Transcriptome profiling of wheat genotypes under heat stress during grain-filling

Journal of Cereal Science 91 (2020) 102895

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Transcriptome profiling of wheat genotypes under heat stress during grain-filling Parimalan Rangan a, b, Agnelo Furtado a, Robert Henry a, * a b

Queensland Alliance for Agriculture and Food Innovation, University of Queensland, St Lucia, QLD, 4072, Australia Division of Genomic Resources, ICAR-National Bureau of Plant Genetic Resources, PUSA Campus, New Delhi, 110012, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Differential expression Grain-filling Heat stress RNA-Seq Transcriptome Triticum aestivum

High temperatures adversely impact productivity and quality (nutritional and functional) of wheat grains. Anthesis and post-anthesis developmental stages are more sensitive to heat stress, with the grain-filling stage being crucial for sustained grain yield and quality. Comparative transcriptome profiling of the developing grain of three wheat genotypes (contrasting for heat stress tolerance) during early- (14-dpa (days post anthesis)) and late-grain filling (30-dpa) was studied to identify key genes involved in imparting heat tolerance. Heat stress during grain-filling (early- and late-) significantly down-regulated key genes in the genotypes, Gregory and Banks, found to be more heat susceptible. Upregulation of the cluster of genes comprising 6-phosphogluconate dehydrogenase, S6 RPS6-2 ribosomal protein, peptidylprolyl isomerase, plasma membrane proton ATPase, Heat shock cognate-70, FtsH protease, RuBisCO activase B, methionine synthase, cytochrome C (class I), and HMW-glutenin in the genotype Fang-60 during heat stress was found to be associated with heat stress tolerance in this genotype. Upregulation of β-glucanase (1,3 & 1,4), triose phosphate isomerase and calnexin at 14-dpa; and downregulation of Naþ/Hþ antiporter, glucose-1-phosphate adenylyltransferase, ips1 riboregulator and AraC family transcriptional regulator at 30-dpa was observed specifically in the heat susceptible genotypes. Hsp-family, ascorbate peroxidase, β-amylase, γ-gliadin-2 and LMW-glutenin were heat stress responsive and were upregulated during stress across 14and 30-dpa in all three genotypes. This study provides insights into genes that may be involved in regulating heat tolerance in a tolerant genotype and those that are responsive to heat in the developing wheat grain of tolerant and susceptible genotypes. The genotype Fang-60 was demonstrated to be a potential source of heat stress tolerance for use in wheat breeding.

1. Introduction Heat and drought are major abiotic stress components affecting crop growth and productivity globally. Genotypes adaptable to such adverse habitats are valuable genetic resources in a variable climate (Henry et al., 2016). Wheat is a major food crop that is likely to be impacted by climate change. Heat stress is considered as primary environmental stress factor influencing wheat productivity and grain quality and in developing countries it is estimated to affect 57% of the land area under wheat (Kosina et al., 2007). Heat stress during the post-heading stage affects grain yield with yield loss of up to 15% (Wiegand and Cuellar, 1981) and reduced quality for various end-uses (Qin et al., 2008). In wheat, every degree Celsius rise from a seasonal mean minimum crop temperature of 15 � C, has been found to cost yield losses of up to 5%; although this threshold may vary based on other environmental factors,

genotypes, and the developmental stage of the plant. Recent reports indicate that every degree rise in the daily minimum temperature has a larger effect (4%) on yield than the daily maximum (2%)(Gupta et al., 2017). High temperatures during grain filling result in wheat grain with significantly altered composition (Shamloo et al., 2017) with implica­ tions for human nutrition. Heat stress modulates multiple metabolic pathways, disrupts cellular and sub-cellular structures, and transportation of metabolites with a primary effect on photosynthesis and thereby impacts on yield, and in extreme cases may challenge the survival of crop plants (Wahid et al., 2007). Histone variant H2A.Z was considered to act as a thermo-sensor in plants during heat stress (Wu et al., 2016) that in turn activates or down-regulates a cascade of events for thermo-tolerance as a response to heat. In wheat, oxidative damage to chloroplasts; CO2-exchange rate (CER); disruption of the photosystem II complex; and altered specificity of RuBisCO towards oxygenation are all effects of heat stress linked to

* Corresponding author. Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, QLD, 4072, Australia. E-mail addresses: [email protected], [email protected] (P. Rangan), [email protected] (A. Furtado), [email protected] (R. Henry). https://doi.org/10.1016/j.jcs.2019.102895 Received 15 September 2019; Received in revised form 4 December 2019; Accepted 4 December 2019 Available online 7 December 2019 0733-5210/© 2019 Elsevier Ltd. All rights reserved.

