh SE,
Katz S, Las G, Alroy J, Wu M, Py BF, Yuan J, Deeney JT, Corkey BE, Shirihai OS
(2008) Fission and selective fusion govern mitochondrial segregation and elimination by
autophagy. EMBO J 27:433-446
72
Twig G, Graf SA, Wikstrom JD, Mohamed H, Haigh SE, Elorza A, Deutsch M, Zurgil N,
Reynolds N, Shirihai OS (2006) Tagging and tracking individual networks within a
complex mitochondrial web with photoactivatable GFP. Am J Physiol Cell Physiol
291:C176-C184
Van Gestel K, Verbelen JP (2002) Giant mitochondria are a response to low oxygen pressure in
cells of tobacco (Nicotiana tabacum L.). J Exp Bot 53:1215-1218
Vance JE (2014) MAM (mitochondria-associated membranes) in mammalian cells: lipids and
beyond. Biochim Biophys Acta 1841:595-609
Vogelman TC, Bornman JF, Yates DJ (1996) Focusing of light by leaf epidermal cells.
Physiologia Plantarum 98:43-56
Vogelmann TC (1993) Plant tissue optics. Ann Rev Plant Physiol Plant Mol Biol 44:231-251
Weigel D, Glazebrook J (2006) Transformation of agrobacterium using the freeze thaw method.
CSH Prot 7:1031-1036
Westermann B (2008) Molecular machinery of mitochondrial fusion and fission. J Biol Chem
283:13501-13505
Westermann B (2011) Organelle dynamics: ER embraces mitochondria for fission. Curr Biol
21:R922-R924
Yoon Y, Krueger EW, Oswald BJ, McNiven MA (2003) The mitochondrial protein hFis1
regulates mitochondrial fission in mammalian cells through an interaction with the
dynamin-like protein DLP1. Mol Cell Biol 23:5409-5420
Yoon Y, Pitts KR, McNiven MA (2001) Mammalian dynamin-like protein DLP1 tubulates
membranes. Mol Biol Cell 12:2894-905
Yoshinaga K, Arimura S, Niwa Y, Tsutsumi N, Uchimiya H, Kawai-Yamada M (2005)
Mitochondrial behaviour in the early stages of ROS stress leading to cell death in
Arabidopsis thaliana. Annals of Botany 96:337-342
Youle RJ, van der Bliek AM (2012) Mitochondrial fission, fusion, and stress. Science
337:1062-1065
Yu T, Robotham JL, Yoon Y (2006) Increased production of reactive oxygen species in
hyperglycemic conditions requires dynamic change of mitochondrial morphology. PNAS
103:2653-2658
Yu T, Sheu SS, Robotham JL, Yoon Y (2008) Mitochondrial fission mediates high glucoseinduced cell death through elevated production of reactive oxygen species. Cardiovasc
Res 79:341-351
73
Zhang X, Hu J (2009) Two small protein families, DYNAMIN-RELATED PROTEIN3 and
FISSION1, are required for peroxisome fission in Arabidopsis. Plant J 57:146-159
Zhang X, Hu J (2010) The Arabidopsis chloroplast division protein DYNAMIN-RELATED
PROTEIN5B also mediates peroxisome division. Plant Cell 22:431-442
Zhang XC, Hu JP (2008) FISSION1A and FISSION1B proteins mediate the fission of
peroxisomes and mitochondria in Arabidopsis. Mol Plant 1:1036-1047
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APPENDIX I
SUMMARY OF MITOCHONDRIAL MORPHOLOGY DISCUSSED
Light grown ± Sugar
Dark Grown, No
Sugar
Round
Enlarged or Swollen
Tubular
Dark to Light
Elongated
Twisted
Unevenly Constricted
Beaded
Beads-on-a-string Constriction
Figure I-1 Effects of light, dark and sugar on mitochondrial morphology
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Short period of
hypoxia (~45
min - 1h)
Ringed ± End Fusion
Ringed, Thin Middle Matrix Extended Elongate
Irregular Polygon,
Flattened ± Middle Matrix
Elongated Loop ± End Fusion
Flattened with Branching
Long period of
hypoxia (~
≥2h)
Giant Fusion
Figure I-2 Morphological effects of O2 depletion on mitochondria
