Drought priming induces thermo-tolerance to post-anthesis high-temperature in offspring of winter wheat

Accepted Manuscript Title: Drought priming induces thermo-tolerance to post-anthesis high-temperature in offspring of winter wheat Author: Xiaxiang Zhang Xiulin Wang Jianwen Zhong Qin Zhou Xiao Wang Jian Cai Tingbo Dai Weixing Cao Dong Jiang PII: DOI: Reference:

S0098-8472(16)30050-8 http://dx.doi.org/doi:10.1016/j.envexpbot.2016.03.004 EEB 3040

To appear in:

Environmental and Experimental Botany

Received date: Revised date: Accepted date:

11-1-2016 9-3-2016 11-3-2016

Please cite this article as: Zhang, Xiaxiang, Wang, Xiulin, Zhong, Jianwen, Zhou, Qin, Wang, Xiao, Cai, Jian, Dai, Tingbo, Cao, Weixing, Jiang, Dong, Drought priming induces thermo-tolerance to post-anthesis high-temperature in offspring of winter wheat.Environmental and Experimental Botany http://dx.doi.org/10.1016/j.envexpbot.2016.03.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title: Drought priming induces thermo-tolerance to post-anthesis high-temperature in offspring of winter wheat Authors: Xiaxiang Zhang1, Xiulin Wang1, Jianwen Zhong1, Qin Zhou1, Xiao Wang1; Jian Cai1, Tingbo Dai1, Weixing Cao 1, Dong Jiang*1 Affiliations: 1National Technology Innovation Center for Regional Wheat Production / National Engineering and Technology Center for Information Agriculture / Key Laboratory of Crop Physiology and Ecology in Southern China, Ministry of Agriculture, Nanjing Agricultural University, P. R. China *Correspondence author: Dong Jiang Correspondence address: College of Agriculture, Nanjing Agricultural University, No. 1 Weigang Road, Nanjing 210095, P. R. China Tel. & Fax: +86 25 8439 6575 E-mail: [email protected]

Highlights 

Drought priming on wheat parents induced thermo-tolerance in their offspring

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Up-regulated anti-oxidation and Pn contributed to the thermo-tolerance acquaintance

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Modified protein expressions caused less Pn reduction of primed offspring plants

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Robust signaling and HSPs played vital roles in the drought-to-heat tolerance

Abstract High temperature stress is a worldwide environmental constraint on crop production. Exposure of plants to a stress event could induce tolerance in them and even in their offspring to subsequent stresses. This study was to test the hypothesis that drought priming of parent plants could trigger a cross tolerance to hightemperature stress in their offspring. Winter wheat plants were used and were firstly subjected to a drought stress during grain filling, and their offspring were exposed to a post-anthesis high-temperature stress. Grain yield, photosynthesis, anti-oxidation, and proteomic profile in wheat flag leaves of the offspring were determined. The results showed a less yield loss in the next generation of the wheat plants that had received drought priming, which could be ascribed to the improved photosynthesis because of the up-regulated expression of proteins involved in the light reaction and in the Calvin cycle, and to the enhanced anti-oxidation capacity as exemplified by the decreased contents of MDA and H2O2 because of the improved activities of SOD and POD. In addition, the proteomic analysis suggested that the enhanced thermo-tolerance could also be attributed to the more robust signal perception and transduction, better maintenance of protein structures, up-regulation of sucrose synthesis and accumulation of heat shock proteins.

Keywords: Drought priming; heat stress; photosynthesis; thermo-tolerance; wheat (Triticum aestivum L.)

1. Introduction Plants in the field often suffer from abiotic stresses depressing their growth, development and productivity (Suzuki et al., 2014). Drought and heat stresses are the major adversities (Rollins et al., 2013). Furthermore, drought and heat waves are getting more frequent due to global warming (Mackova et al., 2013). Wheat, as one of the most important staple food crops, is reported to be very susceptible to hightemperature stresses which often occur during grain filling in early summer and result in great yield loss (Barnabas et al., 2008). Therefore, improving tolerance to hightemperature stresses during grain filling is of great significance to crop production. It has been found that priming of plants, e.g. pre-exposing them to a biotic or abiotic stress, can induce tolerance in them to subsequent stresses (Bruce et al., 2007). Primed plants display faster and/or stronger activation of various defense responses to the re-imposed attack of biotic or abiotic stresses (Conrath et al., 2006). These phenomena may be ascribed to the changes in some key signaling processes, transcription factors and epigenetic modification (Bruce et al., 2007). For instance, heat priming of winter wheat plants during vegetative growth effectively improved their thermo-tolerance during grain filling by enhancing their anti-oxidation and maintaining the function of their photosynthetic apparatuses (Wang et al., 2014). Recently, we found that drought priming not only improved resistance to post-anthesis drought stress but also enhanced thermo-tolerance by increasing the ABA level and decreasing energy disperse in wheat leaves (Wang et al., 2015). It is interesting that the priming effects on plants could be passed on to the next generation (Molinier et al., 2006). Research on Arabidopsis thaliana indicated that changes in environmental factors increased genomic flexibility in the plant leading to the potential of adaptive evolution (Molinier et al., 2006). In rice, pre-exposure to

sub-lethal heat improved thermo-tolerance of the plant at the vegetative stage within and across generations (Shi et al., 2015). This stress imprint was also observed in tobacco, radish and alfalfa (Bruce et al., 2007; Choi and Sano, 2007; Vu et al., 2015). However, little is known if it is true of wheat. Drought and heat stresses, as the major abiotic stresses that depress crop production, often occur simultaneously, especially in the semi-arid or drought-stricken areas (Rizhsky et al., 2004). Plants close their leaf stomata to avoid transpiration, which reduces CO2 assimilation in leaf and lowers the photosynthetic rate under drought and heat stresses (Barnabas et al., 2008; Farooq et al., 2011). In addition, both drought and heat stresses cause damage to membrane integrity and lipid peroxidation in leaf, leading to stress-induced premature senescence (Silva et al., 2010). Based on these physiological processes in plants, we hypothesized that drought priming of parent plants could trigger cross tolerance to high-temperature stress in their offspring. To test this hypothesis, we pre-treated the parent plants of wheat with drought during grain filling, and then subjected their offspring to a post-anthesis hightemperature stress. The variations in gas exchange, the quantum yield of PSII reaction centers, the antioxidant system, and the proteomic changes in the flag leaf were monitored. The results would help to better understand the mechanisms of thermotolerance in offspring plants.