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litre ANOVA™ pots were filled with UQ23 potting mix and placed on a wheel supported workbench with a capillary mat base for automated irrigation inside the glass house chamber. The UQ23 potting mix consist of 70% composted pine bark (0–5 mm) and 30% coco peat. Every cubic metre of potting mix was augmented with 1 kg Yates flow trace, 1 kg iron sulfate heptahydrate, 0.4 kg Superphosphate, 0.03 kg copper sulfate and 1 kg Gypsum with a final pH of 5.5–6.5 using FeSO4 (if pH is high) or Dolomite (if low). Three seeds were sown per pot and three such pots were used per treatment per true biological replicate and in total, three true biological replicates were used for each genotype/treatment and the same number of samples were retained for transcriptome analysis without pooling. The use of mean RPKM values from transcriptome data generated with three true independent biological replicates, when sub­ jected to statistical treatment for identifying significantly differentially expressed genes, relaxes the requirement of qRT-PCR validation; since the error degree of freedom in our transcriptome study is high enough (greater than 12) to give the required statistical confidence. Plants were grown and maintained under glass-house conditions with day and night temperature at 20 � C and 18 � C respectively at an RH of 40–50% unless otherwise specified (Fig. 1). The glasshouse facility supported with a set of staff for its maintenance, is a central facility support provided by The University of Queensland, Australia for various research activities. Fifteen days after sowing when the seeds were germinated, percent of germination was calculated, and the plants were fertilized with osmo­ cote slow release (3–4 m) fertilizer granules at the rate of 30 gm per 4 L pot. The whole experiment was conducted in a randomized block design with three biological replicates.

Abbreviations CER CO2 exchange rate DAD defender against cell death Dpa days post anthesis ED pathway Entner-Doudoroff pathway ERA1 enhanced response to aba1 HSC-70 heat shock cognate-70 HSF heat shock factor HSP heat shock protein HSR1 heat stress RNA1 PGD3 6-phosphogluconate dehydrogenase, chloroplastic RCA RuBisCO activase ZWF glucose-6-phosphate dehydrogenase

photosynthesis. The role of post-transcriptional modification of rubisco activase and post-translational modification of heat-shock factor 1 (HSF1) (Kourtis et al., 2015) and enhanced response to aba1 (ERA1) (Wu et al., 2016) during heat stress response (HSR) have been well documented. Gene expression in the developing wheat grain is critical to the determination of the final composition (nutritional and functional qualities) and yield of this key food crop (Henry et al., 2018). Next generation sequencing (NGS) based transcriptomic studies have revo­ lutionized the study of seed development and helped discover novel genes for bread-making (Furtado et al., 2015); hardness (Nirmal et al., 2016); and milling quality (Nirmal et al., 2017) and led to the discovery of a C4 photosynthetic pathway (Rangan et al., 2016a, 2016b; Henry et al., 2017; Hu et al., 2018) specifically located in the inner pericarp of wheat grains. Studies on the response of wheat to heat stress have mainly focused on specific genes, traits or metabolic pathways. Although some transcriptomic studies in wheat have explored grain yield and quality (Furtado et al., 2015; Rangan et al., 2016b, 2017); global analyses of gene expression in relation to stress tolerance are very limited and are array based and mostly compare a single genotype under control and stress conditions. The present study was conducted to examine differences in the gene expression pattern between stress tolerant and susceptible genotypes to improve understanding of the molecular mechanisms of heat stress tolerance. Heat stress in general is unique among stress factors in that it cannot be overcome through external crop management practices like soil or plant or seed treatments at a commercial scale; rather it requires iden­ tification of genotypes avoiding (early maturing) or tolerating heat stress. Identifying genotypes with heat tolerance based upon an under­ standing on the mechanisms of heat tolerance may be crucial in limiting severe loss of crop yield and quality resulting from highly variable climate patterns (Henry et al., 2016). The effect of heat stress on wheat crop especially during anthesis and grain filling has been widely shown to include reduced grain yield, grain size, and the number of grains per ear. In this study, the response of the transcriptome to heat stress was followed in three genotypes, Banks (AUS20599), EGA Gregory (AUS34283) and Fang 60 (AUS24511), with the latter one being found to be especially heat tolerant (Rangan et al., 2019).

