RUNNING HEAD CORRESPONDING AUTHOR INFORMATION …5 Name: Ana Paula Alonso 6 Address: Department of Molecular Genetics, The Ohio State University, 1060 Carmack Road, 7 Columbus, OH

RUNNING HEAD 1 Sorghum metabolism under elevated CO2 and drought 2 3 CORRESPONDING AUTHOR INFORMATION 4 Name Ana Paula Alonso 5 Address Department of Molecular Genetics The Ohio State University 1060 Carmack Road 6 Columbus OH 43210 USA 7 Phone +1 (614) 688-7404 8 Fax +1 (614) 247-8937 9 E-mail alonso19osuedu 10 11 Name Marcos S Buckeridge 12 Address Laboratory of Plant Physiological Ecology (LAFIECO) Department of Botany Institute 13 of Biosciences University of Sao Paulo 05508-090 Sao Paulo Brazil 14 Phone +55-11-3091-7592 15 Fax + 55-11-3091-7547 16 E-mail msbuckuspbr 17 18 RESEARCH AREA 19 Biochemistry and Metabolism 20 21 AUTHORrsquoS E-MAILS 22 Amanda P de Souza (amandapsouzagmailcom) Jean-Christophe Cocuron 23 (cocuron1osuedu) Ana Carolina Garcia (acgarcia93gmailcom) 24 25 SUMMARY 26 The effect of drought on sorghum during grain filling was reduced under elevated CO2 plant 27 fitness and grain quality were maintained while the metabolism of different organs was adjusted 28 29 30 31 32 33 34

Plant Physiology Preview Published on September 2 2015 as DOI101104pp1501054

CHANGES IN WHOLE-PLANT METABOLISM DURING GRAIN-FILLING STAGE IN 35 SORGHUM BICOLOR L (MOENCH) GROWN UNDER ELEVATED CO2 AND DROUGHT 36 37 38 Amanda P De Souza1 Jean-Christophe Cocuron23 Ana Carolina Garcia1 Ana Paula Alonso2 39 Marcos S Buckeridge1 40 41 1 Laboratory of Plant Physiological Ecology (LAFIECO) Department of Botany Institute of 42 Biosciences University of Sao Paulo 05508-090 Sao Paulo Brazil 43 2 Department of Molecular Genetics The Ohio State University Columbus OH 43210 USA 44 3 Center for Applied Plant Sciences The Ohio State University Columbus OH 43210 USA 45 Corresponding authors 46 47 48

FOOTNOTES 50 51 This work was supported by Fundaccedilatildeo de Amparo agrave Pesquisa do Estado de Satildeo Paulo 52 and Microsoft Research (FAPESP Grant ndeg 201152065-3) and by the Ohio State 53 UniversityFAPESP joint mobility Grant ndeg 201350316-4 54 55 Present address for Amanda P Souza Institute for Genomic Biology University of 56 Illinois at Urbana-Champaign ndash 1206 W Dr Gregory Drive Urbana IL United States 57 61801-3832 58 59 Corresponding authors alonso19osuedu msbuckuspbr 60 61 62 63

ABSTRACT 66 67 Projections indicate an elevation of the atmospheric CO2 concentration ([CO2]) 68 concomitantly with an intensification of drought for this century increasing the 69 challenges to food security On the one hand drought is a main environmental factor 70 responsible for decreasing crop productivity and grain quality especially when occurring 71 during the grain-filling stage On the other hand elevated [CO2] is predicted to mitigate 72 some of the negative effects of drought Sorghum (Sorghum bicolor L Moench) is a C4 73 grass that has important economical and nutritional values in many parts of the world 74 Although the impact of elevated [CO2] and drought in photosynthesis and sorghum 75 growth has been well documented the effects of the combination of these two 76 environmental factors on plant metabolism have yet to be determined To address this 77 question sorghum plants (cv BRS 330) were grown and monitored at ambient (400 78 micromolmol-1) or elevated [CO2] (800 micromolmol-1) for 120 days and submitted to drought 79 during the grain-filling stage Leaf photosynthesis respiration and stomatal conductance 80 were measured at 90 and 120 days after planting and plant organs (leaves culm roots 81 prop roots and grains) were harvested Finally biomass and intracellular metabolites 82 were assessed for each organ As expected elevated [CO2] reduced the stomatal 83 conductance which preserved soil moisture and plant fitness under drought 84 Interestingly the whole-plant metabolism was adjusted and protein content in grains was 85 improved by 60 in sorghum grown under elevated [CO2] 86

87 88 Key-words C4 plant global climate change metabolite profiling biomass grain 89 composition 90 91 92 Abbreviations [CO2] atmospheric CO2 concentration DAP days after planting GC-93 MS gas chromatography - mass spectrometry LCMS-MS liquid chromatography - 94 tandem mass spectrometry OTC open-top chamber TCA cycle tricarboxylic acid 95 cycle UHPLC ultra-high pressure liquid chromatography 96 97 98 99

INTRODUCTION 100 101

Global food demand is projected to increase up to 110 by the middle of this 102 century (Alexandratos and Bruinsma 2012 Tilman et al 2011) particularly due to a rise 103 in world population that is likely to plateau at about 9 billion people (Godfray et al 2010) 104 Additionally the average concentration of atmospheric CO2 ([CO2]) has increased 175 105 micromolmol-1 per year between 1975 and today reaching 400 micromolmol-1 in April 2015 106 (NOAA 2015) According to the A2 emission scenario from the United States 107 Environmental Protection Agency (EPA) in absence of explicit climate change policy 108 atmospheric CO2 concentrations will reach 800 micromolmol-1by the end of this century The 109 increasing atmospheric [CO2] is resulting in global climate changes such as reduction in 110 water availability and elevation in temperature These factors are expected to heavily 111 influence the food production in the next years (Godfray and Garnett 2014 Magrin et al 112 2014) 113

Sorghum (Sorghum bicolor L Moench) is a C4 grass considered a staple food 114 grain for millions of the poorest and most food-insecure people in the semi-arid tropics of 115 Africa Asia and Central America serving as an important source of energy proteins 116 vitamins and minerals (Taylor et al 2006) Moreover this crop is used for animal feed 117 and as industrial raw material in developed countries such as the United States which is 118 the main world producer (FAO 2015) With a fully sequenced genome (Paterson et al 119 2009) and over 45000 accessions representing a large geographic and genetic 120 diversity sorghum is a good model system to study the impact of global climate changes 121 in C4 grasses 122

The increase in [CO2] in the atmosphere which is the main driver of global 123 climate changes (Meehl et al 2007) is predicted to boost photosynthesis rates and 124 productivity in a series of C3 legumes and cereals mainly due to a decrease in the 125 photorespiration process (Grashoff et al 1995 Long et al 2006) On the contrary due 126 to its capacity to concentrate CO2 in bundle sheath cells and reduce the photorespiration 127 to virtually zero C4 plants are unlikely to respond to the elevation of atmospheric [CO2] 128 (Leakey 2009) However even for C4 plants elevated [CO2] can ameliorate the effects 129 caused by drought and maintain higher photosynthetic rates due to an improvement in 130 efficiency of water use that is achieved by the reduction in stomatal conductance 131 (Leakey et al 2004 Markelz et al 2011) 132

The rate of photosynthesis as well as the redistribution of photoassimilates 133 accumulated in different plant tissues during the day andor during the vegetative growth 134 is crucial to grain development and later to its filling (Schnyder 1993) Due to this 135 relationship any environmental stress such as drought occurring during the reproductive 136 phase has the potential to result in poor grain filling and losses in yield (Blum et al 137 1997) For instance postanthesis drought can cause up to 30 decrease in yield 138 (Borrell et al 2000) It is also known that elevated [CO2] drought high temperature and 139 any combinations of these stresses can lead to significant changes in grain composition 140 (Taub et al 2008 Da Matta et al 2010 Uprety et al 2010 Madan et al 2012) 141 suggesting diverse metabolic alterations andor adaptations that occur in the plant when 142 it is cultivated in such conditions 143

Although the impact of elevated [CO2] and drought on photosynthesis and growth 144 of sorghum have been well documented (Wall et al 2001 Conley et al 2001 Ottman 145 et al 2001) no attention has been given to the impact of the combination of these two 146 environmental changes on plant metabolism and composition Regarding physiology 147 studies on growth of sorghum under elevated [CO2] and drought showed an increase of 148 the net assimilation rate of 23 due to a decrease of 32 in stomatal conductance (Wall 149 et al 2001) This resulted in sorghums ability to use water 17 more efficiently (Conley 150 et al 2001) An improvement on the final overall biomass under elevated [CO2] and 151 drought has also been described (Ottman et al 2001) but without a significant effect in 152 grain yield (Wall et al 2001) 153