Table I-1 Cytosolic sugar, light and oxygen effects on mitochondrial morphology
Condition
Effect on Morphology
Cytosolic Sugar Increase
fragmented
No Exogenous Sugar
enlarged, tubular
Light Exposure
fragmented
Dark Grown
enlarged to tubular
Hypoxia
tubular, ring shaped, giant
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APPENDIX II
MUTANTS AND TRANSGENIC LINES
Table II-1 Arabidopsis transgenics and mutants used and discussed in this thesis
FP-fusion(s) Expressed
Utilization for work in this thesis and potential follow-up work
any1 mito-GFP RER
GFP-β-ATPase
RFP-HDEL
Investigation of ER role in mitochondrial pleomorphy and fission in response to HL
stress
any1 mito-GFP Yperoxi
GFP-β-ATPase
YFP-PTS1
Investigation of mitochondria-peroxisome interactions and morphological correlations
in response to HL stress
any1 mito-GFP Yperoxi
RER
GFP-β-ATPase
YFP-PTS1
RFP-HDEL
Investigation of mitochondria, peroxisome, chloroplast and ER interactions during HL
stress
apm1-13 x YFIS1A
GFP-PTS1
pfis1a-YFP-FIS1A
Investigation of FIS1 localization and role in fission
BIGYIN p35S-Eos-FIS1A
mEosFP-FIS1A under
35S promoter
Investigation of mitochondria and peroxisome interactions and fission
BIGYIN pfis1a-EosFIS1A
mEosFP-FIS1A
under native promoter
Investigation of mitochondria and peroxisome interactions and fission
CX-GFP
Calnexin signal peptide
fused to GFP
To create a base vector whereby GFP could be C-terminally fused to a nucleotide
sequence; to create another expression probe for visualizing the ER
CX-YFP
Calnexin signal peptide
fused to YFP
To create a base vector whereby YFP could be C-terminally fused to a nucleotide
sequence; to create another expression probe for visualizing the ER
CX-RFP
Calnexin signal peptide
fused to YFP
To create a base vector whereby RFP could be C-terminally fused to a nucleotide
sequence; to create another expression probe for visualizing the ER
DAL1-Eos
DAL1-mEosFP
Investigation of fission machinery translocated from mitochondria to peroxisomes for
hierarchical fission events
DAL2-Eos
DAL2-mEosFP
Investigation of fission machinery translocated from mitochondria to peroxisomes for
hierarchical fission events
drp3a mito-GFP
GFP-β-ATPase
Investigation of mitochondrial pleomorphy during impaired fission
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Name
55
77
78
Name
FP-fusion(s) Expressed
Utilization for work in this thesis and potential follow-up work
drp3a RER
RFP-HDEL
Investigation of ER pleomorphy during impaired mitochondrial fission
drp3a Yperoxi
YFP-PTS1
Investigation of peroxisomal pleomorphy during impaired fission
elm1-1 RER
GFP-β-ATPase
RFP-HDEL
Investigation of ER-mitochondria interactions during impair mitochondrial fission
elm1-1 Yperoxi
GFP-β-ATPase
YFP-PTS1
Investigation of mitochondria-peroxisome interactions and pleomorphy
FIS1B-RFP
p35S-FIS1B-RFP
Investigation of mitochondria and peroxisome interactions and fission
mito-GFP RER
GFP-β-ATPase
RFP-HDEL
Investigation of ER role in mitochondrial pleomorphy and fission
mito-GFP Yperoxi
GFP-β-ATPase
YFP-PTS1
Investigation of mitochondria-peroxisome interactions and morphological correlations
mito-GFP Yperoxi RER
GFP-β-ATPase
YFP-PTS1
RFP-HDEL
Investigation of mitochondria, peroxisome, chloroplast and ER interactions
p35S-mEos-FIS1A
mEosFP-FIS1A under
35S promoter
Investigation of mitochondria and peroxisome interactions and fission
pah1 pha2 mito-GFP
GFP-β-ATPase
Investigation of effects of ER disruption on mitochondrial pleomorphy
pahl pah2 EosHDEL