2.2. Heat stress treatment Potted plants were irrigated twice a day (morning and evening) during normal growth phase conditions using a Sterling 12 port controller (Superior Controls Inc., USA) with adjustable drippers fitted onto the capillary mat on which the ANOVA™ pots are placed. With the onset of booting and when awns were visible above the flag leaf (0.5–1.0 cm), the individual plantlets were tagged. From this stage, anthesis occurs four days later; hence to calculate date of anthesis, we have added four days to the tagging date in finalizing the timings for the heat stress experiment i.e., 18 days and 34 days from tagging was the 14dpa and 30 dpa stage respectively (Furtado et al., 2015). Booting started around the seventh week from the date of sowing. The pots were shuffled and rearranged so that at least eight spikes from each genotype in each true biological replicate for each time point of study viz., 14 and 30 days-post-anthesis (dpa) was available from both control and heat stress conditions. The potted plants were subjected to heat stress for three days (starting from 9AM) by shifting the plants to another cubicle inside the same glass house that was pre-set to 38 � C and 20 � C for day and night temperature respectively with a 12-h shift with RH at 40–50% condi­ tions (Blumenthal et al., 1995). Although plants were watered manually three times a day in the heat stress treatment cubicle, potted plants were placed on a bench covered with capillary mat to retain moisture for a maximal time to avoid drought stress for the plants due to the high temperature inside the cubicle. Tagged wheat spikes from control and heat stressed plant in true biological triplicates during early (11-14dpa) or late (27-30dpa) grain filling were harvested at the 14 and 30dpa stage for transcriptomic studies (Fig. 1). Awns and the top and bottom 25% of the spikes were chopped off, and the middle 50% portion of the spikes (awns removed) was snap frozen under liquid nitrogen (LN2) for further analyses (Furtado, 2014). After sampling from the high temperature cubicle, the potted plants were shifted back to normal conditions for observation of recovery (refer Figs. 2 and 3). Harvested grains from this study were used to understand the impact of this grain filling heat stress on grain physical characteristics, grain length, grain width, grain thickness, and 1000-grain weight (Rangan et al., 2019).

2. Materials and methods 2.1. Plant material Three hexaploid wheat genotypes, Banks (AUS20599), EGA Gregory (AUS34283) and Fang-60 (AUS24511) were procured from the Austra­ lian genebank. The seeds of three genotypes, Banks, EGA Gregory and Fang-60 used in our study were originally procured from Australian genebank bearing accession numbers as mentioned previously. Four 2

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Fig. 1. Plan for sampling of wheat spikes from con­ trol and heat stressed plants for the three genotypes studied viz., Banks, EGA Gregory, and Fang-60. Purple line: normal temperature (control); red line: high temperature for stress; black circles: sampling points (14 dpa control; 14 dpa stress; 30 dpa control; 30 dpa stress); blue square with dashed lines: highlight the set of samples taken from heat stressed plants at early- or late-grain filling stage and corresponding control plants. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2. Impact of heat stress on the spikes of early-grain filling (11-14-dpa) stage, as observed during recovery at control conditions. A: Bleaching in awns (including its structural deformation) and glumes was visible (indicated by arrows) in susceptible genotype; B: only 70–80% of awns are bleached (indicated by arrows) while the glumes are still green in colour; C: overall glasshouse view showing bleaching effect in susceptible genotypes when compared to control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3. Impact of heat stress on the grains/spikes of late-grain filling (27-30-dpa) heat stress as observed during recovery at control conditions. A: Bleaching in grains of susceptible genotype at stressed condition in comparison to control; B: only 50–60% of awns are bleached (shown in arrows) while the glumes are still green in colour; C: overall glasshouse view showing bleaching effect in susceptible genotypes when compared to the control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

concentrations ranged from 363 to 4849 ng μL 1 when measured using Bioanalyzer. A minimum of 10 μg of total RNA was used for tran­ scriptome sequencing at Translational Research Institute facility, The University of Queensland, St Lucia. Indexed TruSeq libraries were pre­ pared for the 36 RNA samples and 150bp paired-end sequencing was performed using a HiSeq 4000 platform (Illumina Inc., USA). The transcriptome data was made available for download from ArrayExpress in raw read format with the accession number E-MTAB-6140.