2. Materials and methods 2.1 Drought priming treatment in the parent generation The semi-field experiment was conducted in the Experimental Station of Nanjing Agricultural University, Nanjing (32°08' N and 118°51' E), Jiangsu Province, P. R. China in 2011- 2012. A local broadly grown winter wheat cultivar, Yangmai 16

(Triticum aestivum L.), was grown in plastic pots (22cm in height and 25cm in diameter). Each pot was filled with 7.5 kg of clay soil mixed with 0.9 g N, 0.36 g P2O5, and 0.9 g K2O. Another 1.6 g N per pot was top-dressed at the jointing stage. The sowing rate was 15 seeds per pot, and the seedlings were thinned to six at the three-leaf stage. At 10 d after anthesis, half of the pots were withheld from watering for the drought priming treatment. The treat lasted 7 d, and then the plants were rewatered and grown under field conditions till harvest. Grains of each treatment were separately harvested for the offspring experiments. Grains from three pots for each treatment were harvested as three biological replicates. Soil relative water content (SRWC) and leaf relative water content (LRWC) after drought priming and the effect of priming on 1000-kernel weight of the parent plants were listed in supplementary Table 1. 2.2 High-temperature stress treatment in the offspring generation The harvested grains were sowed in the next wheat growth season of 2012-2013. The overall experimental conditions were the same as the experiment described above. At 10 d after anthesis, half of both drought-primed (D-) and non-primed (ND-) offspring were subjected to high-temperature stress with a day/night temperature of 35/27°C in a growth chamber. The rest plants were moved into another chamber with a day/night temperature of 28/20°C. The high-temperature stress lasted for 5 d. After the treatment, all the plants were moved out and grown under semi-field condition till harvest. On the last day of the high temperature stress event, the flag leaves of each treatment were sampled and immediately preserved in liquid nitrogen, and then stored at -80 ˚C until analysis of enzyme activity and proteomics. The conditions of the climate chambers over the period of post-anthesis high temperature event and the climate data of the two wheat growing seasons were provided in the supplementary

files (Fig S1, S2 and S3). In total, four treatments were established: D-C, post-anthesis drought priming of the parent generation + no post-anthesis heat stress of the offspring generation; ND-C, no post-anthesis drought priming of the parent generation + no post-anthesis heat stress of the offspring generation; D-H, post-anthesis drought priming of the parent generation + high-temperature stress after anthesis during the offspring generation; ND-H, no post-anthesis drought priming of the parent generation + post-anthesis heat stress of the offspring generation. 2.3 Gas exchange and SPAD values Gas exchange of flag leaf was measured on the last day of post-anthesis heat stress, using a LI-6400 system (LI-COR Inc., USA) equipped with a standard 2×3 cm chamber with light-emitting diode (LED) light sources. All the measurements were taken at a CO2 concentration of ca.380 μmol mol-1 under a light level of 1000 μmol m2

s-1 and a constant flow rate of 500 μmol s-1. SPAD values of flag leaves were

measured using the Minolta SPAD meter (Konica Minolta Optics Inc., Tokyo, Japan). 2.4 Leaf chlorophyll fluorescence image On the same day of gas exchange measurement, the flag leaves used for gas exchange measurement were taken for chlorophyll fluorescence image analysis. The chlorophyll fluorescence images of Fv/Fm (maximum quantum efficiency of PSII photochemistry) were taken using a FluorImager (CF Imager, Technologia Ltd, Colchester, Essex, UK). Plants were dark-adapted for about half an hour before measuring. Leaves were put in the middle of the stage of the FluorImager and were secured with a clip. The chlorophyll a fluorescence parameters were obtained by analyzing the images with the FluorImager software (FluorImager 2.2) following the user manual.

2.5 Activities of antioxidant enzymes, contents of MDA and H2O2 The activities of superoxide dismutase (SOD, EC 1.15.1.1) and peroxidase (POD, EC 1.11.1.7) in fresh flag leaves were measured following our previous methods (Tan et al., 2008). The content of MDA (malondialdehyde) was measured by a spectrophotometer at 532 and 600 nm according to the method of Du and Bramlage (1992).The content of H2O2 was measured following the methods of Sui et al. (2007) by monitoring the absorbance of titanium peroxide complex at 410 nm. 2.6 Protein extraction and 2-DE analysis The protein extraction of flag leaves was performed following a modified version of the trichloroacetic acid (TCA) acetone precipitation method (Ding et al., 2010; Hurkman and Tanaka, 1986). Two to three grams of leaf samples were finely ground in liquid nitrogen, and precipitated overnight after adding 10 volumes of precold acetone with 10% TCA (w/v), 1 mM PMSF and 10 mM DTT at -40˚C. The extract was then centrifuged at 20, 000 g for 15 min at 4˚C with a refrigerated highspeed centrifuge (Avanti J-30I, Beckman Coulter, USA). The pellet was washed with cold acetone containing 1 mM PMSF and 10 mM DTT, and incubated at -40˚C for 1 h, and then centrifuged at 20, 000 g for 15 min at 4˚C. This procedure was performed in three repetitions. Afterwards, the pellet was washed again with ethanol/ether (V:V, 1:1) containing 1 mM PMSF and 10 mM DTT, and incubated at -40˚C for 1 h, and centrifuged at 20, 000 g for 15 min at 4˚C. The purified protein pellets were dried at room temperature, and then dissolved for 1 h in solubilization buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 1 mM PMSF and 10 mM DTT, and centrifuged at 35, 000 g for 20 min at 4˚C. The supernatants were collected and protein concentrations were determined with the Bradford method (Bradford, 1976).

Seventeen cm Immobiline DryStrip gels (Bio-Rad, Hercules, CA, USA) with linear pH range 4-7 were used for isoelectric focusing (IEF) in the first dimension of the electrophoresis. Rehydration and focusing were performed with the PROTEAN i12 IEF system (Bio-Rad) under 50 mA per strip at 20 ˚C, following the program: 12 h of rehydration at 50V in rehydration buffer (7M urea, 2M thiourea, 4% (w/v) CHAPS, 0.5% (v/v) IPG buffer, 10mM DTT, and 0.1% bromophenol blue); 1 h with 500 V; 1 h with 1000 V; 2 h with 8000 V; and 85 000Vh with 8000 V. After isoelectro focusing, the strips were equilibrated for 15 min in SDS equilibration buffer solution (6M urea, 37.5mM Tris–HCl (pH 6.8), 20% (v/v) glycerol, 2% (w/v) SDS, and 1% (w/v) DTT), followed by equilibration with buffer containing 135mM iodoacetamide for 15 min. After equilibration, SDS-PAGE was conducted on a PROTEAN Plus Dodeca apparatus (Bio-Rad) with 10% polyacylamide gels (250×200×1 mm), and then the gels were stained with silver nitrate. The 2-DE gels were scanned using a GS-800 Calibrated Densitometer image system (Bio-Rad) and analyzed with the PDQuest software (version 8.0, Bio-Rad). Manual inspection and editing were conducted to verify the auto-detected and matched results. Proteins with average spot intensities that increased or decreased by a two-fold between the treatments according to t-test, and significance was determined at the p<0.05 levels. The stained protein spots were excised manually from the gels, digested by trypsin, and analyzed by a MALDI-TOF/TOF mass spectrometer (ABI 4800;

Framingham,

MA,

USA).