mito-GFP
GFP-β-ATPase
mEosFP-HDEL
Investigation of effects of ER disruption on mitochondrial pleomorphy
pah1 pah2 RER mitoGFP
RFP-HDEL
GFP-β-ATPase
Investigation of effects of ER disruption on mitochondrial pleomorphy
pfis1a-mEos-FIS1A
mEosFP-FIS1A under
native promoter
Investigation of mitochondria and peroxisome interactions and fission
RFP-FIS1B
p35S-RFP-FIS1B
Investigation of mitochondria and peroxisome interactions and fission
VPS26A-Eos
VPS26A-mEosFP
Investigation of ER-mitochondrial MCS and interactions
VPS26A-RFP
VPS26A-RFP
Investigation of ER-mitochondrial MCS and interactions
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78
Name
FP-fusion(s) Expressed
Utilization for work in this thesis and potential follow-up work
VDAC-RFP
VDAC-RFP
Investigation of ER-mitochondrial MCS and interactions (ex. tethering)
YFIS1A x FNRGFP
pfis1a-YFP-FIS1A
FNRGP
Investigation of FIS1 localization
YFIS1A x mito-GFP
pfis1a-YFP-FIS1A
GFP-β-ATPase
Investigation of FIS1 localization and role in mitochondrial fission
YFIS1A x mCherryPTS1
pfis1a-YFP-FIS1A
mCherry-PTS1
Investigation of FIS1 localization and role in peroxisome fission
Table II-2 References for mutants and probes used
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Mutant/Probe
Reference
any1
Fujita et al., 2013
apm1-13
Mano et al., 2004
BIGYIN
Scott et al., 2006
elm1-1
Arimura et al., 2008
FIS1B
Lingard et al., 2008
mCherryPTS1
Ching et al., 2012
mEosFP
Mathur et al., 2010
mito-GFP
Logan and Leaver, 2000
pah1 pah2
Eastmond et al., 2010
pfis1a-YFP-FIS1A
Ruberti, 2014
RER
Sinclair et al., 2009
RFP
Shockey et al., 2006
Yperoxi
Mathur et al., 2002
55
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APPENDIX III
PRIMER INFORMATION
The following is a list of primers and plasmids created and discussed in thesis. Unless stated
otherwise, all vectors have a pCAMBIA 1300 backbone, expressed under the pCaMV35S and
terminated by the nos terminator.
Table III-1 Primer sequences
Plasmid
dal1-mEosFP
dal2-mEosFP
RFP (Ching et al., 2012)
in 620
pfis1a
mEosFP-fis1a
fis1b-RFP
mEosFP-fis1b
vps26A-RFP
pdrp3a
drp3a-GFP
vdac3-RFP
Primer ID
Forward SpeI JM311
Reverse BamHI JM312
Forward XhoI JM329
Reverse BamHI JM330
Forward BamHI JM345
Reverse SacI JM346
Forward HindIII JM456
Reverse XbaI JM457
Forward NaeI JM458
Reverse SpeI
Forward SalI JM339
Reverse BamHI JM340
Forward NaeI JM425
Reverse SpeI JM426
Forward XbaI JM409
Reverse BamHI JM410
Forward HindIII JM518
Reverse XbaI JM546
Forward NaeI JM520
Reverse SpeI JM547
Forward XbaI JM548
Reverse BamHI JM549
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Sequence (5’ to 3’)
GGACTAGTATGATTCCTTGGGGTGGAGTT
GGATCCGTGACGATATGTCTTAAGCGCGA
CTCGAGATGATACATTTGGCTGGATTTAC
CCGGATCCATGGCGGTAAATTTTCAAAAC
GGATCCATGGCCTCCTCCGAGGACGTCAT
TATGAGCTCTTAGGCGCCGGTGGAGTGGC
AAGCTTCACTACACGCTATCAATACA
TCTAGATGAAGGCGATTTTGAGCTTTG
ATATGCCGGCATGGATGCTAAGATCGGACA
GCGCACTAGTTCATTTCTTGCGAGACATCG
GTCGACATGGACGCGGCGATAGGGAAG
GGATCCCGACGTCGGTATAATGCGTCG
TTGCCGGCATGGACGCGGCGATAGGGAAG
CGGGACTAGTTTAGCTGCGTAATATGGC
GCTCTAGAATGAATTATCTTCTTGGAGC
GGATCCAGATGTCTCTTCCTTGAGCCTG
GAAGCTTTCACCTTCAAATGCGACTAT
GTCTAGACGTTGGATCGGATTTTGAAT
CGCCGGCATGACTATTGAAGAAGTTTC
GGACTAGTTTAGAATCCGTATCCATTTT
CCTCTAGAATGGTTAAAGGTCCAGGACTC
GGATCCGGGCTTGAGAGCGAGAGCAAT
APPENDIX IV
DESCRIPTIONS OF SUPPLEMENTAL MOVIES
The flowing is a list and description of the movies used in this thesis. All movies were
acquired with a scan time of 3.945s per frame and shown as 5 frames per second.