2.3. Sample preparation, RNA extraction and transcriptome sequencing Wheat grains (two samples at a time) were isolated under LN2 and pulverized using TissueLyser II (Qiagen Inc., USA) with prestandardized conditions at 900 oscillations per minute (15 Hz fre­ quency) for 30 s using a pair of pre-chilled (under LN2) stainless steel grinding jars (10 mL) and grinding balls (20 mm) in each jar. Pulverized samples stored at 80 � C till further processing. Approximately, 500 mg of pulverized sample was used to extract RNA in accordance with earlier reports (Furtado, 2014). The quality and quantity of the extracted RNA was assessed using a Nanodrop 8000 (Thermo Fisher Scientific Inc., USA) and an Agilent Bioanalyzer 2100 (Agilent technologies Inc., USA) respectively. For most of the samples, the RIN (RNA integrity number) value was around 9.0, ranging from 7.4 to 10.0 with the RNA

2.4. RNA-Seq, differential gene expression, and functional annotation All computational analyses except functional annotation for the transcriptome sequence data obtained from TRI, UQ was performed using CLC genomics workbench 9.0 (Qiagen Inc., USA) and functional 3

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4

6.92 85.43 1.97 90.85 93.08 14.57 98.03 9.15

G1: Banks; G2: EGA Gregory; G3: Fang-60; ctrl: Control sample; stress: Heat stressed sample; dpa: days post anthesis; FDR: false discovery rate.

347 1256 507 448 24 1073 10 407 5 15 0 12 19 1058 10 395 323 183 497 41 284 143 442 31 39 40 55 10 G1G2 vs G3 G1G2 vs G3 G1G2 vs G3 G1G2 vs G3 7 8 9 10

Ctrl-14dpa Stress-14dpa Ctrl-30dpa Stress-30dpa

% genes upregulated in G3 % genes upregulated in G1G2 Total no. of differentially expressed transcripts Total no. of transcripts upregulated (or unique) in G3 Unique expression in G3 Upregulated in G3 Upregulated in G1G2 (downregulated in G3) Unique expression in G1G2

Total no. of transcripts upregulated (or unique) in G1G2

83.79 94.63 1.99 97.57 94.40 0.65 3867 633 552 3174 125 771 627 34 541 77 7 766 36 6 12 7 0 22 591 28 529 70 7 744 3240 599 11 3097 118 5 3235 599 11 3081 114 5 5 0 0 16 4 0 G1-14dpa G2-14dpa G3-14dpa G1-30dpa G2-30dpa G3-30dpa vs stress vs stress vs stress vs stress vs stress vs stress ctrl ctrl ctrl ctrl ctrl ctrl 1 2 3 4 5 6

% genes downregulated during stress Total no. of differentially expressed transcripts Total no. of transcripts upregulated (or unique) at stress No. of transcripts uniquely expressed at stress No. of transcripts upregulated during stress Total no. of transcripts upregulated (or unique) at control No. of transcripts upregulated in control (downregulated in stress) No. of transcripts uniquely expressed in control (silenced in stress)