The

MASCOT database

search

engine

(http://www.matrixscience.com) was used to search for peptide mass lists from the obtained spectra against the NCBI database (http://www.ncbi.nih.gov/). All the differently expressed proteins were distributed to different subcellular compartments by SUBA32 (Tanz et al., 2013) and functionally categorized by Mapman (Thimm et

al., 2004). The heat map of differently expressed proteins was drawn with HemI 1.0 software (Deng et al., 2014). 2.7 Statistical analysis Data collected in the first growth season (the parent plants) were subjected to one-way ANOVA using SPSS package Ver. 22.0 (SPSS Inc., Chicago, IL, USA), and data collected in second growth season (the offspring plants) were subjected to twoway ANOVA. The Duncan’s multiple range test was used to check the significance of difference between treatments.

3. Results 3.1 Grain yield and yield components High-temperature stress during grain filling significantly decreased grain yield of wheat (Fig. 1). Compared with ND-C, grain yields in ND-H and D-H treatments were 16.46% and 11.05% lower, respectively. The yield loss was mainly by the obviously lowered 1000-kernal weight rather than insignificant change in the number of spikes and kernels per spike. In addition, the yield loss of D-H plants was lower (6.47%) than the ND-H plants. 3.2 Photosynthesis, chlorophyll fluorescence image and SPAD value of leaf There was no significant difference in the net photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO2 concentration (Ci) and transpiration rate (Tr) between the D-C and ND-C plants, indicating that drought priming in the parent generation did not affect gas exchange in the leaf of the offspring generation under non-heat stress condition (Fig. 2). However, the post-anthesis high-temperature stress significantly decreased Pn and gs of the offspring plants. It is interesting that, in relation to ND-C, the reduction of Pn was 29.30% in ND-H, much higher than that in

D-H (18.24%). Tr and gs were insignificantly higher in D-H than in ND-H, while Ci was lower. In addition, the high-temperature stress also depressed the maximal photochemical efficiency (Fv/Fm) of leaf (Fig. 3). However, the Fv/Fm of D-H was slightly higher than that of ND-H. The similar trend was found to the SPAD values. These results indicated that the drought priming in the parent generation effectively alleviated the depression on the photosynthesis in leaf induced by the hightemperature stress applied to the offspring generation. 3.3 The anti-oxidation level of leaf The activities of SOD and POD were significantly higher under high-temperature stresses, with 1.30 and 1.31 fold change respectively, in ND-H in relation to ND-C. The activities of SOD and POD were 9.09% and 32.98% higher respectively in D-H than in ND-H plants (Fig. 4). The contents of MDA and H2O2, indicators of the oxidation level in plant organs, obviously rose to high levels under the hightemperature stress. However, the contents of MDA and H2O2 were notably lower in D-H than in ND-H. The mitigated effects on peroxidation in the offspring leaves induced by the post-anthesis heat stress were obtained via drought priming in the parent generation. 3.4 Proteomics analysis Around 600 protein spots were detected on the 2-DE gels. The representative 2DE gel was shown in Fig. 5, in which 32 differentially expressed spots were identified by the mass spectrometry (Table 1). The plastidial proteins shared the largest fraction of the differentially expressed proteins (40.63%), followed by the cytosolic proteins (21.88%), endoplasmic reticulum proteins (18.75%) and mitochondrial proteins (9.38%). According to the putative physiological function analyzed by the Mapman, the proteins were further classified into 13 categories, related to photosynthesis

(34.38%), stress (15.63%), glycolysis (12.50%), redox (6.25%) and protein (6.25%) and so on. There were 17 spots differentially expressed with more than 2-fold changes between D-C and ND-C, with 15 protein spots up-regulated and 2 down-regulated. The up-regulated protein included Luminal-binding protein 3 (spot 2), heat shock protein 70 (HSP 70, spot 4), ATP synthase CF1 alpha subunit (spots 8, 32) and Fructose-1,6-bisphosphatase (spot 26), Carotenoid cleavage dioxygenase (spot 31), while the down-regulated proteins included Ribulose bisphosphate carboxylase activase B (spot 23) and Glycine decarboxylase P subunit (spot 29). Comparing with ND-C, 6 proteins increased in abundance by more than two times in ND-H, including Glyceraldehyde-3-phosphate dehydrogenase A (spot 29), Carotenoid cleavage dioxygenase (spot 31), and so on. Hsp70-Hsp90 organizing protein (HOP, spot 9) decreased in abundance ND-H vs. ND-C. These above all mentioned proteins could be related to the effect of drought priming on parents and high-temperature stress on offspring, respectively. The differentially expressed proteins between D-H and ND-H treatments should be closely related to the enhanced thermo-tolerance in offspring because of the drought priming on parent plants. Here, the category of photosynthesis-related proteins (11 spots) accounted for the largest share of the differentially expressed proteins between D-H and ND-H plants (Fig. 6). Up-regulation by the drought priming treatment on parent plants was observed of almost all the proteins including the transketolase (spots 6 and 7), ATP synthase CF1 alpha subunit (spot 8), Rubisco large subunit-binding protein (spot 14), ATPase beta subunit (spot 21), ribulose bisphosphate carboxylase activase (spot 23), Ferredoxin-NADP(H) oxidoreductase (spot 25), rubisco activase alpha form precursor (spot 30) and ATP synthase alpha

subunit (spot 32) (Fig. 7). The down-regulated proteins included the glyceraldehyde3-phosphate dehydrogenase A (spot 28) and glycine decarboxylase P subunit (spot 29). The second largest category was the stress-related proteins (5 spots), including the luminal binding protein (spots 2 and 3), HSP (spots 4 and 5) and HOP (spot 9), which were all up-regulated by the drought-priming. Proteins involved in glycolysis were also up-regulated by the drought priming, such as enolase (spot 17) and phosphoglycerate mutase (spots 18, 19 and 20). 6.25% of the differently expressed proteins were identified as redox-related. Protein disulfide isomerase (spots 12 and 13) increased in abundance in D-H plants in relation to ND-H plants. In addition, mitochondrial processing peptidase subunit (spot 22) identified as the protein-related category increased in in abundance D-H plants compared with ND-H, while the elongation factor G (spot 1) decreased.