Few studies have been monitoring metabolic pathways in plants under elevated 154 [CO2] (Aranjuelo et al 2013 Li et al 2008) and drought (Silvente et al 2012 Nam et 155 al 2015 Wenzel et al 2015) Further to our knowledge there are only two reports in 156 which metabolite profiles or metabolic pathways were investigated under the 157 combination of these two environmental conditions (Sicher and Barnaby 2012 Zinta et 158 al 2014) Although it is widely accepted that whole-plant metabolism and composition 159 can impact grain filling and yield metabolic studies conducted so far have focused on a 160 specific plant organ For instance Sicher and Barnaby (2012) analyzed the metabolite 161 profile of leaves from maize plants that were grown under elevated [CO2] and drought 162 but did not show how those environmental changes could have affected the metabolism 163 of other tissues (eg culm roots) nor how they might have influenced the biomass or 164 grain composition 165

In order to address how the combination of elevated [CO2] and drought can 166 modify the whole-plant metabolism as well as biomass composition in sorghum this 167 study aimed to (i) evaluate photosynthesis growth and yield (ii) underline the 168 differences in biomass composition and primary metabolite profiles among leaves culm 169 root prop roots and grains and (iii) determine the effect of elevated [CO2] and drought in 170 the primary metabolism of each organ 171 172 RESULTS 173 174 Elevated [CO2] did not change the rate of photosynthesis in sorghum plants subjected to 175 drought but modified panicle size and timing of initiation 176 177 Sorghum plants were grown in open top chambers (OTCs) under ambient or 178 elevated [CO2] and submitted to drought during the grain formation and filling (Fig 1) In 179 both treatments the availability of water in the soil was similar during the entire 180 vegetative phase (Fig2) At 60 DAP when the drought treatment started the soil 181 moisture in pots at ambient [CO2] decreased faster than those at elevated [CO2] At 90 182 and 115 DAP soil moisture in pots at elevated [CO2] was approximately 40 and 183 1200 higher than in pots at ambient [CO2] respectively 184 Despite the differences in soil moisture between ambient and elevated [CO2] no 185 significant changes were observed in leaf photosynthesis and leaf respiration (Fig 3A 186 and Fig 3C) However stomatal conductance in plants grown under elevated [CO2] was 187 approximately 53 lower than ambient [CO2] at 90 DAP (Fig 3B) Elevated [CO2] 188 treatment did not affect the height of plants but significantly increased their panicle size 189 by 26 to 28 between 94 and 117 DAP (Fig 4) Furthermore the higher concentration 190 of CO2 promoted a delay of one week on panicle initiation (Fig 4B) 191 192 Elevated [CO2] significantly improved protein content in grains but did not change yield 193 under drought 194 195

To evaluate whether biomass and its biochemical composition were affected by 196 elevated [CO2] and drought plants were harvested at 90 and 120 DAP Only leaf and 197 prop root biomasses increased under elevated [CO2] (Table I) At 120 DAP leaf biomass 198 was 48 higher in plants grown at elevated [CO2] mostly as a result of a decrease in 199

leaf senescence Indeed the biomass of shed leaves under elevated [CO2] was 90 200 lower than in ambient [CO2] (data not shown) While a significant increase in leaf 201 biomass was observed at the end of the plant cycle the prop root biomass showed a 202 transitory increase at 90 DAP At this date prop roots of plants under elevated [CO2] 203 displayed 94 more biomass than plants under ambient [CO2] However this difference 204 did not persist until 120 DAP (Table I) The biomass of grains which correspond to yield 205 did not change significantly between treatments 206

Although the biomass biochemical composition of different organs was not 207 modified by elevated [CO2] it promoted an increase of 35 and 59 in total protein 208 content of grains at 90 and 120 DAP respectively (Table I) In contrast some transitory 209 reduction in total protein content and total fatty acids under elevated [CO2] was observed 210 at 90 DAP in roots and grains respectively 211 212 Intracellular metabolism among organs the first step to assess how grain biochemical 213 composition is altered by elevated [CO2] under drought 214 215 The grain-filling stage in plants is largely dependent on the plant metabolic status 216 (Schnyder 1993) Nevertheless there are no studies that describe the primary 217 metabolite profiles of different plant organs and assess the question of how they are 218 interconnected In order to obtain evidence that supports how each organ can contribute 219 to observed changes in grain biochemical composition under elevated [CO2] and drought 220 the intracellular amounts of primary metabolites of leaves culm roots prop roots and 221 grains were quantified by LC-MSMS at 90 and 120 DAP 222 In total we identified and quantified 76 metabolites in leaves and culm 77 223 metabolites in roots and grains and 75 metabolites in prop roots at 90 and 120 DAP in 224 plants grown under ambient and elevated [CO2] (Supplemental Table SI) Principal 225 component analysis (PCA) of the entire dataset revealed that differences in the amounts 226 of metabolites were sufficient to distinguish metabolism among the organs even 227 between prop roots and roots (Fig 5) Irrespective to the dates and treatments some 228 metabolites were found to be in higher quantities in specific organs (Supplemental Fig 229 S1) 230

Cysteine levels were the highest in grains when compared to other organs This 231 amino acid is the only metabolite donor of sulfur for the production of other sulfur-232 containing compounds (Noji and Saito 2003) Interestingly GABA a metabolite that has 233

been shown to mitigate drought stress in grasses (Krishnan et al 2013) was 234 preferentially accumulating in the grains rather than other plant organs such as leaves 235 and roots Polyols such as glycerol and sorbitol were also found to be the highest in 236 grains These have been previously shown to be involved in osmotic stress responses 237 (Bohnert and Shen 1999) ADP-glucose a precursor for starch biosynthesis was only 238 detectable in the grains (Supplemental Fig S1) The majority of the amino acids were 239 especially accumulated in higher levels in the grains of 90 DAP plants submitted to 240 drought and elevated [CO2] 241

The plants were harvested between 1000 am and 200 pm therefore 242 photosynthesis was very active As anticipated leaves showed higher amounts of 243 metabolites related to the Calvin cycle (ie 2 or 3-phosphoglycerate ribulose-15-244 bisphosphate pentose phosphates sedoheptulose-7-phosphate erythrose-4-245 phosphate) and nucleotide triphosphates In culms some metabolites related to the 246 respiration process such as citrate and isocitrate were in higher quantities Tryptophan 247 a precursor of a large variety of secondary metabolites as well as the precursor of the 248 plant hormone indole-3-acetic acid (IAA) (Kriechbaumer and Glawischnig 2005 Ljung et 249 al 2002) was also accumulated preferentially in the culm The amount of soluble 250 sugars (fructose glucose and sucrose) was greater in prop roots mdash structures 251 described to aid anchorage of the plant to the soil (Gregory 2006) mdash than in any other 252 organ 253

254 Intracellular metabolism is modified by elevated [CO2] in plants grown under drought 255 256

To investigate whether the metabolism of each organ was modified by elevated 257 [CO2] and through different grain-filling stages (90 and 120 DAP) we performed a series 258 of PCA analyses taking into account all metabolites present in a given organ and tested 259 the significance related to CO2 treatment and the grain developmental stage All organs 260 showed significant differences between ambient and elevated [CO2] With exception of 261 prop roots they also had significant differences related to the grain developmental 262 stages (Fig 6) 263

In leaves most of the amino acids decreased from 90 to 120 DAP at ambient 264 [CO2] whereas they were at similar amounts in both dates under elevated [CO2] (Fig 265 7A) Leaves under elevated [CO2] had higher amounts of nucleotide monophosphates 2 266 or 3-phosphoglycerate and sucrose-6-phosphate at 90 DAP in comparison to the leaves 267

grown under ambient [CO2] However no differences between treatments were found at 268 120 DAP Both ambient and elevated [CO2] plants showed a reduction in metabolites 269 related to glycolytic processes in leaves at 120 DAP compared to 90 DAP On the other 270 hand sugars sugar-alcohols and intermediaries of the tricarboxylic acid (TCA) cycle 271 increased in both CO2 treatments between 90 and 120 DAP Metabolites related to the 272 Calvin cycle were lower under elevated [CO2] at 90 DAP but higher at 120 DAP 273

Similar to leaves the majority of the amino acids decreased in culms between 90 274 and 120 DAP However in culms the reduction was observed in both CO2 treatments 275 Sugars and sugar-alcohols decreased at 120 DAP in both conditions Also similar to 276 what was observed in leaves intermediaries of the TCA cycle (fumarate malate citrate 277 isocitrate trans-aconitate cis-aconitate) in the culm of both ambient and elevated [CO2] 278 increased between 90 and 120 DAP Remarkably metabolites related to sucrose 279 metabolism (UDP-glucose sucrose fructose-6-phosphate glucose-6-phosphate 280 sucrose-6-phosphate) were higher in the culm at 90 DAP under elevated [CO2] and 281 decreased to lower levels than ambient [CO2] at 120 DAP (Fig 7B) 282