Among the 3867 genes significantly differently expressed in the Banks genotype, 84% were silenced or down regulated during heat stress and 16% were upregulated during heat stress (Table 1; spreadsheet 01 of Supp 1.xlsx and Supp 2.xlsx). In EGA Gregory, 633 genes were differ­ entially expressed, with 95% being downregulated during heat stress (Table 1; spreadsheet 02 of Supp 1.xlsx and Supp 2.xlsx). Conversely, differential gene expression analyses between control and stress in the tolerant genotype (Fang-60) at 14dpa revealed 552 genes were differ­ entially expressed; of which, only 2% were downregulated during heat stress and 98% were upregulated (Table 1; spreadsheet 03 of Supp 1.xlsx and Supp 2.xlsx) This very different response to heat stress may be associated with the much greater heat tolerance reported for this ge­ notype (Rangan et al., 2019). The overall percentage of differentially expressed genes that were downregulated during heat stress among the genotypes studied was an apparent indication on their level of tolerance. A list of gene ids, along with descriptions, are provided in individual spread sheets of Supplementary file 1 (Supp 1.xlsx) while gene ids with the respective fold changes are provided in individual spread sheets of Supplementary file 2 (Supp 2.xlsx). Phenotypic data on grain physical characteristics (grain length, width and thickness, and 1000-grain weight) measured at maturity from the grains harvested through this same experiment (Rangan et al., 2019) suggested that, Banks and EGA Gregory genotypes were relatively sus­ ceptible, and Fang-60 more resistant to heat stress. The difference in the impact of heat stress between tolerant and susceptible genotype during early-grain filling is clearly visible (Fig. 2). The deformed structure of awns and bleaching of awns and glumes can be seen in susceptible ge­ notype; while only part of the awns (70–80%) were bleached and the

Number of genes or transcripts significantly differentially expressed at FDR 0.01 using Baggerley’s test

3.1. Control vs heat stress at 14dpa

Genotypes/ treatment/ and stages

3. Results

Group comparison

Table 1 Summary statistics of differentially expressed genes during heat stress treatment (11–14 dpa and 27–30 dpa independently) at a false discovery rate (FDR) cut-off value of 0.01 using Baggerley’s test.

% genes upregulated during stress

annotation studies using BLAST2GOv3.0.2 (BioBam, Spain). Paired end reads of 150bp length were imported into CLC genomics workbench, quality check, trimming, and post-trimming quality check was per­ formed with default parameters provided in the software. Reads were initially mapped against the mitochondrial and chloroplast genome se­ quences of hexaploid wheat and the unmapped reads were collected and used for further analyses. Unmapped reads from the 36 samples were aligned independently using TaGI (Triticum aestivum Gene Indices) as the reference dataset (221,925 sequences, release 12.0) for RNA-Seq analyses. The resulting RNA-Seq experimental files were used for differential gene expression analyses comparing the mean of three true biological replicates from control and stressed samples at two different time points for all the three genotypes to identify significantly differentially expressed genes due to heat stress within each genotype. Statistical analyses for the differential gene expression dataset was performed using the Baggerley’s test available within CLC genomic workbench tools and subjected to false discovery rate (FDR) correction with a cutoff p-value of 0.01 was used to identify the statistically differentially expressed genes. Analyses was performed manually between the three genotypes at individual time points to identify the cluster of genes that were significantly differentially expressed and potentially linked to heat stress tolerance by cross comparing the three genotypes contrasting for heat stress. The mapped reference transcripts that were significantly differen­ tially expressed from both the statistical tests were extracted and a subset was formed for each genotype and at each time point between control and stress conditions. The subset of transcripts that are signifi­ cantly differentially expressed (for details please refer Supp 1.xlsx) was subjected to functional annotation studies using BLAST2GOv3.2 for gene ontology term mapping. Annotated transcripts were also mapped and linked through KEGG pathway mapping option using BLAST2GO software tools to identify differentially regulated metabolic pathways.

