1
Running head: In vivo respiration in CEF-PSI mutants 1
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3
4
Corresponding author: 5
Miquel Ribas-Carbo 6
Universitat de les Illes Balears 7
Crta. Valldemossa Km 7.5 8
07122 Palma de Mallorca 9
Spain 10
Tel: +34 971173168 11
Fax: +34 971173184 12
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15
16
Focus Issue on Metabolism 17
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Plant Physiology Preview. Published on October 19, 2016, as DOI:10.1104/pp.16.01025
Copyright 2016 by the American Society of Plant Biologists
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Impaired cyclic electron flow around Photosystem I disturbs high-light respiratory 19
metabolism1 20
Igor Florez-Sarasa2, Ko Noguchi2, Wagner L. Araújo, Ana Garcia-Nogales, Alisdair R. 21
Fernie, Jaume Flexas, Miquel Ribas-Carbo* 22
23
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 24
Potsdam-Golm, Germany (I.F.-S, A.R.F.); School of Life Sciences, Tokyo University of 25
Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan (K.N.); Grup de Recerca 26
en Biologia de les Plantes en Condicions Mediterranies, Departament de Biologia, 27
Universitat de les Illes Balears, Carretera de Valldemossa Km 7.5, 07122 Palma de 28
Mallorca, Spain (J.F., M.R.-C.); Max-Planck Partner Group at the Departamento de 29
Biologia Vegetal, Universidade Federal de Viçosa, Viçosa, Minas Gerais 36570-000, 30
Brazil (W.L.A.); Área de Ecología, Dpto. Sistemas Físicos, Químicos y Naturales, 31
Universidad Pablo de Olavide, Cta. de Utrera km 1, 41013 Sevilla, Spain (A.G.-N.) 32
33
Author contributions 34
I.F-S., K.N. and M. R-C conceived the research plan and designed the experiments. I.F-35
S. and K.N. performed all the respiration measurements. I.F-S., K.N and A. G-N. 36
performed all the photosynthesis measurements and sampling for protein and metabolic 37
analyses. M.R-C. and J.F. supervised the respiration and photosynthesis experiments 38
and discussed the data. W.L.A. performed the GC-MS metabolite profiling analysis and 39
data generated was analyzed by I.F.S together with A.R.F. Western blot and pyridine 40
nucleotide analyses were performed by I.F.S. I.F-S. and K.N. wrote the manuscript. 41
A.R.F., J.F. and M.R-C assisted with the writing of the manuscript. All authors read and 42
approved the final manuscript after critical revision. 43
44
One sentence summary: 45
The cytochrome oxidase pathway mediates photorespiratory glycine oxidation and 46
amino acid synthesis under severe light stress, thus reducing photoinhibition when 47
alternative oxidase is not induced. 48
49
50
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Footnotes: 51
52 1This work was supported by funding the Spanish Ministry of Science and Innovation 53
(MICINN) project BFU2008-1072/BFI and BFU2011-23294 (J.F., M.R.-C., I.F.-S.), the 54
Max Planck Society (A.R.F. and W.L.A.) and the Alexander von Humboldt Foundation 55
(I.F.-S.). 56 2These authors contributed equally to this work. 57
58
*Author to whom correspondence should be sent: Email: [email protected] 59
60
The author(s) responsible for distribution of materials integral to the findings presented 61
in this article in accordance with the policy described in the Instructions for Authors 62
(www.plantphysiol.org) is: Miquel Ribas-Carbo (e-mail: [email protected]) 63
64
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ABSTRACT 65
The cyclic electron flow around photosystem I (CEF-PSI) increases ATP/NADPH 66
production in the chloroplast acting as an energy balance mechanism. Higher export of 67
reducing power from the chloroplast in CEF-PSI mutants has been correlated with 68
higher mitochondrial alternative oxidase (AOX) capacity and protein amount under high 69
light (HL) conditions. However, in vivo measurements of AOX activity are still required 70
to confirm the exact role of AOX on dissipating the excess of reductant power from the 71
chloroplast. Here, CEF-PSI single and double mutants were exposed to short-term HL 72
conditions. Chlorophyll fluorescence, in vivo activities of the cytochrome oxidase (νcyt) 73
and AOX (νalt) pathways, levels of mitochondrial proteins, metabolite profiles and 74
pyridine nucleotides levels were determined under normal growth and HL conditions. 75
νalt was not increased in CEF-PSI mutants while AOX capacity was positively 76
correlated with photoinhibition, probably due to a ROS-induced increase of AOX 77
protein. The severe metabolic impairment observed in CEF-PSI mutants, as indicated by 78
increase in photoinhibition and changes in the levels of stress-related metabolites, can 79
explain their lack of νalt induction. By contrast, νcyt was positively correlated with 80
photosynthetic performance. Correlations with metabolite changes suggest that νcyt is 81
coordinated with sugar metabolism and stress-related amino acid synthesis. 82
Furthermore, changes in glycine/serine and NADH/NAD+ ratios were highly correlated 83
to νcyt. Taken together, our results suggest that νcyt can act as a sink for the excess of 84
electrons from the chloroplast probably via photorespiratory glycine oxidation thus 85
improving photosynthetic performance when νalt is not induced under severe HL-stress. 86
87
88
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INTRODUCTION 89
Respiration in leaf tissues is influenced by their photosynthetic metabolism while 90
respiratory metabolism affects photosynthesis. This forward-reverse interaction between 91
photosynthesis and respiration has received a great attention during the last decades 92
(Krömer, 1995; Atkin et al., 2000; Raghavendra and Padmasree, 2003; Flexas et al., 93
2006; Yoshida and Noguchi, 2010; Araujo et al., 2014a; Obata et al., 2016). Essentially, 94
while photosynthesis provides carbohydrates for glycolysis, mitochondrial metabolism 95
in illuminated leaves supports photosynthesis, photorespiration, nitrogen metabolism 96
and export of redox equivalents (Yoshida and Noguchi, 2010). Among different redox 97
shuttles, the malate valve operates between chloroplasts, cytosol and mitochondria 98
(Scheibe, 2005) thus allowing the oxidation of chloroplast reductants by the 99
mitochondrial electron transport chain (mETC) (Yoshida and Noguchi, 2010). 100
Particularly under high light (HL) conditions, the alternative components of the mETC 101
have been suggested to accomplish non-phosphorylating oxidation of the excess of 102
reductants from the chloroplast (Yoshida and Noguchi, 2010). 103
104
The plant mETC is highly branched as compared to mitochondria from other eukaryotic 105
cells given that it contains different alternative components that bypass the main 106
complexes of the oxidative phosphorylation pathway- the cytochrome c oxidase (COX) 107
pathway (Rasmusson et al., 2008). Among these energy-bypass systems, the mETC is 108
branched at the ubiquinone (UQ) pool from which the electrons of ubiquinol are used 109
by the alternative oxidase (AOX) to reduce oxygen to water without proton 110
translocation (Moore & Siedow, 1991). In this way, AOX activity can bypass complex 111
III and COX (Complex IV) greatly reducing the efficiency of ATP synthesis by the 112
mETC. Several years ago, it was shown that the AOX pathway competes with the COX 113
pathway for the electrons of the UQ pool (Hoefnagel et al., 1995; Ribas-Carbo et al., 114
1995). These findings imply that the actual in vivo electron partitioning between the 115
COX and AOX pathways can be determined by oxygen isotope fractionation during 116
respiration but not using chemical inhibitors of the two pathways (Day et al., 1996; 117
Ribas-Carbo et al., 2005). While several theories have been formulated about the role of 118
AOX in plants (Gupta et al., 2009; Rasmusson et al., 2009; Van Aken et al., 2009; 119
Vanlerberghe, 2013), there is still little confirmation of such roles by measuring AOX 120
activity in vivo as determined by oxygen isotope fractionation (Yoshida and Noguchi, 121
2010; Vanlerberghe, 2013). 122
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123
Transgenic approaches altering mitochondrial metabolism have provoked different 124
effects on photosynthesis in plants with suppressed tricarboxylic acid (TCA) cycle 125
enzymes (reviewed in Nunes-Nesi et al., 2011; Araujo et al., 2014a) or mETC 126
components (Dutilleul et al., 2003; Sweetlove et al., 2006; Galle et al., 2010; Florez-127
Sarasa et al., 2011; Dahal et al., 2014). Genetic mutations in the main components of 128
the respiratory chain such as Complex I have drastic effects on photosynthesis and 129
photorespiration (Dutilleul et al., 2003; Priault et al., 2006; Galle et al., 2010) and 130
changes on the in vivo electron partitioning were also observed under drought stress and 131
cell death (Galle et al., 2010; Vidal et al., 2007). In respect to the alternative 132
components of the mETC, uncoupling protein (UCP) mutants in Arabidopsis have 133