4. Discussion The inheritance of stress tolerance by the next generation has been reported in several plant species (Bruce et al., 2007; Choi and Sano, 2007; Vu et al., 2015). However, little is known if heat stress tolerance and drought-to-heat stress cross tolerance pass from one generation to the next in wheat. In the present study, the parent plants of wheat were primed with drought during grain filling, and their offspring were subjected to a post-anthesis high-temperature stress. Drought priming was observed to transfer thermo-tolerance from the parent plants to their offspring, as exemplified by increase in grain yield, photosynthesis and anti-oxidation and proteomic profile in leaf of D-H, as compared with ND-H. Post-anthesis heat stress resulted in significant loss in grain yield due to the

obvious decrease in 1000-kernel weight of wheat (Fig. 1). However, the droughtprimed offspring (D-H) showed less reduction in 1000-kernel weight than the nondrought-primed offspring (ND-H) did. This was consistent with the study on potato, which also proved that drought priming increased tuber yield of the plants (Ramirez et al., 2015). The grain yield of wheat is predominantly accounted for by the concurrent photosynthates during grain filling. The quantity of the concurrent photosynthates depends on the performance of photosynthesis in leaf. (Wang et al., 1997) It is known that photosynthesis is a process susceptible to heat stress, and the photosystem II (PSII) is the most sensitive components of the photosynthetic apparatus in response to heat stresses (Camejo et al., 2005; Yan et al., 2012). In this study, the post-anthesis heat stress significantly decreased the photosynthetic rate, and Fv/Fm which reflects the maximum quantum efficiency of PSII photochemistry. However, the D-H plants showed much higher Pn and Fv/Fm than the ND-H plants (Fig. 2 and Fig. 3). This indicated that drought-priming of the parent generation effectively alleviated the depression on photosynthesis induced by the high-temperature stress in the offspring generation. These results were in accordance with previous studies that higher Pn and Fv/Fm were detected in drought-primed plants under a recurrent stress condition (Walter et al., 2011). Photosynthesis is mainly inhibited by high photorespiration, low activity of Rubisco, and damage to chloroplast ultrastructure, thylakoid membrane and the PSII center (Allakhverdiev et al., 2008; Chen et al., 2012). The proteome profile in the present experiment well explained the performance of photosynthesis in response to the drought priming of the parent plants and the high-temperature stress on their offspring. The glycine decarboxylase P subunit (spot 29) functioning in the

photorespiration process was less expressed in D-H than in ND-H (Fig. 7), indicating that the photorespiration loss caused by the high-temperature stress was less serious in the offspring of the drought-primed parents than those from the non-primed parents. Rubisco subunits play pivotal roles in determining the relative rate of photosynthesis to photorespiration, and they are the major evaluation factors for improvement of crop yield (Ellis and Van Der Vies, 1988). It was reported that reduced photosynthesis under heat could be counter- balanced by the activation of Rubisco (Rollins et al., 2013). Here, the Rubisco large subunit-binding protein, Ribulose bisphosphate carboxylase activase B and Rubisco activase (spots 14, 23 and 30), were up-regulated in D-H as compared with ND-H. The transketolase (TKL, spots 6 and 7), converting sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate to D-xylulose-5-phosphate in the Calvin cycle (Flechner et al., 1996), was also enhanced in D-H. The light dependent reaction of photosynthesis includes processes of light absorption, photosynthetic electron transport and photophosphorylation, resulting in the conversion of light energy into chemical energy in the form of ATP (Knaff and Arnon, 1969; Kramer et al., 2004). Ferredoxin- NADP(H) oxidoreductase (FNR) is a key enzyme in transferring electron from ferredoxin (Fd) to NADP(H), which is the last step in the photosynthetic electron transport chain (Benz et al., 2009). In this study, the proteins involved in electron transport (spot 25) and ATP synthesis (spots 8, 21 and 32) were also found up-regulated in D-H than in ND-H. Therefore, the enhanced activities of key enzymes in light reaction and in the Calvin cycle, and the depressed photorespiration could contribute to the less depressed photosynthesis in leaf under the high-temperature stress in the offspring of primed parents than that of non-primed parents, as shown in Fig. 7.

Depression of photosynthesis is reported to usually parallel with the burst of the reactive oxygen species (ROS) production under abiotic stresses (Kurepin et al., 2015), since the block of photosynthetic electron transport in reaction centers of PSI and PSII results in the production of ROS due to the over-accumulated electrons with O2 (Oukarroum et al., 2015; Wang et al., 2011). Plants have developed antioxidant pathways to prevent ROS overproduction via both non-enzymic and enzymic antioxidants. The enzymic antioxidants include SOD, POD, catalase (CAT), and so on (Noctor and Foyer, 1998). Here, the activities of SOD and POD were significantly higher, which was consistent with the much lower contents of MDA and H2O2 in D-H than ND-H (Fig. 4). These results indicated that drought-priming of the parents effectively enhanced antioxidant capacity in flag leaves to better cope with the postanthesis heat stress occurring in the offspring generation. In accordance to previous studies, oxidative capacity could be strongly stimulated by heat stress priming (Silva et., 2010; Wang et al., 2014). Here, it is also observed that other processes involved in the drought-to-heat tolerance passed on to the next generation, including signaling, accumulation of HSPs, sucrose synthesis and protein structure modification. Stress signal perception and transduction were the original responses of plants to stresses, which further induce the consequent molecular responses (Knight and Knight, 2001). Calreticulin (CRT) is a ubiquitously expressed Ca2+-binding protein in plants, and it has many cellular functions, including regulation of Ca2+ homeostasis, protein synthesis, and molecular chaperone activity (Jia et al., 2008). Previous Studies indicate that CRT is also specifically associated with several kinds of HSPs in resisting heat stress (Jethmalani and Henle, 1998; Wang et al., 2004). In the present study, CRT (spot 11) was found more up-regulated in D-H than in ND-H, indicating that CRT could play a

protective role in coping with the heat stress occurred in the offspring generation. HSPs play important roles in stress resistance, functioning as molecular chaperones that maintain the function structure of proteins, prevent the aggregation of non-native proteins, and remove potentially harmful polypeptides (Kotak et al., 2007; Timperio et al., 2008; Wang et al., 2004). The HSP 70 family is one of the five major families of HSPs/chaperones. Hsp70-Hsp90 organizing protein (HOP) is an abundant, stress-induced protein associated with HSP90 and HSP70, modulates activities of these chaperone proteins (Carrigan et al., 2006; Johnson et al., 1998). In this study, the HSP 70 family (spots 4, 5) and the HOP (spot 11) were also up-regulated in D-H than in ND-H. In addition, the luminal binding protein (BiP) is a widely distributed and highly conserved endoplasmic reticulum protein that is involved in the co-translational folding of nascent polypeptides, and in the recognition and disposal of misfolded polypeptides (Kalinski et al., 1995; Koizumi, 1996). It is also known that the protein disulfide isomerase (PDI) catalyzes the formation of the native disulfide bonds and the rearrangement of the disulfide bonds, and PDI plays an important role in constructing active proteins (Sullivan et al., 2003). Here, the expression of both the BiP (spots 2 and 3) and the PDI (spots 12 and 13) were significantly more enhanced in D-H than in ND-H. Therefore, the up-regulation of these stress-related proteins could better maintain the synthesis, structure and function of proteins under hightemperature stress in drought-primed offspring plants. The fructose-1,6-bisphosphatase (spot 26) is a key enzyme of sucrose synthesis, the up-regulation of this protein favored the accumulation of sucrose in D-H plants compared with ND-H plants. Moreover, the vacuolar H+-ATPase (V-ATPase, spot 10) functioning in both protein targeting and ion homeostasis (Schumacher et al., 1999)