16.21 5.37 98.01 2.43 5.60 99.35

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glumes are still green in colour in the resistant genotype. Of the total of 1256 differentially expressed genes identified during heat stress at 14dpa between the two susceptible genotypes and the tolerant one (Table 1; spreadsheet 05 of Supp 1, and Supp 2.xlsx), nearly 85% were upregulated in Fang-60 while only 15% were upregulated in the sus­ ceptible genotypes, Banks and EGA Gregory. This comparison across genotypes indicated the involvement of key genes; Histone H2B, pepti­ dylprolyl isomerase, 6-phosphogluconate dehydrogenase, DAD (defender against cell death), S6 RPS6-2 ribosomal protein, plasma membrane proton ATPase, and others (Table 2) that were unique to Fang-60 and might be linked to heat stress tolerance at early (14 dpa)-grain filling (Supp 1.xlsx, spreadsheet “14dpa-strs”; for fold change and RPKM values, please refer “Supp 2.xlsx”). Genes that were expressed more than 100-fold higher in Fang-60 in comparison with Banks and EGA Gregory were: HMW glu­ tenin, Ribosomal protein S8, β-amylase, Histone H2B.2, γ-gliadin, Photo­ system I subunit 5, nicotianamine synthase 3, 60S ribosomal protein L28, β-tubulin 3, methionine synthase, 50S ribosomal protein L9, and 60S ribo­ somal protein L30 (Table 2). In addition to the differential gene expression analyses with Banks and EGA Gregory in one group and Fang-60 on the other group; the genes that were uniquely expressed or upregulated (minimum 100-fold) during heat stress with respect to control as analysed independently in all the three genotypes were also identified (Table 2). This comparison highlighted the role of key genes like heat shock proteins (hsp), small HSPs, FtsH protease, Heat shock Cognate-70, Polyubiquitin, WNT1 inducible signalling pathway genes, C-8,7-sterol isomerase, AsnC gene family, RuBisCO activase B, ascorbate peroxidase, peptidylprolyl isomerase, HSP70, stress-inducible membrane pore protein, Photosystem II 10 kDa polypeptide, galactinol synthase, and others, that might play a crucial role in regu­ lating heat stress tolerance (Supp 1.xlsx; spreadsheets: “Banks-14dpa”, “EGA-Gregory-14dpa”, and “Fang-60-14dpa”; for fold change and RPKM values, please refer “Supp 2.xlsx”). Oligopeptidase-A like, β-amylase, γ-gliadin, AsnC family transcriptional regulator, ascorbate peroxidase, HSP70, and stress-inducible membrane pore protein (Table 2) genes, were upregulated across all the genotypes and hence identified to be stress responsive (inducible) and their functional role in heat tolerance mechanism warrants further study. The grain softness protein encoding gene was expressed more than 100-fold higher during heat stress only in the susceptible genotype (EGA-Gregory) at both 14- and 30-dpa (Table 2) with the implications for heat stress or response not clear yet. Also upregulated (more than 100-fold) genes in the susceptible genotype EGA-Gregory at 14-dpa were: β-glucanase (1,3 & 1,4), calnexin, triose phosphate isomerase, acyl-CoA binding protein, and pyridoxal dependent decarboxylase (Table 2). The expression of these genes might be associated with the failure of these genotypes in with­ standing heat stress.

Table 2 Significantly differentially regulated genes (with at least 100-fold difference) between the genotypes (Banks, EGA Gregory, and Fang-60)# under control and stress conditions (for complete list of gene ids and description of fold change see “Supp 1.xlsx” or “Supp 2.xlsx” respectively). 14 AND 30 DPA

ONLY 14 DPA

ONLY 30 DPA

Heat shock cognate-70

FtsH protease

plasma membrane proton ATPase Peptidylprolyl isomerase methionine synthase

6-phosphogluconate dehydrogenase Photosystem I subunit 5

FtsY precursor (signalrecognition particle-docking protein) DHHC zinc finger domain containing protein thioesterase

Only in tolerant genotype

Cytochrome C (class I) S6 RPS6-2 ribosomal protein HMW glutenin

Photosystem II, 10 kDa polypeptide Rubisco activase B Chloroplast small HSPs

acidic ribosomal protein P2 temperature-induced lipocalin-2

Amino acid permeaseassociated region ferredoxin RNA recognition motif family protein WNT1 inducible signalling pathway Histone H2B & Histone H2B.2 Ribosomal protein S8 60S ribosomal protein L28, L30 50S ribosomal protein L9 C-8,7-sterol isomerase Secretin/TonB short domain precursor DAD (defender against cell death) nicotianamine synthase 3 β-tubulin 3 Glycine rich protein like (PE-PGRS family) Polyubiquitin galactinol synthase

Expressed in both tolerant and non-tolerant genotypes LMW-glutenin γ-gliadin 2 β-amylase Type 2 non-specific lipid transfer protein Ascorbate peroxidase HSPs and small HSP

3.2. Control vs heat stress at 30dpa

Stress inducible membrane protein AsnC family transcriptional regulator Oligopeptidase-A like