shown altered photosynthesis due to a limitation on photorespiration (Sweetlove et al., 134
2006). Regarding the AOX, an important role in optimizing photosynthesis has been 135
reported in both Arabidopsis thaliana and tobacco (Yoshida et al., 2011; Florez-Sarasa 136
et al., 2011; Gandin et al., 2012; Dahal et al., 2014), including one study of the AOX 137
activity in vivo under HL conditions (Florez-Sarasa et al., 2011). Using AOX transgenic 138
plants, as an approach to determine its in vivo role, it has recently been confirmed that 139
AOX is involved in preserving photosynthetic capacity under drought stress by reducing 140
chloroplast overreduction and photodamage (Dahal et al., 2014; 2015). 141
142
As another approach to unravel the role of AOX in photosynthesis, respiratory 143
properties have been examined in photosynthetic mutants including the cyclic electron 144
flow around photosystem I (CEF-PSI) mutants and the FtsH2 metalloprotease required 145
for the repair of damaged photosystem II (PSII) (Yoshida et al. 2007; 2008). In the 146
study of Yoshida et al. (2007), the authors reported a correlation between higher export 147
of excess reductant power from the chloroplast with higher AOX capacity and protein 148
amount in A. thaliana mutants with altered CEF-PSI following HL treatment. These 149
mutants showed higher AOX capacity and protein amount even under low light 150
conditions. Importantly, a general lack of relationship between the AOX protein content 151
and its in vivo activity has been reported in HL stress experiments using A. thaliana 152
plants (Florez-Sarasa et al., 2011). Therefore, AOX in vivo activity measurements are 153
crucial for elucidating the possible role of AOX on dissipating the excess of reductant 154
power from the chloroplast. 155
156
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Since various primary metabolites are involved in interactions between chloroplasts and 157
mitochondria such as photorespiration and reductant transport, levels of primary 158
metabolites may be changed following deficiencies in photosynthesis or respiration and 159
by environmental perturbations. We have previously demonstrated that a large number 160
of metabolites increased in leaves of A. thaliana growing in or transiently exposed to 161
HL (Florez-Sarasa et al., 2012). The AOX1a deficiency in A. thaliana altered the levels 162
of certain metabolites, such as sugars and sugar phosphates, in the shoots under low 163
nitrogen stress (Watanabe et al., 2010). However, changes in primary metabolite levels 164
have not yet been related with in vivo partitioning between COX and AOX pathways in 165
photosynthetic (i.e. CEF-PSI) mutants. 166
167
Here, we investigated the effects of HL treatment on the in vivo activities of COX and 168
AOX pathways in leaves of A. thaliana mutants defective in CEF-PSI. Our hypothesis 169
is that CEF-PSI mutants will present higher in vivo flux through AOX for dissipating 170
the excess of reducing power from the chloroplast under HL conditions. We also 171
examined photosynthetic properties using chlorophyll fluorescence and determined 172
mitochondrial proteins and primary metabolite levels, in an attempt to clarify the 173
underlying mechanisms regulating the in vivo activities of COX and AOX pathways 174
under HL. 175
176
RESULTS 177
178
Chlorophyll fluorescence parameters under growth light (GL) and after high light 179
(HL) treatment 180
Under GL conditions, photochemical (qP) and non-photochemical (qN, NPQ) 181
quenching were similar in wild-type (gl1) plants and single mutants (pgr5 and crr4-3) 182
(Fig. 2). qN and NPQ were also unaltered in double mutants (pgr5 crr4-3) compared to 183
the other lines (Fig. 2B and 2D) while qP was clearly lower in this genotype (Fig. 2A). 184
The quantum efficiency of PSII (ΦPSII) and the electron transport rate (ETR) were 185
similar in gl1, crr4-3 and pgr5 while both were lower in the pgr5 crr4-3 double mutants 186
(Fig. 2C and 2E). In addition, maximum efficiency of PSII (Fv/Fm) was also lower in 187
double mutants compared to the other three lines (Fig. 2F) and, in agreement, the 188
percentages of total (% TPI) and chronic (% CPI) photoinhibition in the double mutants 189
were significantly higher than in the other lines (Table I). 190
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191
Under HL conditions, qP was similarly decreased in all lines while NPQ and qN were 192
increased (Fig. 2A, 2B and 2D). During HL treatment, qN was significantly decreased 193
only in double mutants (Fig. 2B and 2D). NPQ was slightly but significantly reduced in 194
pgr5 and crr4-3 at 8h of HL treatment while it was much more reduced in double 195
mutants already at 4h of HL (Fig. 2B and 2D). ΦPSII and ETR were generally lower in 196
both pgr5 and double mutant compared to gl1 and crr4-3 (Fig. 2C and 2E). The ETR in 197
both gl1 and crr4-3 increased after 2h of HL and then decrease afterwards; this initial 198
increase was lacking in pgr5 and double mutant plant (Fig. 2E). On the other hand, 199
Fv/Fm was decreased in all lines after HL treatment (Fig. 2F). However, while Fv/Fm 200
displayed a similar decrease in gl1 and crr4-3, it was more reduced in pgr5 and the 201
double mutant, the later showing the lowest values after HL treatment (Fig 2F). Finally, 202
total % TPI was significantly higher in pgr5 and double mutants than in gl1 and crr4-3, 203
mainly due to higher % CPI (Table I). Among all the genotypes, double mutants 204
exhibited the highest values of % TPI and % CPI (Table I). Following the HL-205
induction, the % CPI was kept constant in all genotypes with the exception of the 206
double mutants in which it was further increased at 4h of HL (Table I). Finally, the 207
percentage of dynamic photoinhibition (% DPI) presented a similar pattern as qN and 208
NPQ in all genotypes. Thus, %DPI was increased and kept high along the HL treatment 209
in gl1 and crr4-3 plants; whereas it was further increased in pgr5 and double mutants 210
after 2h of HL but then decreased at 4h and 8h of HL (Table I). 211
212
In vivo activities of the COX and AOX pathways under GL and after HL 213
treatment 214
The rates of respiration in Figure 3 are presented on a dry weight (DW) basis due to the 215
differences in the specific leaf weight (leaf DW/Area) observed between lines (data not 216
shown). Under GL conditions, total respiration (Vt) as well as in vivo cytochrome 217
oxidase (νcyt) and alternative oxidase (νalt) pathway activities were similar in all four 218
genotypes (Fig. 3). However, the alternative oxidase capacity (Valt), which is a proxy of 219
the AOX protein content, was two-fold higher in double mutants compared to the other 220
lines (Fig. 3D). 221
222
Under HL, Vt significantly increased in all genotypes peaking at 2h and then decreased 223
towards the 8h after HL stress. However, the pgr5 and double mutants presented a 224
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lower HL-induction of Vt than gl1 and crr4-3 plants (Fig. 3A). Notably, the double 225
mutants were the less responsive genotype thus reaching similar Vt rates at 8h as in GL 226
conditions (Fig. 3A). The pattern of HL-induction of νcyt was similar to that of Vt (Fig. 227
3B). On the other hand, νalt was almost unchanged in all lines, with the exception of a 228
significant increase only in gl1 after 2h of high light treatment (Fig. 3C). However, Valt 229
exhibited a general induction pattern with the exception of the crr4-3 plants that kept 230
constant rates until 8h of HL treatment. Valt was significantly increased only in pgr5 231
(1.5 fold-increase) and double mutants (1.3 fold-increase) after 4 and 8h of HL 232
treatment (Fig. 3D), with the double mutants always exhibiting the highest Valt (Fig. 233
3D). 234
235
Relationships between photosynthetic parameters and in vivo respiratory activities 236
after HL treatment 237
The photosynthetic parameters obtained from chlorophyll fluorescence measurements 238
were correlated to the respiratory parameters determined by oxygen isotope 239
fractionation during respiration across genotypes under HL conditions (Table II, 240
Supplemental Fig. 1). Vt was positively and significantly correlated with the ETR, qN, 241
NPQ and Fv/Fm indicating both a direct link between respiration and photosynthetic 242
electron transport chain activity as well as its degree of photoinhibition under HL 243
conditions. Indeed, the % TPI was negatively and significantly correlated to Vt, with the 244
% CPI being the main contributor to the negative correlation to photoinhibition. It was 245
clearly observed that νcyt, but not νalt, was linked to the chloroplast electron transport 246
chain activity and its degree of photoinhibition under HL conditions (Table II). On the 247
other hand, almost all of the photosynthetic parameters significantly correlated with Valt. 248
Notably, the correlations of the photosynthetic parameters with Valt were in the opposite 249
direction to those observed with Vt and νcyt, thus being negative to photosynthetic 250