The amounts of soluble sugars (sucrose glucose fructose) and some sugar-283 alcohols in roots were higher under ambient [CO2] especially at 90 DAP Under elevated 284 [CO2] the levels of these sugars increased only at 120 DAP (Fig 7C) Roots from plants 285 grown at elevated [CO2] also showed a higher content of hexose phosphates and 286 nucleotide triphosphates at 120 DAP As previously observed for leaves and culm the 287 overall metabolism of amino acids was reduced at 120 DAP in roots under both 288 conditions Overall the intermediaries of glycolysis and TCA cycle were reduced at 120 289 DAP compared to 90 DAP in both CO2 treatments 290

In prop roots an overall increase of organic acids hexose phosphates and 291 sugar-alcohols was measured at 120 DAP under ambient [CO2] but not under elevated 292 [CO2] (Fig 7D) Intriguingly this response is different from what was found in roots 293 Additionally the levels of sugars and metabolites related to sucrose metabolism 294 decreased at 120 DAP in prop roots under elevated [CO2] whereas they were 295 maintained at similar levels under ambient [CO2] (Fig 7D) This response is also 296 opposite to what was observed in roots where the amounts of sugars increased at 120 297 DAP under elevated [CO2] (Fig 7C) 298

The metabolites in grains were generally lower at 120 DAP than at 90 DAP in 299 both treatments indicating a reduction in the overall grain metabolism (Fig 7E) 300

Remarkably the majority of metabolites related to intermediaries of glycolysis TCA 301 cycle and amino acids were higher under elevated [CO2] at 90 DAP 302

303 DISCUSSION 304 305 Elevated [CO2] changes the physiological status and development of sorghum under 306 drought 307 308

Losses in grain quality and yield are observed when drought stress occurs 309 especially during flowering time (Blum et al 1997) For instance postanthesis drought 310 can cause up to 30 decrease in grain yield (Borrell et al 2000) However elevated 311 [CO2] commonly ameliorates the effects of environmental stresses (Ottman et al 2001 312 Leakey et al 2006 Geissler et al 2009 Naudts et al 2013 Sicher and Barnaby 2012 313 Arenque et al 2014 Zinta et al 2014) reducing the impact on photosynthesis biomass 314 production and loss in productivity 315

In C4 plants the mitigation of the effect of drought stress promoted by elevated 316 [CO2] is thought to be a consequence of the reduction in stomatal conductance which 317 leads to an increased in water use efficiency (Leakey 2009) For sorghum previous 318 studies revealed that the improvement in water use efficiency stimulated by elevated 319 [CO2] in plants cultivated under drought stress increased the rate of photosynthesis and 320 final overall biomass (Wall et al 2001 Conley et al 2001 Ottman et al 2001) As 321 consistently described in the literature our results show that stomatal conductance 322 under elevated [CO2] was lower than under ambient [CO2] at 90 DAP (Fig 3B) However 323 this pattern was reversed at 120 DAP where plants at elevated [CO2] displayed higher 324 stomatal conductance than at ambient [CO2] This reversion may reflect the effect of 325 drought on plants at ambient [CO2] Due to the lower stomatal conductance soil 326 moisture was conserved during the period of growth under elevated [CO2] (Fig 2) 327 producing a less severe effect for drought Nevertheless it is important to mention that 328 the large difference of up to 1200 in soil moisture observed between elevated and 329 ambient [CO2] might not reflect values detected under field conditions These high 330 values may be due to an interference of OTCs ventilation on the sensors installed at only 331 15-20cm from the soil surface 332

In spite of the observed effect in stomatal conductance no significant differences 333 in photosynthesis between ambient and elevated [CO2] were measured (Fig 3A) Even 334

at the end of the experiment when plants at ambient [CO2] faced quite low amount of 335 water in the soil (Fig 2) both treatments showed similar photosynthetic rates However 336 leaf biomasses were different between treatments Indeed under elevated [CO2] leaf 337 senescence was reduced leading to a higher leaf biomass accumulation at 120 DAP 338 (Table I) The reduction in leaf senescence under elevated [CO2] (data not shown) 339 indicates that this treatment alleviates some of the drought effects In sorghum drought 340 stress during and after flowering stages (as applied in our experiment) can cause 341 premature leaf senescence (Rosenow and Clark 1995) and it is well known that the 342 retention of green leaves during the grain-filling stage generally results in higher grain 343 yield (Valdez et al 2011 Jordan et al 2012) In our experimental design the greater 344 amount of green leaves under elevated [CO2] did not have an impact in yield (Table I) 345 but can be rather related to the observed changes in grain quality as it will be further 346 discussed 347

Even without a significant impact in yield plants under elevated [CO2] had their 348 panicle development delayed by seven days At the same time their panicle size was 349 significantly increased (Fig 4B) The late inflorescence development was already 350 reported in previous experiments with sorghum (Marc and Gifford 1983) These authors 351 showed similar seven-day delays in inflorescence development from plants cultivated 352 under 500 micromol CO2mol air-1 and postulated that CO2 can exerts some direct effect on 353 inflorescence since no other changes in plant development were observed 354

Another interesting change related to growth was the transitory increase in 355 biomass accumulation in prop roots at 90 DAP under elevated [CO2] (Table I) Since 356 drought can reduce the growth of prop roots (Nielsen 2002) the higher biomass found 357 under elevated [CO2] at 90 DAP might also be a consequence of the lower effect of 358 drought under this treatment At 120 DAP the biomass of prop roots in both ambient and 359 elevated [CO2] were reduced compared to 90 DAP and no differences were found 360 between the two treatments (Table I) During the grain filling stage there is a 361 mobilization of storage compounds from other organs in order to supply carbon and 362 nitrogen to grain In sorghum this storage mobilization can reach up to 40 in optimal 363 conditions and up to 52 under drought stress (Beheshti and Fard 2010) This is one 364 possible explanation for the observed reductions of 49 and 67 in the biomass of prop 365 roots under ambient and elevated [CO2] respectively 366

Elevated [CO2] preserves the quality of grains under drought through the maintenance of 368 whole plant metabolism 369

370 Plant responses to environmental factors is not only limited to physiological 371

behavior but it also involves a coordinated and complex network of different 372 organizational levels (ie physiological metabolic and transcriptional) that may take 373 place in different organs It has been previously demonstrated that plant metabolism can 374 change to maintain whole-plant homeostasis under stress preventing massive 375 alterations in physiological and growth parameters For instance De Souza et al (2013) 376 showed for Miscanthus x giganteus that even when no significant changes in 377 photosynthesis and dry weight were found growth under elevated [CO2] promoted a 378 reduction in starch content in leaves roots and rhizomes of this C4 plant 379

In the experiment reported here one of the most remarkable differences found 380 between ambient and elevated [CO2] was the one related to the protein content in grains 381 Even though there were no changes in yield the protein content per mass in grains was 382 higher at elevated [CO2] (Table I) At 120 DAP we found averages of 5 and 8 of 383 protein in grains under ambient and elevated [CO2] respectively According to 384 EMBRAPA Maize and Sorghum (2015) the cultivar used in this experiment (BRS 330) 385 displays around 10 of protein content in its grains under field conditions Thus the 386 protein content found in plants under elevated [CO2] was closer to what is usually 387 observed in this cultivar indicating that elevated [CO2] ameliorates the effects of drought 388 leading to a preservation of grain quality 389

Corroborating the mitigation effects of drought under elevated [CO2] the 390 conservation of water in the soil observed in this treatment most likely extended the 391 activity of metabolites related to the Calvin cycle sucrose and amino acid metabolisms 392 in leaves (Fig 7A) This condition also promoted a higher accumulation of metabolites 393 related to sucrose metabolism in culms at 90 DAP (Fig 7B) and the maintenance of 394 metabolites related to energy metabolism such as hexose phosphates and nucleotide 395 triphosphates in roots until 120 DAP (Fig 7C) Additionally the appearance of 396 metabolites associated to drought was delayed in roots and prop roots under elevated 397 [CO2] No responses related to drought were found in prop roots under elevated [CO2] 398 during this experiment The increase in levels of sugars and sugar-alcohols in roots 399 which is a consequence of drought response (Bohnert and Sheveleva 1998 Pavli et al 400 2013) occurred only at 120 DAP under elevated [CO2] In contrast high levels of these 401

metabolites were found under ambient [CO2] as soon as at 90 DAP (Fig 7C) Under 402 ambient [CO2] prop roots displayed a later response to drought compared to roots An 403 increase in the contents of organic acids and sugar-alcohols which are metabolites 404 regarded as characteristic of drought response in sorghum (Pavli et al 2013) were 405 observed at 120 DAP in this organ (Fig 7D) It is also important to note that our 406 metabolomics study revealed a maximal level of all the amino acids in grain under 407 elevated [CO2] at 90 DAP (Fig 7E Supplemental Table I) Altogether these responses 408 accounted for the maintenance of grain metabolism under elevated [CO2] and 409 consequently higher protein contents in grains Moreover the higher biomass of prop 410 roots and the larger mobilization of sugars in both culm and prop roots from 90 to 120 411 DAP (Table I Fig 7B and Fig 7D) at elevated [CO2] also contributed to the higher 412 protein levels in grains Indeed the high amounts of fructose glucose and sucrose found 413 in prop roots (Supplemental Fig S1) and the decrease of its biomass from 90 to 120 414 DAP suggests that this organ can serve as a storage organ in addition to its function as 415 plant support 416