Only in non-tolerant genotype Grain softness protein

Differential gene expression analyses between control and heat stress at the late-grain-filling (30 dpa) stage for the three genotypes viz., Banks, EGA Gregory, and Fang-60 are summarized in Table 1. The genotype Banks differentially expressed 3174 genes; while much lower numbers of genes 125 and 771 genes, respectively were found for the genotypes EGA Gregory and Fang-60 (Table 1; spreadsheet 06, 07, 08 of Supp 1, and Supp 2.xlsx). Of these genes, approximately 95% were down regu­ lated (with a few genes completely switched off) during heat stress in both Banks and EGA Gregory; while less than one per cent of genes were downregulated in the tolerant genotype Fang-60 (Table 1) validating the heat tolerant nature of the Fang-60 genotype during grain-filling in line with earlier reports (Blumenthal et al., 1995). Post-heat stress recovery observations during late-grain filling (Fig. 3) clearly indicate the tolerant nature of the Fang-60 genotype, while the susceptible genotype exhibited severe bleaching in grains when compared between the con­ trol and stressed grain samples (Fig. 3A). Conversely, in the tolerant genotype, only 50–60% of awns were bleached (Fig. 3B) while the grains

Triose phosphate isomerase Calnexin Acyl-CoA-binding protein β-glucanase (1,3 & 1,4) Pyridoxal dependent decarboxylase

#

Aspartic proteinase Naþ/Hþ antiporter AraC family, transcriptional regulator Glucose-1-PO4 adenylyl transferase ips1, riboregulator

Ten comparisons made: 1. Control (14-dpa) vs Stressed (14-dpa) for Banks; 2. Control (14-dpa) vs Stressed (14-dpa) for EGA Gregory; 3. Control (14-dpa) vs Stressed (14-dpa) for Fang-60; 4. Control (30-dpa) vs Stressed (30-dpa) for Banks; 5. Control (30-dpa) vs Stressed (30-dpa) for EGA Gregory; 6. Control (30dpa) vs Stressed (30-dpa) for Fang-60; 7. Manual comparison of the differen­ tially expressed genes between Banks, Gregory and Fang-60 independently for 14 dpa (from 1, 2, and 3); 8. Manual comparison of the differentially expressed genes between Banks, Gregory and Fang-60 independently for 30-dpa (from 4, 5, and 6); 9. Banks and EGA Gregory vs Fang-60 for stressed samples at 14-dpa; and 10. Banks and EGA Gregory vs Fang-60 for stressed samples at 30-dpa.

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are still greener in both control and heat stressed samples. An overall view of the glasshouse during recovery clearly distinguishes the sus­ ceptible genotypes from the control and tolerant genotype (Fig. 3C). Differential expression between susceptible (Banks and EGA) and tolerant (Fang-60) genotypes at 30dpa was also studied. The results indicate that around 90% of differentially expressed genes were upre­ gulated in the tolerant genotype during heat stress at 30dpa, while only 10% were upregulated in the non-tolerant ones (Table 1; spreadsheet 10 of Supp 1, and Supp 2.xlsx). Potentially, type 2 non-specific lipid transfer protein precursor, peptidylprolyl isomerase, S6 RPS6-2 ribosomal protein, and plasma membrane proton ATPase genes (Table 2) being uniquely expressed in tolerant genotype – Fang-60 – might help impart tolerance to heat stress during late grain filling. Other genes that had exhibited higher fold change (>100) in Fang-60 were thioesterase, HMW glutenin, β-amylase, γ-gliadin, acidic ribosomal protein P2, temperature-induced lip­ ocalin-2, LMW-glutenin, and methionine synthase (Table 2; Supp 1.xlsx, spreadsheet “30dpa-strs”; for fold change values, please refer to “Supp 2. xlsx”). Most of the genes that were uniquely expressed during heat stress in Fang-60 were common during early (14 dpa) as well as late (30 dpa) -grain filling indicating their possible sustained role to withstand heat stress across grain filling stages. Importantly, more than 100-fold downregulation during heat stress at 30-dpa in Banks and EGA Greg­ ory when compared to the tolerant genotype Fang-60, was observed for the four genes Naþ/Hþ antiporter, AraC family transcriptional regulator, glucose-1-phosphate adenylyl transferase, and ips1 riboregulator (Table 2) suggesting that these genes might be important for heat stress tolerance at 30-dpa. Cytochrome C class I, γ-gliadin, ascorbate peroxidase, FtsY precursor, DHHC zinc finger domain containing protein, HSP40 (regulates HSP70), multiple small HSPs, Heat shock cognate 70 kDa, and peptidylprolyl isom­ erase (Table 2) genes were uniquely expressed or upregulated (>100fold) during heat stress at 30dpa when compared across all the three genotypes independently with respect to their control. These genes might play a key role in imparting heat stress tolerance in wheat during late grain filling (Supp 1.xlsx; spreadsheets: “Banks-30dpa”, “EGAGregory-30dpa”, and “Fang-60-30dpa”; for fold change and RPKM values, please refer to “Supp 2.xlsx”). Among these genes; heat shock proteins and small HSP, type 2 non-specific lipid transfer protein precursor, β-amylase, γ-gliadin, LMW-glutenin, and ascorbate peroxidase were upre­ gulated across all the genotypes during heat stress at both 14- and 30dpa (Table 2) and can be classed as heat stress-responsive irrespective of their role in tolerance.