activity (i.e. ETR) and positive to photoinhibition (% TPI and % CPI). 251
252
Mitochondrial electron transport (mETC) chain protein levels under GL and after 253
HL treatment 254
Different mETC proteins were immunodetected by western blot analyses in whole leaf 255
extracts, including AOX, cytochrome oxidase subunit II (COX), and uncoupling protein 256
(UCP). In addition, porin levels were also determined as a control for mitochondrial 257
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protein loading, and remain similar in all the genotypes and light conditions (Fig. 4 and 258
Supplemental Fig. 2). Band intensities were quantified for all the proteins, and AOX, 259
COXII and UCP protein levels were obtained after correction by the corresponding 260
porin band intensities and then normalized to the levels of the gl1 plants under GL (i.e. 261
gl1 levels for all the proteins are 100%). Two independent experiments were performed 262
(see Materials and Methods section for further details) and the average of the two 263
relative quantifications is presented together with the image of the most representative 264
blot (Fig. 4). The levels of AOX protein were approximately two-fold higher in double 265
mutants than in the other genotypes under GL conditions (Fig. 4), which coincides with 266
the highest AOX capacity observed in this plants (Fig. 3D). By contrast, COX protein 267
levels were lower in the double mutants and UCP levels were similar in all genotypes 268
(Fig. 4). Following HL treatment, the levels of UCP and COX remain relatively 269
constant in all the genotypes as compared to their respective levels under GL, with the 270
exception of COX decreases observed after 8h of HL treatment in gl1 and crr4-3 plants 271
(Fig. 4). On the other hand, the levels of AOX protein were increased in all genotypes 272
after HL treatment, the crr4-3 presenting the lowest increases. After 8h of HL treatment, 273
pgr5 and double mutants presented the highest AOX levels i.e.-an approximately 3-fold 274
increase (Fig. 4). 275
276
Metabolite profiling and pyridine nucleotide levels under GL and after HL 277
treatment 278
A total of 49 metabolites were identified by using gas chromatography-mass 279
spectrometry (GC-MS) including several amino acids, organic acids, some sugars and 280
sugar alcohols (Fig. 5 and Supplemental Table SI). Mutant plants display significant 281
differences in the relative levels of some metabolites under GL conditions as compared 282
to the wild-type (gl1) plants (Fig. 5 and Supplemental Table SI). In all mutant lines, the 283
levels of aspartate, glutamate, and malate were significantly lower as compared to gl1 284
plants while glycerate was significantly higher. In pgr5 plants, the levels of arginine, 285
serine, GABA, fumarate, 2-oxo-butyrate and shikimate were significantly lower than in 286
gl1. On the other hand, the levels of ß-alanine, glycine, ornithine, spermidine, serine, 287
tryptophan, glucose, and glyceraldehyde-3-phosphate were significantly higher in crr4-288
3 than in gl1 plants, while the levels of arginine, asparagine, threonine, 2-oxoglutarate, 289
shikimate and myo-inositol were lower. Finally, the double mutants displayed higher 290
levels of beta-alanine, isoleucine, phenylalanine, 4-hydroxy-proline, serine, tryptophan, 291
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tyrosine, citrate, isocitrate, dehydroascorbate, galactonate and lactate. Among the above 292
mentioned metabolites significantly affected in all the mutant plants, only few 293
metabolites were changed by more than two-fold. Asparagine and glutamate were 0.5- 294
and 0.4-fold lower in crr4-3, whereas glycerate and glycine were 2.5- and 3.3-fold 295
higher than gl1, respectively. Tyrosine and tryptophan levels were 2.3- and 8.4-fold 296
higher in double mutants than in gl1. 297
298
After HL treatment, most of the metabolites were increased except for some organic 299
acids showing significant decreases such as ascorbate, glycerate and shikimate (Fig. 5 300
and Supplemental Table SI). Some metabolites displayed dynamic responses over time 301
after HL treatment (i.e. first increased and then decreased or vice versa) such as 302
mannitol, glucose, galactose, succinate, shikimate and lysine. Nevertheless, several 303
metabolites presented a genotype-specific response to the HL treatment such as glycine, 304
ascorbate among others. These metabolite changes are described below according to 305
their compound class. 306
307
Amino acids showed the highest increases after HL treatment with the exception of 308
cysteine, GABA, glutamine, isoleucine, leucine and putrescine which remained largely 309
unchanged. Glycine was the metabolite exhibiting the highest HL-induction, being 310
approximately 13-fold increase in gl1 and crr4-3 at 8h of HL. However, glycine levels 311
were increased approximately five-fold in pgr5 mutant and it was marginally increased 312
by less than two-fold in the double mutants after HL treatment. Proline was the second 313
most induced metabolite under HL, thus being progressively and similarly increased in 314
all genotypes until 8h of HL. Tyrosine levels increased after HL treatment in all 315
genotypes, and notably double mutants showed the highest levels after 8h of HL 316
(approximately 10-fold). Several other amino acids were showing about two-fold 317
increases after HL treatment. Alanine, ß-alanine and threonine were significantly and 318
continuously increased after HL treatment in all genotypes. Arginine, homoserine and 319
methionine were also significantly increased in all genotypes but to a lesser extent in 320
pgr5 and double mutants after 8h of HL. Glutamate was significantly increased in all 321
genotypes but to a lesser extent in gl1; aspartate showed a similar although less 322
pronounced response to glutamate. Valine was increased after 2h of HL and then 323
maintained at high levels in all genotypes. A similar trend was observed in 324
phenylalanine HL-response in general, although pgr5 and double mutants presented 325
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lower increases. Lysine levels increased after 2h of HL treatment and subsequently 326
decreased, this pattern being observed in all genotypes although the induction being less 327
pronounced in the double mutants. Asparagine displayed a similar pattern as lysine 328
except in gl1 plants. Finally, 4-hydroxy-proline, serine, spermidine and tryptophan 329
levels significantly increased after HL treatment in all genotypes but generally by less 330
than 2-fold. Ornithine level significantly increased after 8h HL only in gl1 and crr4-3 331
lines. 332
333
In contrast to the HL-induced changes in amino acids levels, organic acids displayed 334
some significantly and pronounced decreases, whereas increases of this compound class 335
were few. For instance, malate and 2-oxoglutarate levels were continuously increased 336
after HL treatment in all genotypes although the response of 2-oxoglutarate was less 337
pronounced in pgr5 and double mutants. Similarly, dehydroascorbate was increasing 338
over time in all genotypes but the response was much lower in the double mutants. 339
Also, citrate and fumarate levels presented slight but significant increases after HL in all 340
lines and the levels in the double mutants again displayed a milder, if any. Succinate 341
levels increased after 2h and then decreased towards the 8h of HL treatment; this pattern 342
was observed in all genotypes but the induction was less pronounced in the double 343
mutants. An inverse pattern was observed for shikimate for which the levels decreased 344
at 2h after HL in all genotypes but then increased at 4 and 8h after HL. The ascorbate 345
level was greatly and continuously decreased in gl1 and crr4-3 following HL treatment. 346
In pgr5 mutants, significant decreases in ascorbate were only observed after 4 and 8h 347
while a significant decrease was observed in double mutants only after 8h of HL. 348
Glycerate also significantly decreased to similar levels in all genotypes after HL 349
treatment, although the levels in crr4-3 were much higher under GL conditions. As 350
reported above, the levels of galactonate, isocitrate and lactate were significantly higher 351
in the double mutants in all conditions, and these metabolites were not generally altered 352
after HL treatment. Pyruvate levels remained mostly unchanged after HL treatment in 353
all the lines and the same was observed for 2-oxo-butyrate and benzoate except for 354
slight increases in the double mutant after 8h of HL. 355
356
With respect to the sugars and sugar alcohols, galactose, glucose and mannitol levels 357
peaked after 2h and then decrease towards the 8h of HL treatment in all genotypes and 358
again double mutants display a lower HL-response. Erythritol was also increased 359
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although to a lesser extent and mostly at the end of the HL treatment in all genotypes. A 360