Even though the cultivation under elevated [CO2] diminished the effects of 417 drought metabolic responses related to this stress can be observed in both treatments 418 at 120 DAP The contents of sugars and sugar-alcohols in leaves and roots increased in 419 both treatments from 90 to 120 DAP (Fig 7A and Fig 7C) Similarly the levels of some 420 organic acids primarily the intermediaries of the TCA cycle were greater in leaves and 421 culm (Fig 7A and Fig 7B) The increase in concentration of these metabolites has 422 previously been suggested to be associated to drought responses in sorghum (Pavli et 423 al 2013) 424

Along plant development the overall metabolic response indicated a reduction in 425 plant metabolism from 90 to 120 DAP with a decrease in amino acid contents in leaves 426 and culm a concomitant decrease in metabolites related to sucrose metabolism in culm 427 and prop roots and a reduction in intermediaries of glycolysis and TCA cycle in roots 428 (Fig 7) 429

430 CONCLUSIONS AND PERPECTIVES 431 Our results showed that although little physiological effects were observed the 432 cultivation of sorghum under elevated atmospheric CO2 alleviated the loss in grain 433 quality caused by drought during the grain-filling stage due to a delay in physiological 434 and metabolic responses to drought To our knowledge this is the first study that 435

demonstrates the simultaneous metabolic responses of the different organs of a plant 436 cultivated under elevated atmospheric CO2 and drought at the same time It also shows 437 for the first time in sorghum how changes in each organ can affect grain composition In 438 this context in the future it would be key to evaluate metabolic fluxes within the plant in 439 order to highlight how different organs integrate to produce whole plant physiological 440 behavior under different environmental conditions Furthermore our results set up a map 441 of metabolic events occurring in different organs of sorghum This information could be 442 used to search candidate genes related to the control of inter-organ communication 443 Thus they might be targeted in plant engineering to help coping with the impacts of the 444 Global Climate Changes on crop grasses 445 446 MATERIALS AND METHODS 447 448 Plant material and growth conditions 449

Seeds of Sorghum bicolor L cv BRS 330 were obtained from EMBRAPA Maize 450 and Sorghum Brazil They were germinated in trays containing Plantmaxreg soil compost 451 (peat Pinus bark and vermiculite) in a greenhouse Fifteen days after planting (DAP) 452 seedlings of similar size were transferred to 40 L pots and randomly distributed through 453 four open-top chambers (OTCs) with 15 x 35 m (diameter x height) as described in De 454 Souza et al (2008) (Fig 1A) Two OTCs were coupled to a CO2 cylinder in order to 455 maintain the chamber internal concentration at approximately 800 micromolmol-1 (elevated 456 [CO2]) The other two chambers were maintained with current atmospheric [CO2] (~ 400 457 micromolmol-1 ambient [CO2]) 458

Every week pots were rotated inside the OTCs and every two weeks they were 459 rotated between OTCs to avoid acclimation to microenvironment Air temperature air 460 relative humidity and CO2 concentration for each OTC were constantly recorded 461 (Supplemental Fig S2) After 25 days soil moisture sensors (10HS ICT International 462 Australia) were installed at 15-20 cm from soil surface in 4 pots of each OTC in which 463 the soil moisture values were recorded every 24 hours Once a week each plant 464 received 400 mL of Hoagland solution (Epstein 1972) as a source of nutrients until the 465 end of the experiment During the vegetative phase (from 0-60 DAP) each pot received 466 15 L of water per day At the beginning of the reproductive phase (after 60 DAP with 467 the initiation of the flag leaf) the amount of water per pot in both treatments was reduced 468 to 045 L (reduction of 70) per day to start the water deficit treatment (Fig 1B) 469

470 Harvests 471

Five plants from each treatment were harvested at 90 and 120 DAP 472 corresponding to developmental stages of immature and mature grains respectively (Fig 473 1B) Each harvested plant was separated into leaves culm roots prop roots and grains 474 immediately frozen in liquid nitrogen and freeze-dried 475

476 Growth measurements 477

Plant height and panicle size was evaluated every week from 31 to 117 DAP and 478 from 66 to 117 DAP respectively using a measuring tape Plant height was considered 479 as being the distance between the root-shoot transition and the tip of the younger leaf 480 and panicle size as the distance between the peduncle-inflorescence transition and the 481 inflorescence tip Biomass accumulation in leaves culm roots prop roots and grains 482 was determined at 90 and 120 DAP in freeze-dried material 483

484 Gas exchange measurements 485

Before each harvest leaf CO2 uptake (A) stomatal conductance (gs) and leaf 486 respiration (Rd) of the flag leaf were measured with a portable open gas-exchange 487 system (LI-6400 XT Li-Cor USA) Leaf temperature was maintained at 26degC light 488 intensity at 1000 micromolm-2s-1 of photosyntetically active radiation (PAR) and CO2 489 concentration at 400 or 800 micromolmol-1 depending of the [CO2] treatment For Rd 490 measurements the leaf was dark-adapted during 30 minutes All measurements were 491 made between 1000 am and 200 pm 492 493 Fatty-acid protein and starch extraction and quantification 494

Fatty-acids proteins and starch were sequentially extracted from 20 mg of 495 freeze-dried grinded sorghum tissues as previously described (Cocuron et al 2014) 496

Fatty acid content was determined by GC-MS Fatty acid methyl esters (FAMEs) 497 were analyzed using a Thermo Trace 1310 gas chromatograph coupled to an ISQ single 498 quadrupole mass spectrometer FAME derivatives were separated using an Omegawax 499 250 capillary (30 m x 025 mm x 025 μm) column from Supelco as previously described 500 (Tsogtbaatar et al 2015) GC-MS data were acquired and processed using Xcalibur 501 software FAME derivatives were identified using NIST 11 library and neat FAME 502 standards purchased from Sigma 503

Proteins and starch were quantified following the steps previously described 504 (Cocuron et al 2014) 505

506 Metabolite extraction 507

Metabolites were extracted from 20 mg of freeze-dried grinded sorghum tissues 508 using boiling water following a protocol previously described (Cocuron and Alonso 509 2014) At the time of the extraction 200 200 and 50 nmol of [U-13C]glucose [U-510 13C]glycine and [U-13C]fumarate were added respectively as internal standards 511

512 LC-MSMS quantification of intracellular metabolites 513

After lyophilization extracts were resuspended in 500 microL of nanopure water and 514 vortexed Two hundred microL of sample was loaded onto a 02 microm nanosep MF centrifugal 515 device in order to quantify the sugars and sugar alcohols The remaining 300 microL was 516 transferred to a 3 kDa Amicon Ultra 05 mL filtering device for the quantification of amino 517 acids phosphorylated compounds and organic acids The samples were centrifuged at 518 14000timesg for 45 minutes at 4degC The intracellular metabolites were separated and 519 quantified as previously described (Cocuron et al 2014) with minor modifications The 520 LC was performed with an UHPLC 1290 from Agilent Technologies Inc The MSMS 521 analyses were performed with a hybrid triple-quadrupoleion trap mass spectrometer 522 QTRAP 5500 from AB Sciex LC-MSMS data were acquired and processed using 523 Analyst 161 software 524

For sugars and sugar alcohols quantification the extracts from leaves roots and 525 grains obtained after centrifugation in nanosep MF filter were diluted 25X and those 526 from culm and prop roots were diluted 50X in acetonitrilewater (6040) solution For 527 amino acids the extracts from leaves and roots were diluted 25X and those from culm 528 prop roots and grains were diluted 50X in 1 mM hydrochloric acid The quantification 529 was made by injecting 5 microL of the diluted sample onto the LC-MSMS column 530

In order to quantify phosphorylated compounds and organic acids the extracts 531 from leaves culm roots prop roots and grains were diluted in ultra-pure water 25 25 532 20 20 and 10X respectively Ten microL were injected onto the column 533