was also up-regulated in the drought-primed offspring plants as compared with the non-primed plants. However, one point needs explanation. Only two chambers were used in this experiment (one for each temperature treatment), although we obtained valid results. Ideally, three or more chambers used per treatment would surely facilitate experiment, which is our future pursuit. In conclusion, this study proved an acquired drought-to-heat cross tolerance in offspring plants conferred by drought priming in parent plants. This could be explained by the improved photosynthesis because of the regulated expressions of proteins involved in light reaction, Calvin cycle and photorespiration processes, and also by the enhanced anti-oxidation capacity. In addition, the more robust signal perception and transduction, better maintenance of protein structures, up-regulation of sucrose synthesis and accumulation of heat shock proteins could also contribute to the enhanced thermo-tolerance in the offspring.

Acknowledgements This study is supported by the projects of the National Natural Science Foundation of China (31325020, 31401326, 31471445), the China Agriculture Research System (CARS-03), the Collaborative Innovation Center of Crop Gene Resources, the Graduate-Student Innovation Program of Jiangsu Province (CXZZ130291), the Jiangsu Collaborative Innovation Center for Modern Crop Production (JCIC-MCP), and the National Non-profit Program by the Ministry of Agriculture (2014039). We thank the editor and the reviewers for their valuable comments on the manuscript.

References Allakhverdiev, S.I., Kreslavski, V.D., Klimov, V.V., Los, D.A., Carpentier, R., Mohanty, P., 2008. Heat stress: an overview of molecular responses in photosynthesis. Photosynth. Res. 98, 541-550. Barnabas, B., Jager, K., Feher, A., 2008. The effect of drought and heat stress on reproductive processes in cereals. Plant Cell Environ. 31, 11-38. Benz, J.P., Stengel, A., Lintala, M., Lee, Y.H., Weber, A., Philippar, K., Gugel, I.L., Kaieda, S., Ikegami, T., Mulo, P., Soll, J., Bolter, B., 2009. Arabidopsis Tic62 and ferredoxin-NADP(H) oxidoreductase form light-regulated complexes that are integrated into the chloroplast redox poise. Plant Cell 21, 3965-3983. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Bruce, T.J.A., Matthes, M.C., Napier, J.A., Pickett, J.A., 2007. Stressful “memories” of plants: Evidence and possible mechanisms. Plant Sci. 173, 603-608. Camejo, D., Rodríguez, P., Angeles Morales, M., Miguel Dell’Amico, J., Torrecillas, A., Alarcón, J.J., 2005. High temperature effects on photosynthetic activity of two tomato cultivars with different heat susceptibility. J. Plant Physiol. 162, 281-289. Carrigan, P.E., Sikkink, L.A., Smith, D.F., Ramirez-Alvarado, M., 2006. Domain:domain interactions within Hop, the Hsp70/Hsp90 organizing protein, are required for protein stability and structure. Protein Sci. 15, 522-532.

Chen, W.R., Zheng, J.S., Li, Y.Q., Guo, W.D., 2012. Effects of high temperature on photosynthesis, chlorophyll fluorescence, chloroplast ultrastructure, and antioxidant activities in fingered citron. Russ. J. Plant Physiol. 59, 732-740. Choi, C.S., Sano, H., 2007. Abiotic-stress induces demethylation and transcriptional activation of a gene encoding a glycerophosphodiesterase-like protein in tobacco plants. Mol. Genet. Genomics 277, 589-600. Conrath, U., Beckers, G.J.M., Flors, V., García-Agustín, P., Jakab, G., Mauch, F., Newman, M.-A., Pieterse, C.M.J., Poinssot, B., Pozo, M.J., Pugin, A., Schaffrath, U., Ton, J., Wendehenne, D., Zimmerli, L., Mauch-Mani, B., 2006. Priming: Getting ready for battle. Mol. Plant Microbe In. 19, 1062-1071. Deng, W., Wang, Y., Liu, Z., Cheng, H., Xue, Y., 2014. HemI: a toolkit for illustrating heatmaps. PLoS One 9, e111988. Ding, C., You, J., Liu, Z., Rehmani, M.I.A., Wang, S., Li, G., Wang, Q., Ding, Y., 2010. Proteomic analysis of low nitrogen stress-responsive proteins in roots of rice. Plant Mol. Biol. Rep. 29, 618-625. Du, Z., Bramlage, W.J., 1992. Modified thiobarbituric acid assay for measuring lipid oxidation in sugar-rich plant tissue extracts. J. Agr. Food Chem. 40, 15661570. Ellis, R.J., Van Der Vies, S., 1988. The Rubisco subunit binding protein. Photosynth. Res. 16, 101-115. Farooq, M., Bramley, H., Palta, J.A., Siddique, K.H.M., 2011. Heat stress in wheat during reproductive and grain-filling phases. Crit. Rev. Plant Sci. 30, 491-507.

Flechner, A., Dressen, U., Westhoff, P., Henze, K., Schnarrenberger, C., Martin, W., 1996. Molecular characterization of transketolase (EC 2.2.1.1) active in the Calvin cycle of spinach chloroplasts. Plant Mol. Biol. 32, 475-484. Hurkman, W.J., Tanaka, C.K., 1986. Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 81, 802-806. Jethmalani, S.M., Henle, K.J., 1998. Calreticulin associates with stress proteins: Implications for chaperone function during heat stress. J. Cell Biol. 69, 30-43. Jia, X.Y., Xu, C.Y., Jing, R.L., Li, R.Z., Mao, X.G., Wang, J.P., Chang, X.P., 2008. Molecular cloning and characterization of wheat calreticulin (CRT) gene involved in drought-stressed responses. J. Exp. Bot. 59, 739-751. Johnson, B.D., Schumacher, R.J., Ross, E.D., Toft, D.O., 1998. Hop modulates hsp70/hsp90 interactions in protein folding. J. Biol. Chem. 273, 3679-3686. Kalinski, A., Rowley, D., Loer, D., Foley, C., Buta, G., Herman, E., 1995. Bindingprotein expression is subject to temporal, developmental and stress-induced regulation in terminally differentiated soybean organs. Planta 195, 611-621. Knaff, D.B., Arnon, D.I., 1969. A concept of three light reactions in photosynthesis by green plants. Proc. Natl. Acad. Sci. 64, 715-722. Knight, H., Knight, M.R., 2001. Abiotic stress signalling pathways: specificity and cross-talk. Trends Plant Sci. 6, 262-267. Koizumi, N., 1996. Isolation and responses to stress of a gene that encodes a luminal binding protein in Arabidopsis thaliana. Plant Cell Physiol. 37, 862-865. Kotak, S., Larkindale, J., Lee, U., von Koskull-Doring, P., Vierling, E., Scharf, K.D.,