minor increase was also observed in glyceraldehyde-3-P levels but only apparent in gl1 361
plants. On the other hand, fructose and sucrose were not altered after HL treatment in 362
any genotype, as well as myo-inositol that was generally lower in crr4-3. 363
364
In addition to single metabolite levels, relevant redox-related ratios were calculated (for 365
details see Material and Methods section) and their HL-response was compared among 366
genotypes. The Glycine/Serine ratio was increasing over time thus reaching nine-fold 367
and ten-fold increase at 8h of HL in gl1 and crr4-3, respectively, while pgr5 showed a 368
reduced response (i.e. reaching a maximum of four-fold increase). More severely 369
dampened, double mutants exhibited a lack of HL-induction on glycine/serine ratio. The 370
malate/aspartate ratio was increased after HL in all the genotypes mostly after 8h of HL. 371
The glutamate/2-oxoglutarate ratio was much higher in gl1 under GL conditions as 372
compared to the other genotypes. The glutamate/2-oxoglutarate ratio decreased after 2h 373
HL treatment in gl1 and crr4-3 but not in pgr5 and double mutants. Finally, the 2-374
oxuglutarate/citrate ratio showed generally increases after HL treatment in all genotypes 375
that were less pronounced in pgr5 and double mutants. 376
377
In order to further investigate on the redox changes in the CEF-PSI mutants, we 378
measured the levels of NAD+, NADH, NADP+ and NADPH, and calculated the 379
corresponding redox ratios (Supplemental Fig. S3) in leaf samples of all genotypes and 380
light conditions. Generally, no statistical differences on pyridine nucleotide levels were 381
observed among genotypes when compared in each light condition. Nonetheless, some 382
trends were observed after HL treatment; the levels of NAD+ and NADH tended to 383
increase in all genotypes after HL treatment (Supplemental Fig. S3). After 8h of HL, gl1 384
showed the highest levels of NAD+ as compared to all other genotypes and conditions, 385
while double mutants displayed the highest levels of NADH. As for the NADH, the 386
highest levels of NADPH we detected in double mutants after 8h of HL. With regard to 387
the redox-related ratios, gl1 plants generally displayed the lowest NADH/NAD+ and 388
NADPH/NADP+ ratios while double mutants displayed the highest (Supplemental Fig. 389
S3). 390
391
Relationships between metabolite levels and respiratory activities after HL 392
treatment 393
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In order to obtain insights into the metabolic regulation or metabolic processes involved 394
in the different respiratory response to HL observed in the CEF around PSI mutants, 395
Pearson correlation coefficients were calculated between the fold-changes of metabolite 396
levels and those of respiratory activities parameters across all four genotypes. 397
Respiratory data were normalized to the mean of gl1 plants under GL conditions as was 398
done for the relative metabolite levels calculations, thus allowing 15 point correlations 399
using the means for each genotype and light treatment. Positive (red) and negative 400
(blue) significant values (P<0.05) of the r-Pearson coefficients are shown in Figure 6. 401
All correlation plots derived from significant (P<0.05) correlations are shown in 402
Supplemental Figure 4. 403
404
Changes in several amino acids, as affected by the genotype and the HL treatment, 405
correlated positively and significantly with changes in total respiration rates (Vt), 406
including glycine, lysine, alanine, phenylalanine, homoserine, methionine, and valine. 407
Only isoleucine changes were negatively correlated to Vt. Regarding organic acids, 408
TCA cycle intermediates such as fumarate, 2-oxoglutarate, succinate and pyruvate were 409
also positively correlated to Vt, while glycerate was negatively correlated to it. On the 410
other hand, sugars such as glucose, galactose, sucrose and the sugar alcohol mannitol 411
were positively correlated to Vt. With regard to redox ratios, glycine/serine and to a 412
lesser extent 2-oxoglutarate/citrate ratios were positively correlated to Vt. In addition, 413
only NADH/NAD+ ratios were significantly and negatively correlated with in vivo 414
respiratory activities. 415
416
Notably, most of the observed significant correlations to Vt were mainly driven by νcyt 417
which in addition was showing positive correlation to arginine, threonine and malate, 418
and negative to ascorbate and benzoate. On the other hand, changes in νalt were 419
presenting only positive correlation to mannitol and negative correlations to glutamine 420
and leucine. Among all the correlations presented in Figure 6, glucose and glycine/ 421
serine ratio with Vt and νcyt were showing the highest Pearson coefficient. 422
423
DISCUSSION 424
The high light (HL) treatment caused a severe stress response in pgr5 and double 425
mutants 426
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15
The photosynthetic response of the wild-type (gl1) and the crr4-3 single mutant plants 427
after the HL treatment was generally similar while a severely impaired photosynthetic 428
response was observed in pgr5 and double mutants. The pgr5 mutant is deficient in the 429
ferredoxin (Fd) -dependent CEF-PSI pathway (Munekage et al., 2002), while crr4-3 is 430
deficient in the NAD(P)H dehydrogenase (NDH)-dependent pathway (Kotera et al., 431
2005). While the Fd-dependent CEF-PSI is thought to be the predominant pathway in 432
C3 photosynthesis, the NDH complex may act as a safety valve that prevents over-433
reduction of the stroma (Shikanai, 2007). Although the quantum efficiency of PSII 434
(ΦPSII) and photochemical quenching (qP) were greatly decreased in wild-type (gl1) 435
plants and the crr4-3 after 2h of HL treatment, both genotypes presented an enhanced 436
electron transport rate after 2h of HL that was decreased towards the end of the light 437
treatment. The NPQ and qN values were also up-regulated after 2h of HL in all 438
genotypes and this up-regulation was maintained during the HL treatment except for the 439
double mutant. In prg5 crr4-3 at HL, both the linear electron flow and CEF-PSI were 440
suppressed (Shikanai, 2007). Thus the proton gradient across the thylakoid membrane 441
(ΔpH) may be more gradual in the double mutant, and thereby the NPQ and qN values 442
were decreased. This HL-inhibition of photosynthetic activity was related to the high 443
level of photoinhibition denoted by the low Fv/Fm levels (Fig. 2) and increasing levels 444
of calculated photoinhibition (Table I). Indeed, the percentage of total photoinhibition 445
(% TPI) observed in wild-type (gl1) was higher, around 40% (Table I), as compared to 446
previously reported % TPI, i.e. less than 30%, in Arabidopsis Col-0 plants under very 447
similar HL conditions (Florez-Sarasa et al., 2011). We used Arabidopsis gl1 (glabra1) 448
that is deficient in trichome of leaves (Oppenheimer et al., 1991) and plants on the 449
different developmental stage. Since trichomes can attenuate strong light (Steyn et al., 450
2002), this may explain the severity of the stress response observed in this study. In 451
particular, the pgr5 and double mutant (pgr5 crr4-3) were characterized by a very high 452
percentage of chronic photoinhibition (% CPI) after the HL treatment (Table I). A high 453
% CPI is associated to damage of the PSII (Osmond, 1994; Demming-Adams et al., 454
2012) mostly due to increased ROS production at the chloroplast electron transport 455
chain (Pinto-Marijuan and Munné-Bosch, 2014). In this respect, the observed decreases 456
in ascorbate levels and increases dehydroascorbate levels after HL stress (Fig. 4) denote 457
an important ROS-induced antioxidant response, as compared to the previously reported 458
and minor HL-induced changes of these metabolites in Arabidopsis Col-0 plants 459
(Florez-Sarasa et al., 2012). In addition, proline accumulated at very high levels in all 460
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16
genotypes, particularly after 8h (Fig. 4). Proline is involved in ROS-detoxification 461
under many abiotic stresses (Szabados and Savouré, 2010), and notably 4-hydroxy-462
proline levels, a product of proline oxidation, was higher in the double mutants than in 463
the other genotypes. In addition, other stress-related metabolites such as ß-alanine (Stiti 464
et al., 2011) and tyrosine (Yoo et al., 2013) were higher in the double mutants at growth 465
light (GL) and further accumulated after HL, as compared to the other genotypes. 466
Particularly, the most increased metabolite in double mutants was tryptophan, which is 467
a precursor for the synthesis of secondary metabolites such as phytoalexins, 468
glucosinolates and alkaloids, and also of the hormone auxin (Radwanski and Last, 1995; 469
Zhao, 2012). The reduced growth, previously observed in Munekage et al. (2004) and 470