534 Statistical analysis 535

Differences between ambient and elevated [CO2] (P-valuelt005) were tested by 536 Studentrsquos t test (n=3) using JMPreg Statistical Discovery Software version 501 NC 537 USA 538

Metabolites were analyzed by principal component analysis (PCA) using the 539 software Minitabreg version 141 Before PCA all metabolite data were normalized using 540 log2 function The significance of each principal component was tested by a two-way 541 ANOVA test treating grain developmental stages and CO2 concentration as fixed 542 effects 543

Heatmaps were generated using MetaboAnalyst v30 (Xia et al 2009 2012 544 2015) a free online software (wwwmetaboanalystca) Briefly metabolites levels were 545 first normalized using log2 function and then mean-centered and divided by the standard 546 deviation of each variable Finally Wards hierarchical clustering algorithm was used to 547 group metabolites that have the same pattern of distribution 548 549 SUPPLEMENTAL MATERIAL 550 551 Table SI List of metabolites and their quantification in sorghum tissues under ambient 552 and elevated [CO2] 553 Figure S1 Heatmap comparing metabolite levels in different organs 554 Figure S2 Atmospheric [CO2] air temperature and air relative humidity inside the open-555 top chambers 556 557 ACKNOWLEDGMENTS 558 559 We are grateful to The Ohio State University Targeted Metabolomics Laboratory 560 (metabolomicsosuedu) for access to the GC-MS and LC-MSMS equipments funded 561 respectively by the Center for Applied Plant Sciences (CAPS) and the Translational 562 Plant Sciences Targeted Investment in Excellence (TIE) Finally we are thankful to Erin 563 Ponting for her critical reading of this manuscript 564 565 AUTHORS CONTRIBUTION 566 567 APDS designed the experiments contributed to the collection of physiological data 568 performed metabolite extractions and data analyses and wrote the manuscript 569

JCC performed metabolite quantifications by LC-MSMS and contributed to biomass 570 composition data analysis and manuscript revision 571 ACG collected and analyzed the physiological data 572 APA contributed to intracellular metabolite analyses and statistical analyses 573 APA and MSB conceived the project supervised the research and revised the 574 manuscript 575 All authors contributed to data interpretation 576 577 578 579 FIGURE LEGENDS 580 581 Figure 1 (A) Open-top chambers used during the experiment to grow Sorghum bicolor 582 cv BRS 330 at ambient and elevated CO2 (B) Experimental design 583 584 Figure 2 Soil moisture (cm3 of water per cm3 of soil) during the experiment with 585 Sorghum bicolor cv BRS330 at ambient and elevated CO2 n=8 Water deficit treatment 586 started after 60 days after planting (DAP) From 90 to 93 DAP soil moisture sensors 587 from pots at elevated CO2 did not log the data (gray dashed line) 588 589 Figure 3 (A) Light saturated rate of leaf photosynthesis (A) (B) Stomatal conductance 590 (gs) and (C) Dark leaf respiration (Rd) of the youngest fully expanded leaf of Sorghum 591 bicolor cv BRS330 at ambient and elevated CO2 at 90 and 120 days after planting 592 (DAP) Bars are the mean plusmn SD of biological replicates n=3 No significant differences 593 were observed 594 595 Figure 4 (A) Plant height (without panicle) (cm) and (B) Panicle size (cm) of Sorghum 596 bicolor cv BRS330 at ambient and elevated CO2 along the experiment Data points are 597 the mean plusmn SD of biological replicates n=3 Asterisks indicate significant statistic 598 differences between treatments (P lt005) 599 600 Figure 5 Principal component analysis (PCA) of the metabolites identified by LC-MS-601 MS of Sorghum bicolor cv BRS330 at ambient (open symbols) and elevated CO2 602

(closed symbols) at 90 (squares) and 120 days (circles) after planting (DAP) P-values 603 indicate significant statistic differences of PCs 604 605 Figure 6 Principal component analysis (PCA) of the metabolites identified by LC-MS-606 MS of leaves culm roots prop roots and grains of Sorghum bicolor cv BRS330 at 607 ambient and elevated CO2 at 90 and 120 days after planting (DAP) P-values lt005 608 indicate significant statistic differences of PCs 609 610 Figure 7 Heatmaps showing the variation of amount of metabolites in Sorghum bicolor 611 (cv BRS 330) grown under ambient and elevated [CO2] at 90 and 120 DAP in (A) leaves 612 (B) culm (C) roots (D) prop roots and (E) grains Abbreviations 23PGA 2 or 3-613 phosphoglycerate 6PG 6-phophosgluconate ADPGlc ADP-glucose AKG alpha-614 ketoglutarate cis-aco cis-aconitate Deoxyxyl5P deoxyxylulose-5-phosphate E4P 615 erythrose-4-phosphate F16bP Fructose-16-bisphosphate Fru6P fructose-6-616 phosphate GABA gamma-aminobutyric acid Gal1p galactose-1-phosphate Glc6P 617 glucose-6-phosphate GlycerolP glycerol-3-phosphate Man6P mannose-6-phosphate 618 ManGal-ol mannitol or galactitol ManGlc1P mannose- or glucose-1-phosphate 619 OHPro hydroxyproline Pentoses P pentose-phosphates PEP phosphoenolpyruvate 620 Ribul15bP ribulose-15-bisphosphate S7P sedoheptulose-7-phosphate Suc6P 621 sucrose-6-phosphate trans-aco trans-aconitate Tre6P trehalose-6-phosphate UDP-622 Gal UDP-galactose UDPGlc UDP-glucose 623 624 625 626

TABLES 627 628 Table I Biomass (g) and starch fatty acids and proteins contents ( of dry weight) of leaves culm roots prop roots and grains of 629 Sorghum bicolor cv BRS330 at ambient and elevated CO2 at 90 and 120 days after planting (DAP) Values are the mean plusmn SD of 630 biological replicates n=3 Bold values indicate significant statistic differences between treatments (P lt005) nd = not detected 631

632 633 634

Days after planting

Biomass Starch Fatty acids Proteins

Ambient CO2 Elevated CO2 Ambient CO2 Elevated CO2 Ambient CO2 Elevated CO2 Ambient CO2 Elevated CO2

Leaves 90 2354 plusmn 684 3263 plusmn 54 nd nd 149 plusmn 009 142 plusmn 004 144 plusmn 123 168 plusmn 045

120 1775 plusmn 204 2619 plusmn 38 nd nd 109 plusmn 007 123 plusmn 005 163 plusmn 201 191 plusmn 270

Culm 90 1325 plusmn 451 1549 plusmn 296 nd nd 029 plusmn 002 030 plusmn 003 22 plusmn 026 19 plusmn 008

Roots 90 1074 plusmn 354 917 plusmn 203 nd nd 029 plusmn 002 029 plusmn 001 39 plusmn 015 34 plusmn 022

Prop roots

Grains 90 3474 plusmn 314 3008 plusmn 751 574 plusmn 451 516 plusmn 606 191 plusmn 014 132 plusmn 011 75 plusmn 053 101 plusmn 070

120 6195 plusmn 1692 4691 plusmn 1356 599 plusmn 115 573 plusmn 243 209 plusmn 003 210 plusmn 013 51 plusmn 010 81 plusmn 068

plantphysiolorgon June 18 2020 - P

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LITERATURE CITED 635 636 Alexandratos N Bruinsma J (2012) World agriculture towards 20302050 the 2012 637

revision ESA Working paper No 12-03 Rome FAO 638 Aranjuelo I Sanz-Saacuteez A Jaurengui I Irigoyen JJ Araus JL Saacutenchez-Diacuteaz M Erice G 639

(2013) harvest index a parameter conditioning responsiveness of wheat plants to 640 elevated CO2 Journal of Experimental Botany 64 1879-1892 641

Arenque BC Grandis A Pocius O De Souza AP Buckeridge MS (2014) Responses of 642 Senna reticulata a legume tree from the Amazonian floodplains to elevated 643 atmospheric CO2 concentration and waterlogging Trees 28 1021-1034 644

Beheshti AR Fard BB (2010) Dry matter accumulation and remobilization in grain 645 sorghum genotypes (Sorghum bicolor L Moench) under drought stress Australian 646 Journal of Crop Science 4 185-189 647

Blum A Golan G Mayer J Sinmena B (1997) The effect of dwarfing genes on sorghum 648 grain filling from remobilized stem reserves under stress Field Crops research 649 52 43-54 650

Bohnert HJ Sheveleva E (1998) Plant stress adaptations ndash making metabolism move 651 Current Opinion in Plant Biology 1 267-274 652