2007. Complexity of the heat stress response in plants. Curr. Opin. Plant Biol. 10, 310-316. Kramer, D.M., Avenson, T.J., Edwards, G.E., 2004. Dynamic flexibility in the light reactions of photosynthesis governed by both electron and proton transfer reactions. Trends Plant Sci. 9, 349-357. Kurepin, L.V., Ivanov, A.G., Zaman, M., Pharis, R.P., Allakhverdiev, S.I., Hurry, V., Huner, N.P., 2015. Stress-related hormones and glycinebetaine interplay in protection of photosynthesis under abiotic stress conditions. Photosynth. Res. 126, 221-235. Mackova, H., Hronkova, M., Dobra, J., Tureckova, V., Novak, O., Lubovska, Z., Motyka, V., Haisel, D., Hajek, T., Prasil, I.T., Gaudinova, A., Storchova, H., Ge, E., Werner, T., Schmulling, T., Vankova, R., 2013. Enhanced drought and heat stress tolerance of tobacco plants with ectopically enhanced cytokinin oxidase/dehydrogenase gene expression. J. Exp. Bot. 64, 2805-2815. Molinier, J., Ries, G., Zipfel, C., Hohn, B., 2006. Transgeneration memory of stress in plants. Nature 442, 1046-1049. Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 249-279. Oukarroum, A., Bussotti, F., Goltsev, V., Kalaji, H.M., 2015. Correlation between reactive oxygen species production and photochemistry of photosystems I and II in Lemna gibba L. plants under salt stress. Environ. Exp. Bot. 109, 80-88. Ramirez, D.A., Rolando, J.L., Yactayo, W., Monneveux, P., Mares, V., Quiroz, R.,

2015. Improving potato drought tolerance through the induction of long-term water stress memory. Plant Sci. 238, 26-32. Rizhsky, L., Liang, H., Shuman, J., Shulaev, V., Davletova, S., Mittler, R., 2004. When defense pathways collide. The response of Arabidopsis to a combination of drought and heat stress. Plant Physiol. 134, 1683-1696. Rollins, J.A., Habte, E., Templer, S.E., Colby, T., Schmidt, J., von Korff, M., 2013. Leaf proteome alterations in the context of physiological and morphological responses to drought and heat stress in barley (Hordeum vulgare L.). J. Exp. Bot. 64, 3201-3212. Schumacher, K., Vafeados, D., McCarthy, M., Sze, H., Wilkins, T., Chory, J., 1999. The Arabidopsis det3 mutant reveals a central role for the vacuolar H+– ATPase in plant growth and development. Genes Dev. 13, 3259-3270. Shi, W., Lawas, L.M.F., Raju, B.R., Jagadish, S.V.K., 2015. Acquired thermotolerance and trans-generational heat stress response at flowering in rice. J. Agron. Crop Sci. Silva, E.N., Ferreira-Silva, S.L., Fontenele Ade, V., Ribeiro, R.V., Viegas, R.A., Silveira, J.A., 2010. Photosynthetic changes and protective mechanisms against oxidative damage subjected to isolated and combined drought and heat stresses in Jatropha curcas plants. J. Plant Physiol. 167, 1157-1164. Sui, N., Li, M., Liu, X.Y., Wang, N., Fang, W., Meng, Q.W., 2007. Response of xanthophyll cycle and chloroplastic antioxidant enzymes to chilling stress in tomato

over-expressing

glycerol-3-phosphate

acyltransferase

gene.

Photosynthetica 45, 447-454. Sullivan, D.C., Huminiecki, L., Moore, J.W., Boyle, J.J., Poulsom, R., Creamer, D., Barker, J., Bicknell, R., 2003. EndoPDI, a novel protein-disulfide isomeraselike protein that is preferentially expressed in endothelial cells acts as a stress survival factor. J. Biol. Chem. 278, 47079-47088. Suzuki, N., Rivero, R.M., Shulaev, V., Blumwald, E., Mittler, R., 2014. Abiotic and biotic stress combinations. New Phytol. 203, 32-43. Tan, W., Liu, J., Dai, T., Jing, Q., Cao, W., Jiang, D., 2008. Alterations in photosynthesis and antioxidant enzyme activity in winter wheat subjected to post-anthesis water-logging. Photosynthetica 46, 21-27. Tanz, S.K., Castleden, I., Hooper, C.M., Vacher, M., Small, I. and Millar, H.A., 2013. SUBA3: a database for integrating experimentation and prediction to define the SUBcellular location of proteins in Arabidopsis. Nucleic Acids Res. 41, D1185-D1191. Thimm, O., Bläsing, O., Gibon, Y., Nagel, A., Meyer, S., Krüger, P., Selbig, J., Müller, L.A., Rhee, S.Y., Stitt, M., 2004. Mapman: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. Plant J. 37, 914-939. Timperio, A.M., Egidi, M.G., Zolla, L., 2008. Proteomics applied on plant abiotic stresses: role of heat shock proteins (HSP). J. Proteomics 71, 391-411. Vu, W.T., Chang, P.L., Moriuchi, K.S., Friesen, M.L., 2015. Genetic variation of transgenerational plasticity of offspring germination in response to salinity

stress and the seed transcriptome of Medicago truncatula. BMC Evol. Biol. 15, 59. Wang, W., Vinocur, B., Shoseyov, O., Altman, A., 2004. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci. 9, 244-252. Wang, X., Cai, J., Jiang, D., Liu, F., Dai, T., Cao, W., 2011. Pre-anthesis hightemperature acclimation alleviates damage to the flag leaf caused by postanthesis heat stress in wheat. J. Plant Physiol. 168, 585-593. Wang, X., Cai, J., Liu, F., Dai, T., Cao, W., Wollenweber, B., Jiang, D., 2014. Multiple heat priming enhances thermo-tolerance to a later high temperature stress via improving subcellular antioxidant activities in wheat seedlings. Plant Physiol. Biochem. 74, 185-192. Wang, X., Vignjevic, M., Liu, F., Jacobsen, S., Jiang, D., Wollenweber, B., 2015. Drought priming at vegetative growth stages improves tolerance to drought and heat stresses occurring during grain filling in spring wheat. Plant Growth Regul. 75, 677-687. Wang, Z., Fu, J., He, M., Yin, Y., Cao, H., 1997. Planting density effects on assimilation and partitioning of photosynthates during grain filling in the latesown wheat. Photosynthetica 33, 199-204. Yan, K., Chen, P., Shao, H., Zhao, S., Zhang, L., Zhang, L., Xu, G., Sun, J., 2012. Responses of photosynthesis and photosystem II to higher temperature and salt stress in sorghum. J. Agron. Crop Sci. 198, 218-225.