Yamamoto et al. (2011), and stress-related phenotype of the double mutants may be 471
related to the tryptophan accumulation although further research will be needed to 472
unravel the nature of this metabolic alteration. 473
474
The AOX pathway activity in vivo was not higher in CEF-PSI mutants thus 475
partially refuting the initial hypothesis 476
The AOX is hypothesized to be activated in order to dissipate the excess of reducing 477
equivalents under HL stress (Yoshida et al., 2007). Therefore, a higher in vivo AOX 478
activity (νalt) was expected in pgr5 and double mutants due to an even higher excess of 479
reductants in the chloroplast than gl1 and crr4-3 plants. Surprisingly, νalt in the pgr5 and 480
double mutant was not significantly changed after HL treatment, while it was 481
significantly increased in gl1 plants after 2h of HL as previously reported in 482
Arabidopsis Col-0 plants (Florez-Sarasa et al., 2011; 2012). By contrast, the AOX 483
capacity was more HL-induced in pgr5 after than in gl1 plants as previously reported 484
(Yoshida et al., 2007). More importantly, double mutants presented the highest AOX 485
capacity (Valt) even under GL conditions, and it was also significantly increased after 486
HL. Furthermore, Valt presented the highest positive correlation to % CPI, while 487
negative correlations with photosynthetic activity, i.e. ETR (Table II), were observed. 488
These results suggest that the production of ROS associated to the high %CPI 489
(Demmig-Adams et al., 2012) probably increased the ROS-mediated signal that induces 490
the AOX expression (Rhoads et al., 2006) and as a consequence its capacity. Indeed, the 491
levels of AOX protein were higher in double mutants under GL conditions and were 492
further induced after HL treatment (Fig. 4), thus following very similar patterns as Valt 493
(Fig. 3D). On the other hand, the slightly better photosynthetic performance (i.e. higher 494
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17
ETR, NPQ, and lower % CPI), as compared to gl1, observed in crr4-3 mutants may 495
partially explain their lack of Valt response following HL treatment. However the nature 496
of the absence of such a response of Valt in crr4-3 remains unclear. That said it appears 497
reasonable to assume that the milder HL-response of the AOX protein levels is the 498
limiting factor of the Valt and νalt response in crr4-3 mutants. The ROS-mediated 499
signaling pathway that induces AOX expression is not fully understood yet (Liu et al., 500
2013). Notably, the AOX was mostly induced in green but not in white areas from 501
leaves of yellow variegated mutants exposed to HL stress (Yoshida et al., 2008) thus 502
denoting that chloroplast photooxidative stress was influencing AOX protein synthesis. 503
The AOX expression and capacity was previously well correlated in CEF-PSI mutants 504
under HL stress (Yoshida et al., 2007), although this does not indicate the in vivo 505
activity (Florez-Sarasa et al., 2011). In this respect, data presented in Figures 3 and 4 are 506
in the line of previous observations, thus confirming that induction of both AOX 507
capacity and protein levels is not always associated to an increase on in vivo AOX 508
activity (i.e.- as in the double mutants). Indeed, it has been demonstrated that AOX is 509
inhibited by lipid peroxidation products (Winger et al., 2005) which are likely to be 510
produced under the photooxidative stress conditions applied in this study. 511
512
In vivo COX instead of AOX activity mediated the mitochondrial response to 513
severe HL stress and was highly coordinated with photosynthetic performance 514
As mentioned above, the νalt was not increased in double mutants under severe HL 515
stress conditions probably due to a ROS-inactivation of the AOX. Moreover, the νalt 516
was also decreasing in gl1 plants from its levels at 2h towards 4h and 8h of HL (Fig. 3). 517
On the other hand, COX pathway activity (νcyt) was significantly induced in all lines 518
after HL treatment (Fig. 3). The observed νcyt induction was not due to COX protein 519
level modifications since the levels of immunodetected COXII in all genotypes were 520
either similar or lower in HL than in GL conditions; these results indicate that COX 521
protein level did not limit mETC activity and increased flux through COP may lead to 522
greater turnover of COX protein. Moreover, νcyt but not νalt was significantly and 523
positively correlated with photosynthetic activity (ETR) and non-photochemical 524
quenching (NPQ) (Table II, Supplemental Fig. 1). Increases in the linear electron flow 525
of PSII induce a proton gradient across thylakoid membranes (ΔpH) which leads to an 526
induction of NPQ (Shikanai, 2007). Also, νcyt was negatively correlated to total (% 527
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18
TPI) and chronic (% CPI) photoinhibition. Therefore, νcyt was correlated with a better 528
photosynthetic performance under severe HL stress. While ATP provision by νcyt has 529
been reported as an important role for mitochondrial metabolism in leaves under 530
illumination, there is a high need for energy dissipation under HL conditions that can be 531
accomplished by mitochondrial metabolism (Yoshida and Noguchi, 2010). In this 532
respect, it has been proposed that COX pathway can also allow an uncoupled electron 533
transport under situations of chronic oxidative stress through the activation of the 534
uncoupling proteins (UCPs) (Rasmusson et al., 2009). In the present study, UCP protein 535
levels were generally similar among genotypes and no substantial changes were 536
detected after HL treatment (Fig. 4). However, post-translational activation of UCP 537
proteins cannot be discarded. Indeed, UCP has been shown to be activated by lipid 538
peroxidation products (Vercesi et al., 2006) which would inhibit νalt but not νcyt as 539
observed in here. 540
541
In vivo COX activity is highly coordinated with photorespiratory and amino acid 542
metabolism 543
The active CEF-PSI in gl1 plants after 2h of HL can diminish the excess of reducing 544
power by balancing NADPH/ATP levels and therefore keep an active and less ROS-545
damaged chloroplast electron transport chain. The increased ETR observed in gl1 and 546
crr4-3 plants produced NADPH and ATP, not only for carbon assimilation but also for 547
photorespiration and the export of reductants via malate valve (Yoshida and Noguchi, 548
2010). The three processes co-occurring at HL provide NAD(P)H to the mitochondrial 549
electron transport chain via sugar oxidation, glycine decarboxylation and malate 550
oxidation. Under these conditions, both COX and AOX pathways were oxidizing all the 551
reductant power from the chloroplast as observed in gl1 plants after 2h HL. Indeed, in a 552
very recent study in Arabidopsis AOX1a mutants, we have shown that AOX pathway 553
can support photorespiration under high light conditions (Watanabe et al., 2016). 554
However, when the light stress conditions were more severe (indicated by higher levels 555
of photoinhibition) only the COX but not the AOX pathway was showing a response to 556
HL-stress. Moreover, the impairment of the CEF around PSI in double mutants caused 557
severe damage on the photosynthetic electron transport chain already after 2h of HL, as 558
indicated by the highest levels of CPI. Such severe impairment of the chloroplast ETR 559
probably restricted the generation of chloroplast derived substrates/reductants by any of 560
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19
the three processes commented above and thus limited the response of the 561
mitochondrial electron transport chain. Importantly, the statistically significant 562
correlation observed between in vivo respiratory activities and the NADH/NAD+ ratio 563
supports this view (Fig. 6). 564
565
In order to better understand the metabolic links involved in the attenuated respiratory 566
response as a consequence of CEF-PSI impairment under HL, metabolite profiles were 567
analyzed and also their correlation to the respiratory activities in vivo (Fig. 6, 568
Supplemental Fig. 4). The induction pattern of sugar levels such as sucrose, galactose 569
and especially glucose, was highly correlated to νcyt possibly indicating the fueling of 570
respiration by the increase on photosynthetic-derived substrates. In relation to this, 571
pyruvate and TCA cycle intermediates such as succinate, fumarate and 2-oxoglutarate 572
as well as 2-oxoglutarate/ citrate ratio were showing also positive and significant 573
correlations to νcyt. 2-oxoglutarate is a key metabolite in nitrogen metabolism being 574
used as carbon skeleton for amino acid synthesis (Araujo et al., 2014b). In the present 575
study, νcyt was also positively correlated to several amino acids. Taken together, the 576
correlations observed suggest that νcyt may oxidase matrix NADH providing ATP and 577
allowing TCA cycle carbon flow towards amino acid and protein synthesis under severe 578