Movie 3.1 Mitochondrial elongation during water submergence
7 day old elm1-1 Mt-GFP RER plants grown in the light on MS medium with 3% sucrose
were submerged in water under a coverslip. Over the span of the video, just over 15min, small
spherical mitochondria continuously fused and became elongated, tubular structures.
Movie 3.2 Giant mitochondrial fusion
Plants expressing green-to-red photo-convertible mitoEos (mEosFP N-terminally fused to
the β-ATPase subunit) were submerged in water under a coverslip for over 1h until giant
mitochondria formed. One giant mitochondrion was UV irradiated for 30s, which photoconverted it from green to red. The other two were not photo-converted, and therefore remained
green. The red and green giant mitochondria moved together and fused. This is indicated by a
mixing of the green and red fluorophores within the matrix.
Movie 3.3 ER moulding a giant mitochondrion
A cell expressing Gmito and RER is shown. The ER around the mitochondria is flattened
in sheets or cisternae. The mitochondria are also expanded. As the ER rearranges, the
mitochondria on the left also shift into the space created by the surrounding ER. As the ER
narrows, a mitochondrion forms a tubule. When the ER dilates, the mitochondrion does as well,
which creates a beaded appearance.
Movie 3.4 ER-mediated mitochondrial fission
Elongated mitochondria are constricted by the ER. The ER is very dynamic, and
constantly rearranging. The mitochondria move along with the ER. The ER-constricted area
creates a point of weakness and as the mitochondria are pulled, fission occurs. The fragments are
pulled apart and disperse throughout the cell. GFP=mitochondria; RFP=ER.
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Movie 3.5 ER caging chloroplasts, mitochondria and peroxisomes
A triple transgenic plant expressing Gmito Yperoxi RER and chloroplast autofluorescence is shown. Some mitochondria and peroxisomes are seen moving quickly towards
the bottom left of the frame. However, mitochondria and peroxisomes found within ER cages
with chloroplasts are relatively immotile in comparison. The ER appears to contain the other
three organelles.
Consequently,
all
four
organelles
maintain
sustained
interactions.
GFP=mitochondria; YFP=peroxisomes; RFP=ER; red chlorophyll auto-fluorescence false
coloured blue=chloroplasts.
Movie 3.6 Sustained mitochondria-peroxisome interactions
A cluster of mitochondria, peroxisomes and a chloroplast moves together while
maintaining an interaction with each other. Mitochondria and peroxisomes outside of this cluster
move quickly in the direction of the cytoplasmic streaming without
interacting.
GFP=mitochondria; YFP=peroxisomes; blue=chloroplast.
Movie 3.7 Fragmented mitochondria clustering a peroxule
The peroxisomes have extended peroxules. The peroxisome body on the bottom right
maintains contact with the chloroplasts. Small, spherical mitochondria are clustered around the
peroxule. As the peroxule draws in towards the peroxisome body, the mitochondria cluster
moves with it. Note that mitochondria and peroxisomes to the left are moving rapidly without
interacting. GFP=mitochondria; YFP=peroxisomes; blue=chloroplasts.
Movie 3.8 Abundant peroxules in any1
This any1 cotyledon cell has abundant chloroplasts, small, spherical mitochondria and
peroxisomes with peroxules. The organelles are quite clustered and the peroxules are very long.
The peroxules give the impression that they are extending and retracting to grab hold of
something within the cytosol. GFP=mitochondria; YFP=peroxisomes; blue=chloroplasts.
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