Legends of figures Fig. 1 Effects of drought priming of winter wheat during grain-filling on grain yield and yield components under post-anthesis high-temperature stress in the next generation Notes: D-C, drought priming (D) + no post-anthesis high-temperature (C); ND-C, no drought priming (ND) + no post-anthesis high-temperature (C); D-H, drought priming (D) + post-anthesis hightemperature (H); ND-H, no drought priming (ND) + post-anthesis high-temperature (H). Data are means ± SE (n=3). Different lowercase letters indicate the significant difference at p < 0.05 level. D, T and D×T indicate drought priming, temperature treatment and interaction of drought priming by temperature treatment, respectively. * and ** refer to the significant levels of 0.05 and 0.01 respectively, while ns refers to insignificant.

Fig. 2 Effects of drought priming of winter wheat during grain-filling on net photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr) of flag leaf under post-anthesis high-temperature stress in the next generation Notes: D-C, drought priming (D) + no post-anthesis high-temperature (C); ND-C, no drought priming (ND) + no post-anthesis high-temperature (C); D-H, drought priming (D) + post-anthesis hightemperature (H); ND-H, no drought priming (ND) + post-anthesis high-temperature (H). Data are means ± SE (n=3). Different lowercase letters indicate the significant difference at p < 0.05 level. * and ** refer to the significant levels of 0.05 and 0.01 respectively, while ns refers to insignificant.

Fig. 3 Effects of drought priming of winter wheat during grain-filling on SPAD value (A) and dark-adapted image of maximum quantum efficiency of photosystem II (Fv/Fm, B) of flag leaf under post-anthesis high-temperature stress in the next generation Notes: D-C, drought priming (D) + no post-anthesis high-temperature (C); ND-C, no drought priming (ND) + no post-anthesis high-temperature (C); D-H, drought priming (D) + post-anthesis hightemperature (H); ND-H, no drought priming (ND) + post-anthesis high-temperature (H). Data are means ± SE (n=3). Different lowercase letters indicate the significant difference at p < 0.05 level. * and ** refer to the significant levels of 0.05 and 0.01, respectively, while ns refers to insignificant.

Fig. 4 Effects of drought priming of winter wheat during grain-filling on activities of SOD and POD; contents of MDA and H2O2 in flag leaf under post-anthesis high-temperature stress in next generation Notes: D-C, drought priming (D) + no post-anthesis high-temperature (C); ND-C, no drought priming (ND) + no post-anthesis high-temperature (C); D-H, drought priming (D) + post-anthesis hightemperature (H); ND-H, no drought priming (ND) + post-anthesis high-temperature (H). Data are means ± SE (n=3). Different lowercase letters indicate the significant difference at p < 0.05 level.

* and ** refer to the significant levels of 0.05 and 0.01 respectively, while ns refers to insignificant.

Fig. 5 Representative 2-DE gel for the proteome profile of flag leaf Notes: Changes in abundance of proteins as a result of the drought priming and the high-temperature stress are indicated by arrows, and the descriptions of the differentially expressed proteins are listed in Table 1. MW: relative molecular mass; pI: isoelectric point.

Fig. 6 Ratio of differentially expressed proteins in flag leaves between treatments Notes: D-C, drought priming (D) + no post-anthesis high-temperature (C); ND-C, no drought priming (ND) + no post-anthesis high-temperature (C); D-H, drought priming (D) + post-anthesis hightemperature (H); ND-H, no drought priming (ND) + post-anthesis high-temperature (H). The difference in expression levels at the given protein spots between treatments was given as numerical value after log2 and converted to a color scale. Up- and down-regulation was indicated by changes in red and blue coloring, respectively. Proteins were listed and classified with their functions indicated by Mapman.

Fig. 7 Diagram of the regulated processes of photosynthesis in offspring of the drought-primed parent plants in relation to offspring of the non-primed parent plants under post-anthesis high temperature stress Notes: The processes of light reaction, photorespiration and Calvin cycle are separated into three boxes. The regulation levels are shown in the small color-filled boxes. The numeral in each box indicates the differentially expressed protein spot, which is shown in Table 1.

Spikes per pot

14

D ns

a

T ns

a

D×T a ns

T **

a

a

D×T a ns

48 45

12 42

11 9

39

8

36

0 39

D ns

D*

a 1000-kernel weight (g)

D ns

a

36

a

T **

a

0 24

T*

ab

D×T ns

D×T ns

21

bc

b

c

c

33

18

30

15

27

12

0

0 D-C

ND-C

D-H

ND-H

D-C

Treatments Fig. 1

ND-C

Kernels per spike

a

D-H

ND-H

Grain yield (g pot-1)

15

0.32

a

T **

16

T **

a

D×T ns

0.24

b

14

b b

c

12

0.28

D×T ns

0.20

10

0.16

8 0 285

0.12 0.00 8

270 255

a

D ns

D ns

a

T*

T **

D×T ns

D×T *

b

7

ab

b

b c

240

c

6 5 4

225 0

D-C

ND-C

D-H

ND-H

D-C

Treatments Fig. 2

ND-C

D-H

ND-H

3 0

gs (mol H2O m-2s-1)

a

-2 -1

-2 -1 Pn (mmol CO2 m s ) -1

Ci (mol CO2 mol )

D ns

D ns

Tr (mmol H2O m s )

a

18

58

a

a

D ns T **

56

b

D×T **

SPAD

54

c 52 50 48 0

D-C D-C

ND-C ND-H ND-C D-H D-H

ND-H

D-C D-C

Treatments Fig. 3

ND-C D-H ND-C

ND-H D-H

ND-H

 

D×T ns

60

T **

b

3.2

 

2.4

 

c

40 30 0 60 50

D ns

a

D ns

T **

 

T **

D×T *

b

c

c

1.6

a ab

D×T ns

bc

40 30

4.0

D×T ns

b

b 50

a

b

0.8 0.0 0.45 0.42 0.39

b

 

0.36

 

20

0.33

10 0

0.30 0.00 D-C

ND-C D-H

D-C

ND-H

Treatments Fig. 4

ND-C

D-H

ND-H

-1

T **

4.8 MDA (mmol mg protein)

SOD (unit mg-1 protein) POD (unit mg-1 protein min-1)