HL. Proline synthesis requires carbon flow from 2-oxoglutarate and glutamate as well 579
as ATP (Szabados and Savouré, 2010) and remarkably, it was one of the highest 580
accumulated metabolite following HL treatment in all genotypes. Although not being 581
directly correlated to νcyt, a high synthesis of this ROS-protective metabolite can be 582
speculated and which would likely require a high rate of ATP-coupled respiration. In 583
this line, significant and positive correlations of νcyt with phenylalanine may also 584
indicate a high ATP demand for the synthesis of secondary metabolites with an 585
important protective role under HL conditions (Tohge et al., 2013). Also tyrosine was 586
continuously accumulated after HL treatment in very high levels, particularly in double 587
mutants that showed the highest levels after 8h of HL (10-fold increase). Recently a 588
new alternative pathway has been described to link tryrosine and phenylalanine 589
metabolism (Yoo et al., 2013), presenting the possibility that tyrosine accumulation also 590
contributes to the synthesis of the secondary metabolites derived from phenylalanine 591
(Tohge et al., 2013). In addition, given that phenylalanine is a precursor for UQ 592
biosynthesis (Block et al., 2014; Toghe et al., 2014) correlations to an enhanced νcyt 593
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20
under HL appear to reveal direct metabolic links to the mitochondrial electron transport 594
which warrant further investigation. 595
596
Finally, glycine was the metabolite which exhibited the greatest increase following HL 597
treatment as previously observed in Arabidopsis Col-0 plants after HL treatment 598
(Florez-Sarasa et al., 2012) and suggests a high photorespiratory activity (Timm and 599
Bauwe, 2013). However, this increase was much lower in pgr5 and, more strikingly, it 600
was almost abolished in the double mutants as compared to gl1 and crr4-3. These 601
results denote a link between CEF-PSI and photorespiration. Recently, it has been 602
reported that CEF-PSI is increased under photorespiratory and HL conditions, thus 603
denoting the cooperation of both processes as energy balancing mechanisms under HL 604
conditions (Walker et al., 2014). Photorespiration under HL contributes to the 605
consumption of the excess of reducing power thus acting as a photoprotective 606
mechanism to avoid photoinihibiton (Takahashi and Badger, 2011). Thus a high 607
photorespiratory flux, as expected in the HL conditions applied in this study, produced a 608
huge increase in glycine which is a major substrate for mitochondria in illuminated 609
leaves (Dry et al., 1983; Igamberdiev et al., 1997). In agreement, glycine and the 610
glycine/ serine ratio showed amongst the highest correlation coefficients with the νcyt, 611
which suggests that COX pathway can be involved in the re-oxidation of the NADH 612
from glycine decarboxylation. In parallel, νcyt was also highly and negatively correlated 613
to NADH/NAD+ ratios and glycerate which is one of the typical metabolites altered in 614
photorespiratory mutants (Timm and Bauwe, 2013). Interestingly, Sweetlove et al. 615
(2006) found that an uncoupled mitochondrial electron transport by the action of UCP 616
was important to keep the photorespiratory oxidation of glycine to serine, which in turn 617
benefits photosynthetic performance. Taken together, these observations strongly 618
suggest that the νcyt permits the oxidation of NADH derived from a HL-stimulated 619
glycine to serine decarboxylation under severe oxidative stress in cooperation with 620
active UCPs. In this way, the νcyt can act as a sink for the excess of electrons from the 621
chloroplast via photorespiratory glycine oxidation thus improving photosynthetic 622
performance and reducing photoinhibition when νalt is not induced due to severe HL-623
induced oxidative stress. Notably, νcyt was more increased in AOX1a antisense plants 624
than in WT under HL, thus compensating for the lack of νalt increase under HL (Florez-625
Sarasa et al., 2011). 626
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21
627
CONCLUSIONS 628
The in vivo activities of the mitochondrial COX and AOX pathways were studied for 629
the first time in cyclic electron flow around PSI (CEF-PSI) mutants under growth and 630
high light (HL) conditions. In addition, the AOX pathway capacity was also determined 631
and was highly and positively correlated to photoinhibition probably due to the effect of 632
photosynthetic-derived-ROS on the observed induced AOX expression. However, the 633
AOX pathway activity in vivo (νalt) was not higher in CEF-PSI mutants than in wild-634
type (gl1) plants under HL conditions, thus partially refuting our hypothesis. The severe 635
metabolic impairment observed in CEF-PSI mutants may underlie the reason for the 636
lack of AOX activity response (i.e. due to an inactivation by lipid peroxidation products 637
(Winger et al., 2005)), which was indeed engaged in the less severely stressed gl1 plants 638
after 2h of HL treatment, in agreement with previous results (Florez-Sarasa et al., 2011; 639
2012) and suggestions (Yoshida et al., 2007). Despite the severity of the HL stress, the 640
COX activity in vivo (νcyt) was induced in all genotypes and it was tightly linked to their 641
photosynthetic performance. The correlations with metabolite changes suggest that νcyt 642
is highly coordinated with stress-related amino acid synthesis. More strikingly, the 643
severely attenuated response of the glycine to serine ratio in pgr5 and double mutants 644
suggest an impaired photorespiration that was correlated with an attenuated response of 645
the νcyt. In addition, NADH/NAD+ ratios were strongly and negatively correlated with 646
νcyt. We propose that νcyt can act as a sink for the excess of electrons from the 647
chloroplast via photorespiratory glycine oxidation, thus reducing photoinhibition when 648
νalt is not induced under severe HL-stress. These observations open new perspectives for 649
further studies on the in vivo role and regulation of the mETC in leaves under HL-stress. 650
We speculate that such new in vivo role of COX pathway can be partially accomplished 651
through an ATP-uncoupled electron transport (i.e. ROS-activation of uncoupling 652
proteins), although more experiments will be needed to establish the exact mechanism 653
by which this is occurring. 654
655
MATERIAL AND METHODS 656
657
Plant material and growth conditions 658
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22
Plants of Arabidopsis thaliana wild type (ecotype Columbia gl1) and mutants in a 659
growth chamber (Fitotron UIB) under controlled conditions: temperature of 25ºC, 660
relative humidity above 40%, 12 h of photoperiod and light intensity of 80 µmol m-2 s-1 661
(growth light conditions, GL). Plants grown for 4-5 weeks under GL were transferred 662
for 2, 4 and 8 hours to 800 µmol m-2 s-1, considered HL conditions. A. thaliana pgr5 663
(proton gradient reduction) is deficient in the Fd-dependent CEF-PSI pathway 664
(Munekage et al., 2002), while crr4-3 (chlororespiratory reduction) is deficient in the 665
NDH-dependent pathway (Kotera et al., 2005). Also, a double mutant pgr5 crr4-3 was 666
gifted by Prof. Toshiharu Shikanai. The rosettes of the pgr5 and crr4-3 single mutants 667
were slightly smaller than gl1 plants after 4 weeks of growth. On the other hand, double 668
mutants presented pale leaves and growth was more affected and therefore this genotype 669
was grown for 5 weeks in order to reach similar developmental stage (i.e. number of 670
leaves) than the other three genotypes (Figure 1). 671
672
Chlorophyll fluorescence 673
The quantum efficiency of PSII (ΦPSII) was obtained from chlorophyll fluorescence 674
measurements in the light by setting actinic light of a portable pulse amplitude 675
modulation fluorometer (PAM-2000, Walz, Effeltrich, Germany) to 80 or 800 µmol m-2 676
s-1 for growth light and HL measurements, respectively. Chloroplast electron transport 677
rate (ETR) was calculated as the product of ΦPSII x actinic light intensity x 0.84 x 0.5. 678
In addition, photochemical (qP), non-photochemical quenching (qN, NPQ) and 679
maximum quantum efficiency of PSII (Fv/Fm) were obtained from chlorophyll 680
fluorescence measurements in the light and after 30 minutes dark adaptation with the 681
PAM-2000 fluorometer. Also, total, chronic and dynamic photoinhibition were obtained 682
as previously described in Florez-Sarasa et al. (2011) with slight modification; in the 683
present study, the Fv/Fmmax value was obtained from the mean value of Fv/Fm30 under 684
GL for gl1 plants (i.e. % CPI in gl1 plants is 0), in order to compare all the data to the 685
gl1. Ten to twelve replicates were performed per line and experimental condition. 686
687
Respiration measurements 688
Measurements oxygen isotope fractionation during respiration were performed as 689