Bohnert HJ Shen B (1999) Transformation and compatible solutes Scientia 653 Horticulturae 78 237-260 654

Borrell AK Hammer GL Henzel RG (2000) Does maintaining green leaf area in 655 sorghum improve yield under drought II Dry matter production and yield Crop 656 Science 40 1037-1048 657

Cocuron JC Alonso AP (2014) Liquid chromatography tandem mass spectrometry for 658 measuring 13C-labeling in intermediaries of the glycolysis and pentose-phosphate 659 pathway Methods in Molecular Biology 1090 131-142 660

Cocuron JC Anderson B Boyd A Alonso AP (2014) Targeted metabolomics of Physaria 661 fendleri an industrial crop producing hydroxy fatty acids Plant Cell Physiology 55 662 620-633 663

Conley MM Kimball BA Brooks TJ Pinter Jr PJ Hunsaker HJ Wall GW Adam NR La 664 Morte RL Matthias AD Thompson TL Leavitt SW Ottman MJ Cousins AB 665 Triggs JM (2001) CO2 enrichment increases water-use efficiency in Sorghum 666 New Phytologist 151 407-412 667

Da Matta FM Grandis A Arenque BC Buckeridge MS (2010) Impacts of climate 668 changes on crop physiology and food quality Food Research International 43 669 1814-1823 670

De Souza AP Gaspar M da Silva E A Ulian E C Waclawovsky A J Nishiyama-Jr MY 671 dos Santos RV Teixeira MM Souza G Buckeridge MS (2008) Elevated CO2 672 increases photosynthesis biomass and productivity and modifies gene 673 expression in sugarcane Plant Cell and Environment 311116ndash1127 674

De Souza AP Arundale RA Dohleman FG Long SP Buckeridge MS (2013) Will 675 exceptional productivity of Miscanthus x giganteus increase further under rising 676 atmospheric CO2 Agricultural and Forest Meteorology 171-172 82-92 677

EMBRAPA Maize and Sorghum (2015) Cataacutelogo de produtos e serviccedilos ndash Sorgo BRS 678 330 Available at 679 httpwwwcatalogosntcnptiaembrapabrcatalogo20catalogo_de_produtos_e_s680 ervicosarvoreCONTAG01_156_266200615542html Accessed in June 20 681 2015 682

Epstein E (1972) Mineral nutrition of plants principles and perspectives Wiley New 683 York NY USA 684

FAO Food and Agriculture Organization of the United Nations (2015) Statistics of 685 production Available at httpfaostat3faoorghomeE Accessed in May 25 686 2015 687

Geissler N Hussin S Koyro HW (2009) Elevated atmospheric CO2 concentration 688 ameliorates effects of NaCl salinity on photosynthesis and leaf structure of Aster 689 tripolium L Journal of Experimental Botany 60 137-151 690

Godfray HCJ Beddington JR Crute IR Haddad L Lawrence D Muir JF Pretty J 691 Robinson S Thomas S Toulmin C (2010) Food Security the challenge of feeding 692 9 billion people Science 327 812-818 693

Godfray HCJ Garnett T (2014) Food security and sustainable intensification 694 Philosophical Transactions of the Royal Society of Biological Sciences 369 695 20120273 696

Grashoff C Dijkstra P Nonhebel S Schapendonk AHCM Van de Geijn SC (1995) 697 Effects of climate change on productivity of cereals and legumes model 698 evaluation of observed year-to-year variability of the CO2 response Global 699 Change Biology 1 417-428 700

Gregory PJ (2006) Plant Roots Growth activity and interactions with the soil Wiley-701 Blackwell 328p 702

Jordan DR Hunt CH Cruickshank AW Borrell AK Henzell RG (2012) The relationship 703 between the stay-green trait and grain yield in elite sorghum hybrids grown in a 704 range of environments Crop Science 52 1153ndash1161 705

Kriechbaumer V Glawischnig E (2005) Auxin biosynthesis within the network of 706 tryptophan metabolism Journal of Nano and Bio Tech 2 53-58 707

Krishnan S Laskowski K Shula V Merewitz EB (2013) Mitigation of drought stress 708 damage by exogenous application of a non-protein amino acid γndash aminobutyric 709 acid on perennial ryegrass Journal of the American Society for Horticultural 710 Science 138 358-366 711

Leakey ADB (2009) Rising atmospheric carbon dioxide concentration and the future of 712 C4 crops for food and fuel Proceedings of the Royal Society Biological Sciences 713 276 2333-2343 714

Leakey ADB Bernacchi CJ Dohleman FG Ort DR Long SP (2004) Will photosynthesis 715 of maize (Zea mays) in the US Corn Belt increase in future [CO2] rich 716 atmospheres An analyses of diurnal courses of CO2 uptake under free-air 717 concentration enrichment (FACE) Global Change Biology 10 951- 962 718

Li P Ainsworth EA Leakey ADB Ulanov A Lozovaya V Ort DR Bohnert HJ (2008) 719 Arabidopsis transcript and metabolite profiles ecotype-specific responses to 720 open-air elevated [CO2] Plant Cell and Environment 31 1673-1687 721

Ljung K Hull AK Kowalczyk M Marchant A Celenza J Cohen JD Sandberg G (2002) 722 Biosynthesis conjugation catabolism and homeostasis of indole-3-acetic acid in 723 Arabidopsis thaliana Plant Molecular Biology 49249-272 724

Long SP Ainsworth EA Leakey AD Noumlsberger J Ort DR (2006) Food for thought 725 lower-than-expected crop yield stimulation with rising CO2 concentrations 726 Science 312 1918-1921 727

Madan P Jagadish SVK Craufurd PQ Fitzgerald M Lafarge T Wheeler TR (2012) 728 Effect of elevated CO2 and high temperature in seed-set and grain quality of rice 729 Journal of Experimental Botany 63 695-709 730

Magrin GO Marengo JA Boulanger J-P Buckeridge MS Castellanos E Poveda G 731 Scarano FR Vicuntildea S (2014) Central and South America In Barros VR Field CB 732 Dokken DJ Mastrandrea MD Mach KJ Bilir TE Chatterjee M Ebi KL Estrada 733 YO Genova RC Girma B Kissel ES Levy AN MacCracken S Mastrandrea PR 734

White LL (eds) Climate Change 2014 Impacts Adaptation and Vulnerability 735 Part B Regional Aspects Contribution of Working Group II to the Fifth 736 Assessment Report of the Intergovernmental Panel on Climate Change 737 Cambridge University Press Cambridge United Kingdom and New York NY 738 USA pp 1499-1566 739

Marc J Gifford RM (1983) Floral initiation in wheat sunflower and sorghum under 740 carbon dioxide enrichment Canadian Journal of Botany 62 9-14 741

Markelz RJC Strellner RS Leakey ADB (2011) Impairment of C4 photosynthesis by 742 drought is exacerbated by limiting nitrogen and ameliorated by elevated [CO2] in 743 maize Journal of Experimental Botany 62 3235-3246 744

Meehl GA Stocker TF Collins WD Friedlingstein P Gaye AT Gregory JM Kitoh A 745 Knutti R Murphy JM Noda A Raper SCB Watterson IG Weaver AJ Zhao Z 746 (2007) Global Climate projections In Solomon S Qin D Manning M Chen Z-C 747 marquis M Averty KB Tignor M Miller HL (eds) Climate Change 2007 The 748 physical science basis Contribution of working group I to the Fourth Assessment 749 Report of the Intergovernmental Panel on Climate Change Cambridge University 750 Press UK New York NY USA 751

Nam K-H Shin HJ Pack I-S Park J-H Kim H-B Kim C-G (2015) Metabolomic changes 752 in grains of well-watered and drought stressed transgenic rice Journal of the 753 Science of Food and Agriculture DOI 101002jsfa7152 754

Naudts K Van Den Berge J Janssens IA Nijs I Ceulemans R (2013) Combined effects 755 of warming and elevated CO2 on the impact of drought in grassland species Plant 756 and Soil 369 497-507 757

Nielsen RL (2002) Root lodging concerns in corn Purdue University Department of 758 Agronomy Available at 759 httpswwwagrypurdueeduextcornnewsarticles02RootLodge-0711html 760 Accessed in June 20 2015 761

Noji M Saito K (2003) Sulphur Amino Acids Biosynthesis of Cysteine and Methionine 762 In Abrol YP Ahmad A (eds) Sulphur in Plants Springer Netherlands pp 135-144 763

NOAA - National Oceanic and Atmospheric Administration (2015) Available at 764 httpwwwesrlnoaagovgmdccggtrends Accessed in June 30 2015 765

Ottman MJ Kimball BA Pinter Jr PJ Wall GW Vanderlip RL Leavitt SW LaMorte RL 766 Matthias AD Brooks TJ (2001) Elevated CO2 increases sorghum biomass under 767 drought conditions New Phytologist 150 261-273 768