70

a

D ns

-1

a

D ns

H2O2 (mol mg protein)

80

4

pI

7

MW(Kda)

IEF

31 1 6

2 3

100

7

5 13 12

19 4

8 20

9 32

10

50

33 11

22

15 18 17

14

21

16 34

23

24 28

25

29 25

27 26

10 Fig. 5

30

                                         

Fig. 6

   

The photorespiration

The light reaction                                   The Calvin cycle            

Fig. 7

Table 1. List of proteins differently expressed in different treatments analyzed by MALDI TOF-MS Spot ID 1

Protein name

GI accessionThr. MW no. (kDa)/pI a

Score

SC NMPc (%)b

Taxonomy

Homologue Subcellular At Arab. d locations e

Function

AT1G62750 Plastid

Protein. Synthesis. Elongation

G,475559072

79017/4.97 270

11

5

Triticum tauschii

2

Elongation factor chloroplastic Luminal-binding protein 3

474200923

77693/5.08 346

14

7

3

Luminal-binding protein 3

474200923

77693/5.08 228

11

6

4 5 6

HSP70 HSP70 Predicted protein

2827002 2827002 326533372

71385/5.14 308 71385/5.14 452 74032/5.45 980

11 15 21

5 8 9

7 8

Transketolase, chloroplastic 474352176 ATP synthase CF1 alpha subunit 14017569

68881/5.36 438 55318/6.11 383

17 20

8 7

AT5G42020 Endoplasmic reticulum Triticum urartu AT5G42020 Endoplasmic reticulum Triticum aestivum AT3G12580 Cytosol Triticum aestivum AT3G12580 Cytosol Hordeum vulgareAT3G60750 Plastid subsp. Vulgare Triticum urartu AT3G60750 Plastid Triticum aestivum ATCG00120 Plastid

HOP (Hsp70-Hsp90 organizing306811648 protein) 10 Vacuolar proton-ATPase subunit90025017 A 11 Calreticulin, partial 439586

65342/5.78 117

4

2

Triticum aestivum

AT1G12270 Nucleus

68754/5.23 119

9

5

Triticum aestivum

AT1G78900 Vacuole

47180/4.45 108

8

3

16692/5.16 93

16

2

56726/4.99 148 57656/4.83 184

13 11

6 5

Hordeum vulgareAT1G56340 Endoplasmic subsp. Vulgare reticulum Triticum aestivum AT1G77510 Endoplasmic reticulum Triticum aestivum AT1G21750 Plastid Triticum aestivum AT2G28000 Plastid

9

12 Protein disulfide isomerase,47118054 partial 13 Protein disulfide isomerase 1709620 14 RuBisCO large subunit-binding134102 protein subunit alpha, chloroplastic 15 Hypothetical protein475559550 F775_28691 16 ATP synthase beta subunit 525291

Triticum urartu

3

Triticum tauschii

AT3G46780 Mitochondrion

59326/5.56 1158

30

10

Triticum aestivum

AT5G08690 Cytosol

48459/5.49 205

18

5

Triticum aestivum

AT2G36530 Cytosol

18 Phosphoglycerate mutase, partial 32400802

29615/5.43 140

26

4

Triticum aestivum

AT3G08590 Cytosol

19 Phosphoglycerate mutase, partial 32400802

29615/5.43 95

32

6

Triticum aestivum

AT3G08590 Cytosol

20 Phosphoglycerate mutase, partial 32400802

29615/5.43 127

31

5

Triticum aestivum

AT3G08590 Plastid

461744058

Stress. Abiotic. Heat Stress. Abiotic. Heat Stress. Abiotic. Heat PS. Calvin cycle. Transketolase PS. Calvin cycle. Transketolase PS. Light reaction. ATP synthase. Alpha subunit Stress Transport. P- and v-ATPases Signaling. Calcium Redox. Thioredoxin. PDIL Redox. Thioredoxin. PDIL PS. Calvin cycle. Rubisco interacting

10

17 Enolase

Stress. Abiotic. Heat

43836/5.11

110

RNA. Transcription Mitochondrial electron transport / ATP synthesis.F1-ATPase Glycolysis. Cytosolic branch. Enolase Glycolysis. Cytosolic branch. Phosphoglycerate mutase Glycolysis. Cytosolic branch. Phosphoglycerate mutase Glycolysis. Cytosolic branch. Phosphoglycerate mutase

21 ATPase, beta subunit

14017579

10

3

Hordeum vulgare

ATCG00480 Mitochondrion

101849/7.05 153

7

5

Triticum tauschii

AT3G02090 Plastid

PS. Light reaction. ATP synthase. Beta subunit Protein. Targeting. Mitochondria

48012/6.92 135

7

2

Triticum aestivum

AT2G39730 Plastid

PS. Calvin cycle. Rubisco interacting

69718/6.42 153

7

3

40491/6.92 207

17

5

Hordeum vulgareAT3G48420 Plastid subsp. Vulgare Triticum aestivum AT5G66190 Plastid

26 Fructose-1,6-bisphosphatase, 300681469 cytosolic 27 Cytosolic malate dehydrogenase 37928995

37854/5.38 162

10

3

Triticum aestivum

AT1G43670 Cytosol

24560/6.60 135

18

3

Triticum aestivum

AT5G43330 Endoplasmic reticulum

28 Glyceraldehyde-3-phosphate 475620495 dehydrogenase A, chloroplastic 29 Glycine decarboxylase P subunit 2565305

43337/8.63 887

30

9

Triticum tauschii

AT1G12900 Plastid

111975/6.32 223

4

5

x Tritordeum sp.

AT4G33010 Mitochondrion

30 Rubisco activase alpha form32481061 precursor 31 Carotenoid cleavage226858194 dioxygenase

51381/5.96 114

5

2

AT2G39730 Plastid

61685/5.80 85

5

3

Deschampsia antarctica Brachypodium sylvaticum

32 ATP synthase alpha subunit

55287/5.94 185

13

6

Triticum aestivum

ATCG00120 Plastid

22 Putative mitochondrial475623273 processing peptidase subunit beta 23 Ribulose bisphosphate960277 carboxylase activase B 24 Predicted protein 326490295 25 Ferredoxin-NADP(H) oxidoreductase

   

20302473

552976

53899/5.11

206

AT3G63520 Vacuole

Not assigned.no ontology PS. light reaction. Other electron carrier (ox/red). Ferredoxin reductase Major CHO metabolism. Synthesis. Sucrose. FBPase TCA / org transformation. Other organic acid transformations. Cyt MDH PS. Calvin cycle. GAP PS. photorespiration. Glycine cleavage. P subunit PS. Calvin cycle. Rubisco interacting Secondary metabolism. Isoprenoids. Carotenoids. Carotenoid cleavage dioxygenase PS. light reaction. ATP synthase. Alpha subunit