described in Florez-Sarasa et al. (2007) to determine the activities of the cytochrome 690
oxidase (COX) and alternative oxidase (AOX) pathways in vivo. The oxygen isotope 691
fractionation of the AOX pathway required for the electron partitioning calculation 692
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23
(Guy et al., 1989) was determined after 10 mM KCN incubation as previously described 693
(Florez-Sarasa et al., 2007); a mean value of 30.5‰ was used from all the 694
measurements performed in the mutant and wild-type plants because no differences 695
were observed among genotypes (data not shown). On the other hand, the oxygen 696
isotope fractionation of the AOX pathway (Δc) value of 20.9‰ was taken from 697
previously measured in Arabidopsis thaliana leaves (Florez-Sarasa et al., 2007). Four to 698
five replicates per line and experimental condition were performed. 699
700
In addition, the AOX capacity was determined with a Clark-type oxygen electrode as 701
previously described (Florez-Sarasa et al., 2009). Four to ten replicates per line and 702
experimental condition were performed. 703
704
Metabolite profiling and pyridine nucleotides determinations 705
Metabolite extractions were performed as described previously (Lisec et al., 2006) using 706
approx. 150 mg of frozen-powdered leaf tissue. Derivatization and gas chromatography-707
time of flight-mass spectrometry (GC-TOF-MS) analyses were carried out as described 708
previously (Lisec et al., 2006). Metabolites were manually identified using the 709
TagFinder plug-in of the TagFinder software (Luedemann et al., 2008) and the reference 710
library mass spectra and retention indices housed in the Golm Metabolome Database 711
(http://gmd.mpimp-golm.mpg.de (Kopka et al., 2005)). The metabolite ratios were 712
calculated by dividing the signal intensities of the selected masses for the corresponding 713
metabolites and no calibration was performed with known concentrations of the 714
reference compounds; therefore, the ratios obtained are only used to compare their HL-715
responses among the different genotypes but they are not indicating the absolute (actual) 716
ratios. Data were normalized to the mean value of wild-type (gl1) plants at GL 717
conditions (i.e. the value of all metabolites and ratios for gl1 at GL was set to 1). Values 718
presented are the mean ± SE of six replicates, each replicate representing a pool of three 719
rosettes. 720
For pyridine nucleotides, two aliquots of 25 mg each of frozen-powdered leaf tissue (i.e. 721
from the same frozen-powdered used for GC-MS metabolite analysis) were used to 722
determine the reduced (NADH, NADPH) and the oxidized forms (NAD+, NADP+). 723
Extractions and spectrophotometer determinations were performed as described by 724
Kühn et al. (2015). 725
726
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24
Western blotting 727
Aliquots of 20 mg of frozen-powdered leaf tissue (i.e. from the same frozen-powdered 728
used for GC-MS metabolite analysis) was used to extract proteins by adding 100 ul of 729
SDS sample buffer [2% (w/v) SDS, 62.5mM Tris–HCl (pH 6.8), 10% (v/v) glycerol and 730
0.007% (w/v) bromophenol blue] and including 50mM DTT and a protease inhibitor 731
tablet (Roche). Samples were then incubated for 30 min in ice to allow full reduction of 732
AOX protein. Thereafter samples were boiled at 95ºC for c. 5 min and the homogenate 733
was centrifuged at 14000 rpm for 10 min. The supernatant was transferred into a new 734
tube and kept at -20ºC for later analysis. Twenty microliters were loaded and separated 735
on 12% SDS–PAGE gels and transferred to nitrocellulose membranes using wet Mini-736
PROTEAN system of Bio-Rad. The following primary antibodies and dilutions were 737
used for detecting mitochondrial proteins: monoclonal anti-Porin, voltage-dependent 738
anion channel porin (PM035, from Dr Tom Elthon, Lincoln, NE) at 1:5000 dilution; 739
polyclonal anti-AOX, alternative oxidase 1 and 2 (AS04054, Agrisera, Sweden) at 740
1:500 dilution; polyclonal anti-COXII, cytochrome oxidase subunit II (AS04053A, 741
Agrisera, Sweden) at dilution 1:1000; and polyclonal anti-UCP, uncoupling protein 742
(AS121850, Agrisera, Sweden) at 1:1000 dilution. Secondary antibodies linked to 743
horseradish peroxidase were used (Sigma-Aldrich Co.). The signals were detected by 744
chemiluminescence using Pierce™ ECL Western Blotting Substrate (ThermoFischer) 745
and a Luminescent Image Analyzer (G-Box-Chemi XT4, Syngene). The protein band 746
quantifications were performed with GeneTools analysis software from the 747
Luminescent Image Analyzer (G-Box-Chemi XT4, Syngene) according to 748
manufacturer’s instructions. The obtained band intensities for AOX, COX and UCP 749
were corrected for their corresponding porin band intensities and then normalized to the 750
levels of the gl1 plants under GL (i.e. gl1 levels for all the proteins are 100%). Two 751
different immunoblot experiments per protein were performed with very similar results 752
(data not shown), and images shown in Figure 4 and Supplemental Figure 2 belong to 753
one of the two membranes obtained. Samples used in each of the two immunoblot 754
experiments were a mixture of aliquots belonging to two biological replicates from the 755
same six biological replicates used for the GC-MS metabolite analyses-i.e. four of the 756
six biological replicates available were used. The percentage values presented in Figure 757
4 are the mean of the two immunoblot experiments. 758
759
Statistical analysis 760
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25
For statistical analyses of Figures 2, 3, Supplemental Figure 3 and Table I, a one way 761
analysis of variance (ANOVA) with a level of significance of p-value <0.05 was 762
performed with SPSS for Windows 17.0 and Duncan post hoc test was to determine 763
statistically significant differences. Pearson correlations (Figure 6 and Table II) and 764
their significance (p-value <0.05) between the fold-changes in metabolite levels and 765
fold-changes in respiratory or photosynthetic parameters were performed with 766
Microsoft Excel Software 2010. Student’s t-tests were used for statistical analyses in 767
Supplemental Table S1 in order to compare all genotypes under the different light 768
treatments to the gl1 under GL levels. 769
770
ACKNOWLEDGEMENTS 771
We would like to thank all the staff at the Serveis Cientifico-Tecnics of the Universitat 772
de les Illes Balears for their help while running these experiments, with especial 773
mention to Dr Biel Martorell for his technical help on the IRMS. We are also very 774
grateful to Yunuén Avalos Padilla for her assistance during the Western blot 775
experiments. 776
777
778
779
780
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26
Table I. Percentage of total (TPI), chronic (CPI) and dynamic (DPI) photoinhibition in leaves of gl1, crr4-3, pgr5 and pgr5 crr4-3 plants under 781
growth light (GL) and after 2, 4 and 8h of high light (HL) treatment. TPI, CPI and DPI were obtained as previously described in Florez-Sarasa et 782
al. (2011) and the Fv/Fmmax value was obtained from the mean value of Fv/Fm30 under GL for gl1 plants (i.e. % CPI in gl1 plants is 0). Values 783
are means ± SE of 10-12 replicates. Different letters denote significant differences (P < 0.05) between species. 784
785 786 787
Genotype % TPI % CPI % DPI GL 2h HL 4h HL 8h HL GL 2h HL 4h HL 8h HL GL 2h HL 4h HL 8h HL
gl1 2.3±0.5a 38.5±1.9cd 40.2±2.7cd 41.7±3.9d 0.0±0.3a 26.4±1.4cd 27.1±2.3cd 30.1±3.3d 2.3±0.2a 12.1±1.1de 13.1±0.8def 11.6±2.6de
crr4-3 2.9±0.4a 32.8±2.7c 38.1±2.7cd 38.7±3.4cd 0.4±0.4a 20.9±2.0c 25.2±2.1cd 28.3±3.0cd 3.3±0.2a 11.9±0.8de 12.9±0.9def 10.5±1.0cde
pgr5 3.4±0.7a 67.3±3.4e 68.8±2.4e 67.1±3.7e 0.3±0.4a 52.9±3.9e 59.1±2.9ef 59.6±3.7ef 3.0±0.4a 14.4±1.0ef 9.7±1.2cd 7.5±0.8bc
pgr5 crr4-3
14.4±3.9b 81.1±2.2f 83.0±1.2f 79.8±2.4f 8.6±2.8b 64.4±3.4f 77.3±2.0g 74.1±3.4g 5.9±1.1ab 16.7±1.9f 5.7±1.6ab 5.7±1.8ab
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Table II. Pearson correlation coefficients (r) between photosynthetic and respiratory 788
parameters. Twelve data points, corresponding to the means for each parameter of each 789
genotype under the different hours of high light treatment, were used in the correlation 790
analyses. Values in bold indicate statistically significant Pearson r correlations (P < 791
0.05). Vt (total respiration); νcyt (cytochrome oxidase pathway activity); νalt (alternative 792
oxidase pathway activity); Valt (alternative oxidase pathway capacity); ETR (chloroplast 793
electron transport rate); Fv/Fm (maximum quantum efficiency of PSII); qP 794
(photochemical quenching); qN and NPQ (non-photochemical quenching); % TPI, % 795
CPI and % DPI (percentage of total, chronic and dynamic photoinhibition). 796
797 Vt νcyt νalt Valt
ETR 0.74 0.73 0.25 -0.75 Fv/Fm 0.80 0.81 0.22 -0.92
qP 0.29 0.22 0.24 -0.14 NPQ 0.85 0.78 0.39 -0.89 qN 0.66 0.56 0.41 -0.69