Paterson AH Bowers JE Bruggmann R Dubchak I Grimwood J Gundlach H Haberer 769 G Hellsten U Mitros T Poliakov A Schutz J Spannagl M Tang H Eang X 770 Wicker T Bharti AK Chapman J Feltus FA Gowik U Grigoriev IV Lyons E 771 Maher CA Martis M Narechania A Otillar RP Penning BW Salamov AA Wang 772 Y Zhang L Carpita NC Freeling M GIngle AR Hash T Keller B Klein P 773 Kresovih S McCann MC Ming R Peterson DG Rahman M Ware D Westhoff P 774 Mayer KFX Messing J Rokhsar DS (2009) The Sorghum bicolor genome and the 775 diversification of grasses Nature 457551-556 776

Pavli OI Vlachos CE Kalloniati C Flemetakis E Skaracis GN (2013) Metabolite profiling 777 reveals the effect of drought on sorghum (Sorghum bicolor L Moench) 778 metabolism Plant Omics Journal 6 371-376 779

Rosenow DT Clark LE (1995) Drought and lodging resistance for quality sorghum crop 780 In Proceedings of the 50th Annual Corn and Sorghum Industry Research 781 Conference held at Chicago Ill 6ndash7 Dec 1995 Am Seed Trade Assoc 782 Washington DC pp 82ndash97 783

Schnyder H (1993) The role of carbohydrates storage and redistribution in the source-784 sink relations of wheat and barley during grain filling ndash a review New Phytologist 785 123 233-245 786

Sicher RC Barnaby JY (2012) Impact of carbon dioxide enrichment on the responses of 787 maize leaf transcripts and metabolites to water stress Physiologia Plantarum 114 788 238-253 789

Silvente S Sobolev AP Lara M (2012) Metabolite adjustments in drought tolerant and 790 sensitive soybean genotypes in response to water stress Plos One 7 e38554 791

Taub DR Miller B Allen H (2008) Effects of elevated CO2 on the protein concentration of 792 food crops a meta-analysis Global Change Biology 14 565-575 793

Taylor JRN Schober TJ Bean SR (2006) Novel food and non-food uses of sorghum and 794 millets Journal of Cereal Science 44 252-271 795

Tilman D Balzerb C Hillc J Beforta BL (2011) Global food demand and the sustainable 796 intensification of agriculture Proceedings of the National Academy of Sciences 797 10820260-20264 798

Tsogtbaatar E Cocuron JC Sonera MC Alonso AP (2015) Metabolite fingerprinting of 799 pennycress (Thlaspi arvense L) embryos to assess active pathways during oil 800 synthesis Journal of Experimental Botany 66 4267-4277 801

Uprety DC Sen S Dwivedi N (2010) Rising atmospheric carbon dioxide on grain quality 802 in crop plants Physiology and Molecular Biology of Plants 16 215-227 803

Valdez V Deshpande SP Kholova J Hammer GL Borrell AK Talwar HS Hash CT 804 (2011) Stay-green quantitative trait locirsquos effects on water extraction transpiration 805 efficiency and seed yield depend on recipient parent background Functional Plant 806 Biology 38 553ndash566 807

Wall GW Brooks TJ Adam NR Cousins AB Kimball BA Pinter Jr PJ LaMorte RL 808 Triggs J Ottman MJ Leavitt SW Matthias AD Williams DG Webber AN (2001) 809 Elevated atmospheric CO2 improved Sorghum plant water status by ameliorating 810 the adverse effects of drought New Phytologist 152 231-248 811

Wenzel A Frank T Reichenberger G Herz M Engel K-H (2015) Impact of induced 812 drought stress on the metabolite profiles of barley grain Metabolomics 11 454-467 813

Xia J Mandal R Sinelnikov IV Broadhurst D Wishart DS (2012) MetaboAnalyst 20--a 814 comprehensive server for metabolomic data analysis Nucleic Acids Res 40 W127-815 133 816

Xia J Psychogios N Young N Wishart DS (2009) MetaboAnalyst a web server for 817 metabolomic data analysis and interpretation Nucleic Acids Res 37 W652-660 818

Xia J Sinelnikov I Han B Wishart DS (2015) MetaboAnalyst 30 - making metabolomics 819 more meaningful Nucleic Acids Res (DOI 101093nargkv380) 820

Zinta G Abdelgawad H Domagalska MA Vergauwen L Knapen D Nijs I Janssens IA 821 Beemster GTS Asard H (2014) Physiological biochemical and genome-wide 822 transcriptional analysis reveals that elevated CO2 mitigates the impact of combined 823 heat wave and drought stress in Arabidopsis thaliana at multiple organizational levels 824 Global Change Biology 20 3670-3685 825

Figure 1

Figure 1 (A) Open-top chambers used during the experiment to grow Sorghum bicolor cv BRS330 at

ambient and elevated CO2 (B) Experimental design

Figure 2

Figure 2 Soil moisture (cm3 of water per cm3 of soil) during the experiment with Sorghum bicolor cv

BRS330 at ambient and elevated CO2 n=8 Water deficit treatment started after 60 days after planting

(DAP) From 90 to 93 DAP soil moisture sensors from pots at elevated CO2 did not log the data (gray

dashed line)

Figure 3

Figure 3 (A) Light saturated rate of leaf photosynthesis (A) (B) Stomatal conductance (gs) and (C) Dark

leaf respiration (Rd) of the youngest fully expanded leaf of Sorghum bicolor cv BRS330 at ambient and

elevated CO2 at 90 and 120 days after planting (DAP) Bars are the mean SD of biological replicates

n=4 Asterisks indicate significant statistic differences between treatments (P lt005)

Figure 4

Figure 4 (A) Plant height (without panicle) (cm) and (B) Panicle size (cm) of Sorghum bicolor

cv BRS330 at ambient and elevated CO2 along the experiment Data points are the mean SD

of biological replicates n=4 Asterisks indicate significant statistic differences between treatments

(P lt005)

Figure 5

Figure 5 Principal component analysis (PCA) of the metabolites identified by LC-MS-MS of Sorghum

bicolor cv BRS330 at ambient (open symbols) and elevated CO2 (closed symbols) at 90 (squares) and

120 days (circles) after planting (DAP) P-values indicate significant statistic differences of PCs

Figure 6

Figure 6 Principal component analysis (PCA) of the

metabolites identified by LC-MS-MS of leaves culm

roots prop roots and grains of Sorghum bicolor cv

BRS330 at ambient and elevated CO2 at 90 and 120 days

after planting (DAP) P-values indicate significant statistic

differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

Figure 7 Heatmaps showing the

variation of amount of metabolites in

Sorghum bicolor (cv BRS 330) grown

under ambiant and elevated [CO2] at

90 and 120 DAP in (A) leaves (B)

culm (C) roots (D) prop roots and (E)

grains Abbreviations 23PGA 2 or 3-

phosphoglycerate 6PG 6-

phophosgluconate ADPGlc ADP-

glucose AKG alpha-ketoglutarate cis-

aco cis-aconitate Deoxyxyl5P

deoxyxylulose-5-phosphate E4P

erythrose-4-phosphate F16bP

Fructose-16-bisphosphate Fru6P

fructose-6-phosphate GABA gamma-

aminobutyric acid Gal1p galactose-1-

phosphate Glc6P glucose-6-

phosphate GlycerolP glycerol-3-

phosphate Man6P mannose-6-

phosphate ManGal-ol mannitol or

galactitol ManGlc1P mannose- or

glucose-1-phosphate OHPro

hydroxyproline Pentoses P pentose-

phosphates PEP

phosphoenolpyruvate Ribul15bP

ribulose-15-bisphosphate S7P

sedoheptulose-7-phosphate Suc6P

sucrose-6-phosphate trans-aco trans-

aconitate Tre6P trehalose-6-

phosphate UDP-Gal UDP-galactose

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

Parsed CitationsAlexandratos N Bruinsma J (2012) World agriculture towards 20302050 the 2012 revision ESA Working paper No 12-03 RomeFAO

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Aranjuelo I Sanz-Saacuteez A Jaurengui I Irigoyen JJ Araus JL Saacutenchez-Diacuteaz M Erice G (2013) harvest index a parameterconditioning responsiveness of wheat plants to elevated CO2 Journal of Experimental Botany 64 1879-1892

Arenque BC Grandis A Pocius O De Souza AP Buckeridge MS (2014) Responses of Senna reticulata a legume tree from theAmazonian floodplains to elevated atmospheric CO2 concentration and waterlogging Trees 28 1021-1034

Beheshti AR Fard BB (2010) Dry matter accumulation and remobilization in grain sorghum genotypes (Sorghum bicolor L Moench)under drought stress Australian Journal of Crop Science 4 185-189

Blum A Golan G Mayer J Sinmena B (1997) The effect of dwarfing genes on sorghum grain filling from remobilized stemreserves under stress Field Crops research 52 43-54

Bohnert HJ Sheveleva E (1998) Plant stress adaptations - making metabolism move Current Opinion in Plant Biology 1 267-274Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bohnert HJ Shen B (1999) Transformation and compatible solutes Scientia Horticulturae 78 237-260Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Borrell AK Hammer GL Henzel RG (2000) Does maintaining green leaf area in sorghum improve yield under drought II Drymatter production and yield Crop Science 40 1037-1048

Cocuron JC Alonso AP (2014) Liquid chromatography tandem mass spectrometry for measuring 13C-labeling in intermediaries ofthe glycolysis and pentose-phosphate pathway Methods in Molecular Biology 1090 131-142

Cocuron JC Anderson B Boyd A Alonso AP (2014) Targeted metabolomics of Physaria fendleri an industrial crop producinghydroxy fatty acids Plant Cell Physiology 55 620-633

Conley MM Kimball BA Brooks TJ Pinter Jr PJ Hunsaker HJ Wall GW Adam NR La Morte RL Matthias AD Thompson TLLeavitt SW Ottman MJ Cousins AB Triggs JM (2001) CO2 enrichment increases water-use efficiency in Sorghum NewPhytologist 151 407-412

Da Matta FM Grandis A Arenque BC Buckeridge MS (2010) Impacts of climate changes on crop physiology and food quality FoodResearch International 43 1814-1823

De Souza AP Gaspar M da Silva E A Ulian E C Waclawovsky A J Nishiyama-Jr MY dos Santos RV Teixeira MM Souza GBuckeridge MS (2008) Elevated CO2 increases photosynthesis biomass and productivity and modifies gene expression insugarcane Plant Cell and Environment 311116-1127

De Souza AP Arundale RA Dohleman FG Long SP Buckeridge MS (2013) Will exceptional productivity of Miscanthus x giganteusincrease further under rising atmospheric CO2 Agricultural and Forest Meteorology 171-172 82-92

EMBRAPA Maize and Sorghum (2015) Cataacutelogo de produtos e serviccedilos - Sorgo BRS 330 Available athttpwwwcatalogosntcnptiaembrapabrcatalogo20catalogo_de_produtos_e_servicosarvoreCONTAG01_156_266200615542htmlAccessed in June 20 2015

Epstein E (1972) Mineral nutrition of plants principles and perspectives Wiley New York NY USAPubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

FAO Food and Agriculture Organization of the United Nations (2015) Statistics of production Available athttpfaostat3faoorghomeE Accessed in May 25 2015

Geissler N Hussin S Koyro HW (2009) Elevated atmospheric CO2 concentration ameliorates effects of NaCl salinity onphotosynthesis and leaf structure of Aster tripolium L Journal of Experimental Botany 60 137-151

Godfray HCJ Beddington JR Crute IR Haddad L Lawrence D Muir JF Pretty J Robinson S Thomas S Toulmin C (2010) FoodSecurity the challenge of feeding 9 billion people Science 327 812-818

Godfray HCJ Garnett T (2014) Food security and sustainable intensification Philosophical Transactions of the Royal Society ofBiological Sciences 369 20120273

Grashoff C Dijkstra P Nonhebel S Schapendonk AHCM Van de Geijn SC (1995) Effects of climate change on productivity ofcereals and legumes model evaluation of observed year-to-year variability of the CO2 response Global Change Biology 1 417-428

Gregory PJ (2006) Plant Roots Growth activity and interactions with the soil Wiley-Blackwell 328pPubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Jordan DR Hunt CH Cruickshank AW Borrell AK Henzell RG (2012) The relationship between the stay-green trait and grain yieldin elite sorghum hybrids grown in a range of environments Crop Science 52 1153-1161

Kriechbaumer V Glawischnig E (2005) Auxin biosynthesis within the network of tryptophan metabolism Journal of Nano and BioTech 2 53-58

Krishnan S Laskowski K Shula V Merewitz EB (2013) Mitigation of drought stress damage by exogenous application of a non-protein amino acid - aminobutyric acid on perennial ryegrass Journal of the American Society for Horticultural Science 138 358-366

Leakey ADB (2009) Rising atmospheric carbon dioxide concentration and the future of C4 crops for food and fuel Proceedings ofthe Royal Society Biological Sciences 276 2333-2343

Leakey ADB Bernacchi CJ Dohleman FG Ort DR Long SP (2004) Will photosynthesis of maize (Zea mays) in the US Corn Beltincrease in future [CO2] rich atmospheres An analyses of diurnal courses of CO2 uptake under free-air concentration enrichment wwwplantphysiolorgon June 18 2020 - Published by Downloaded from

increase in future [CO2] rich atmospheres An analyses of diurnal courses of CO2 uptake under free-air concentration enrichment(FACE) Global Change Biology 10 951- 962

Li P Ainsworth EA Leakey ADB Ulanov A Lozovaya V Ort DR Bohnert HJ (2008) Arabidopsis transcript and metabolite profilesecotype-specific responses to open-air elevated [CO2] Plant Cell and Environment 31 1673-1687

Ljung K Hull AK Kowalczyk M Marchant A Celenza J Cohen JD Sandberg G (2002) Biosynthesis conjugation catabolism andhomeostasis of indole-3-acetic acid in Arabidopsis thaliana Plant Molecular Biology 49249-272

Long SP Ainsworth EA Leakey AD Noumlsberger J Ort DR (2006) Food for thought lower-than-expected crop yield stimulation withrising CO2 concentrations Science 312 1918-1921

Madan P Jagadish SVK Craufurd PQ Fitzgerald M Lafarge T Wheeler TR (2012) Effect of elevated CO2 and high temperature inseed-set and grain quality of rice Journal of Experimental Botany 63 695-709

Magrin GO Marengo JA Boulanger J-P Buckeridge MS Castellanos E Poveda G Scarano FR Vicuntildea S (2014) Central and SouthAmerica In Barros VR Field CB Dokken DJ Mastrandrea MD Mach KJ Bilir TE Chatterjee M Ebi KL Estrada YO Genova RCGirma B Kissel ES Levy AN MacCracken S Mastrandrea PR White LL (eds) Climate Change 2014 Impacts Adaptation andVulnerability Part B Regional Aspects Contribution of Working Group II to the Fifth Assessment Report of the IntergovernmentalPanel on Climate Change Cambridge University Press Cambridge United Kingdom and New York NY USA pp 1499-1566

Marc J Gifford RM (1983) Floral initiation in wheat sunflower and sorghum under carbon dioxide enrichment Canadian Journal ofBotany 62 9-14

Markelz RJC Strellner RS Leakey ADB (2011) Impairment of C4 photosynthesis by drought is exacerbated by limiting nitrogen andameliorated by elevated [CO2] in maize Journal of Experimental Botany 62 3235-3246

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CHANGES IN WHOLE-PLANT METABOLISM DURING GRAIN-FILLING STAGE IN 35 SORGHUM BICOLOR L (MOENCH) GROWN UNDER ELEVATED CO2 AND DROUGHT 36 37 38 Amanda P De Souza1 Jean-Christophe Cocuron23 Ana Carolina Garcia1 Ana Paula Alonso2 39 Marcos S Buckeridge1 40 41 1 Laboratory of Plant Physiological Ecology (LAFIECO) Department of Botany Institute of 42 Biosciences University of Sao Paulo 05508-090 Sao Paulo Brazil 43 2 Department of Molecular Genetics The Ohio State University Columbus OH 43210 USA 44 3 Center for Applied Plant Sciences The Ohio State University Columbus OH 43210 USA 45 Corresponding authors 46 47 48

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

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90 90120 120 DAP

E Grains

phosphates PEP

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D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

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90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

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90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

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90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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(P lt005)

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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(P lt005)

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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(P lt005)

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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(P lt005)

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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Figure 2

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(P lt005)

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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Article File

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Figure 3

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(P lt005)

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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Figure 4

(P lt005)

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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Article File

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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Article File

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differences of PCs

C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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C RootsB Culm

90 90120 120 DAP90 90120 120 DAP

A Leaves

90 90120 120 DAP

E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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E Grains

phosphates PEP

UDPGlc UDP-glucose

D Prop rootsAmbient

Elevated90 90120 120 DAP90 90120 120 DAP

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RUNNING HEAD CORRESPONDING AUTHOR INFORMATION …5 Name: Ana Paula Alonso 6 Address: Department of Molecular Genetics, The Ohio State University, 1060 Carmack Road, 7 Columbus, OH