% TPI -0.76 -0.80 -0.14 0.88 % CPI -0.80 -0.81 -0.21 0.92 % DPI 0.53 0.39 0.47 -0.60
798 799
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28
FIGURE LEGENDS 800
Figure 1. Photograph of representative wild-type (gl1) and cyclic electron flow around 801
photosystem I (CEF-PSI) mutant plants after 4 weeks of growth. The light treatments, 802
measurements and harvesting were performed after 4 weeks of growth in gl1 and CEF-803
PSI single mutant plants (crr4-3 and pgr5) which were showing slight growth 804
retardation. On the other hand, double mutants (pgr5 crr4-3) presented pale green 805
leaves and a more pronounced growth retardation, and therefore were grown for 5 806
weeks to reach a similar developmental stage as the other genotypes. 807
808
Figure 2. Photosynthetic parameters obtained by chlorophyll fluorescence analysis in 809
wild-type (gl1) and cyclic electron flow around PSI mutants. (A) Photochemical 810
quenching (qP); (B, D) non-photochemical quenching (qN, NPQ); (C) quantum 811
efficiency of PSII (ΦPSII); (E) chloroplast electron transport rate (ETR); and maximum 812
quantum efficiency of PSII (Fv/Fm) were determined in leaves of gl1, crr4-3, pgr5 and 813
pgr5 crr4-3 plants under growth light (0h) and after 2, 4 and 8h of high light (HL) 814
treatment. The chlorophyll fluorescence analysis, parameter calculations, light 815
treatments and genotypes are detailed in material and methods section. Data represent 816
means ± SE of ten to twelve replicates and different letters denote statistically 817
significant differences (P < 0.05). 818
819
Figure 3. In vivo respiratory activities and capacity of the alternative oxidase (AOX) in 820
wild-type (gl1) and cyclic electron flow around PSI mutants. (A) Total respiration (Vt), 821
(B) cytochrome oxidase pathway activity in vivo (νcyt), (C) AOX pathway activity in 822
vivo (νalt) and (D) AOX capacity (Valt) were determined in leaves of gl1, crr4-3, pgr5 823
and pgr5 crr4-3 plants under growth light (0h) and after 2, 4 and 8h of high light (HL) 824
treatment. The respiration analysis, light treatments and genotypes are detailed in 825
material and methods section. Data represent means ± SE of four to ten replicates and 826
different letters denote statistically significant differences (P < 0.05). 827
828
Figure 4. Mitochondrial electron transport chain proteins detected by Western blot in 829
leaves of gl1, crr4-3, pgr5 and pgr5 crr4-3 plants under growth light (GL) and after 4 830
and 8h of high light (HL) treatment. The intensities of the signals from the alternative 831
oxidase (AOX), cytochrome oxidase subunit II (COX) and uncoupling protein (UCP) 832
were normalized to those from the porin and expressed as percentages relative to values 833
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29
of gl1 under GL intensity. The percentage values denoted are the average of two 834
independent blots each using two biological replicates. 835
836
Figure 5. Heat map the relative levels of the GC-MS analysed metabolites in wild-type 837
(gl1) and cyclic electron flow around PSI mutants under growth light and after HL 838
conditions. Metabolites were clustered per class into amino acids, organic acids, sugar 839
and sugar alcohols, and some metabolite ratios were also calculated. Relative metabolite 840
levels of in leaves of gl1, crr4-3, pgr5 and pgr5 crr4-3 plants under all light conditions 841
(see Material and Methods section for further details) were normalized to the mean level 842
of the gl1 plants under GL conditions and then fold-change values were log2 843
transformed (i.e. the level of all metabolites of gl1 plants under GL are 0). In this heat 844
map, red and green colours represent log2 fold-increased and fold-decreased 845
metabolites, respectively. Values are means ± SE of 6 replicates and statistically 846
significant differences to gl1 plants under GL conditions are presented in Supplemental 847
Table SI. 848
849
Figure 6. Pearson correlation coefficients between fold-changes in metabolite levels 850
and in vivo respiratory activities in wild-type (gl1) and cyclic electron flow around PSI 851
mutants. As for the calculation of the metabolite relative levels, respiratory activities 852
were also normalized to those of the gl1 plants under GL condition. Then, all fold-853
change values were log10 transformed and used for the Pearson correlations. The values 854
used for the correlations were the means of all the genotypes under each light condition 855
thus allowing 15 point correlation. Only the metabolites showing statistically significant 856
(P < 0.05) Pearson correlation coefficients to at least one respiratory parameter are 857
presented and their positive (red) and negative (blue) r values are shown. 858
859
SUPPLEMENTAL DATA 860
Supplemental Table SI. Metabolite levels in leaves of gl1, crr4-3, pgr5 and pgr5 crr4-861
3 plants under growth light (GL) and after 2, 4 and 8h of high light (HL) treatment. 862
Supplemental Table SII (xlsx). Parameters used for peak annotation in GC-MS 863
analysis. 864
Supplemental Figure S1. Plots showing all the relationships between photosynthetic 865
and respiratory parameters. 866
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30
Supplemental Figure S2. Images showing membranes blotted from entire SDS-PAGE 867
gels of the immunodetected mitochondrial proteins alternative oxidase (AOX), 868
cytochrome oxidase subunit II (COX), uncoupling protein (UCP) and porin in leaf 869
extracts of gl1, crr4-3, pgr5 and pgr5 crr4-3 plants under growth light (GL) and after 4 870
and 8h of high light (HL) treatment. 871
Supplemental Figure S3. Pyridine nucleotides levels in wild-type (gl1) and cyclic 872
electron flow around PSI mutants. 873
Supplemental Figure S4. Plots showing the statistically significant (P<0.05) 874
relationships between fold-changes in metabolite levels and in vivo respiratory activities 875
in wild-type (gl1) and cyclic electron flow around PSI mutants. 876
877
878
879
Supplemental Table SI. Metabolite levels in leaves of gl1, crr4-3, pgr5 and pgr5 crr4-880
3 plants under growth light (GL) and after 2, 4 and 8h of high light (HL) treatment. Data 881
is presented as means and SE for six biological replicates. Significant differences (p < 882
0.05) to the gl1 under GL are denoted in bold font. 883
884
Supplemental Table SII (xlsx). Parameters used for peak annotation in GC-MS 885
analysis. 886
Supplemental Figure S1. Plots showing all the relationships between photosynthetic 887
and respiratory parameters. Vt (total respiration); νcyt (cytochrome oxidase pathway 888
activity); νalt (alternative oxidase pathway activity); Valt (alternative oxidase capacity); 889
ETR (chloroplast electron transport rate); Fv/Fm (maximum quantum efficiency of PSII); 890
qP (photochemical quenching); qN and NPQ (non-photochemical quenching); %TPI, 891
%CPI and %DPI (percentage of total, chronic and dynamic photoinhibition). 892
893
Supplemental Figure S2. Images showing membranes blotted from entire SDS-PAGE 894
gels of the immunodetected mitochondrial proteins alternative oxidase (AOX), 895
cytochrome oxidase subunit II (COX), uncoupling protein (UCP) and porin in leaf 896
extracts of gl1, crr4-3, pgr5 and pgr5 crr4-3 plants under growth light (GL) and after 4 897
and 8h of high light (HL) treatment. A sample of isolated mitochondria (pre-treated 898
with DTT) from wild-type Arabidopsis leaves (Mito) was loaded in the same gel. All 899
antibodies recognized the corresponding band with the expected molecular weight (see 900
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31
molecular marker lane, MM) that matched the band detected in the isolated 901
mitochondrial sample. 902
903
Supplemental Figure S3. Pyridine nucleotides levels in wild-type (gl1) and cyclic 904
electron flow around PSI mutants. (A) NAD+, (B) NADP+, (C) NADH, (D) NADPH, 905
(E) sums of NAD+ and NADH, (F) sums of NADP+ and NADPH, (G) NADH/NAD+ 906
ratios and (H) NADPH/NADP+ ratios in leaves of gl1, crr4-3, pgr5 and pgr5 crr4-3 907
plants under growth light (0h) and after 2, 4 and 8h of high light (HL) treatment. The 908
pyridine nucleotide determinations, light treatments and genotypes are detailed in 909
material and methods section. Data represent means ± SE of six replicates and different 910
letters denote statistically significant differences (P < 0.05). 911
912
Supplemental Figure S4. Plots showing the statistically significant (P<0.05) 913
relationships between fold-changes in metabolite levels and in vivo respiratory activities 914
in wild-type (gl1) and cyclic electron flow around PSI mutants. Respiratory activities 915
were also normalized to those of the gl1 plants under GL condition. All fold-change 916
values were log10 transformed and plotted. Vt (total respiration); νcyt (cytochrome 917
oxidase pathway activity); νalt (alternative oxidase pathway activity). 918
919
920
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32
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