process”, “translation factor activity, RNA binding”, “endoplasmic re­ ticulum”, and “ribosome” (Supp 6–8) indicating gene ontology enrich­ ments and linking them to heat stress tolerance traits especially in Fang60; which was lacking in the other two genotypes, Banks and EGA Gregory. 4. Discussion It is well known that expression of the heat shock protein family (HSPs) is an important high temperature response mechanism, regulated by heat shock transcription factors (HSF1) during heat stress. The pre­ sent study showed upregulation of various HSPs during heat stress, hsp 101, 82, 70, 40, 26, 22, 20, 18, 17þ, and 16þ (Supp 1, and Supp 2.xlsx) with most of them being expressed in all genotypes irrespective of the tolerance to stress. Monomeric HSF1 is present in cells under ambient conditions and is activated through a trimerization process mediated by a non-coding RNA – heat shock RNA-1 (HSR1) in complex with a trans­ lation elongation factor eEF1A (Shamovsky et al., 2006). In the present study, HSR1 (AF433653) was found to be significantly downregulated in the Banks genotype during heat stress at 30-dpa (Supp 1, and Supp 2. xlsx). Analysis of other significantly differentially expressed genes between control and stress at 14- and 30-dpa across all the three genotypes, emphasised the role of six genes viz., heat shock cognate-70, peptidylprolyl isomerase, plasma membrane proton ATPase, methionine synthase, cyto­ chrome C (Class I), and S6 RPS6-2 ribosomal protein in imparting heat stress tolerance across grain-filling developmental stages in wheat (Table 2). In addition to this, expression of five crucial genes, FtsH protease, 6-phosphogluconate dehydrogenase, Photosystem I subunit 5, Photosystem II (10 kDa polypeptide), and RuBisCO activase B (Table 2) in the tolerant genotype (Fang-60) under heat stress at 14-dpa correlated well with sustained grain filling. For the first time, we have established the importance of 6-phosphogluconate dehydrogenase gene expression under stress for heat tolerance. This might be due to its regulatory role in balancing between the oxidative pentose phosphate pathway (OPP), Calvin-Benson (C3) cycle and Entner-Doudoroff (ED) pathways (Chen et al., 2016) for sustained grain filling even while the plant is under heat stress. This result also supports the functional annotation studies for the enrichment of annotation terms with “Biosynthetic process” and “Car­ bohydrate metabolic process” (Supp 3–5). Irrespective of the genotypes’ tolerance or susceptibility, the following five genes hsp, ascorbate peroxidase, β-amylase, γ-gliadin-2 and LMW-glutenin (Table 2) were heat stress responsive and upregulated across 14- and 30-dpa in all three genotypes; while their role in heat tolerance needs further study. Upregulation of β-glucanase (1,3 & 1,4), triose phosphate isomerase, and calnexin at 14-dpa; downregulation of Naþ/Hþ antiporter, glucose-1-phosphate adenylyltransferase, ips1 ribor­ egulator, and AraC family transcriptional regulator (Table 2) at 30-dpa; was associated with heat stress susceptibility This indicates the role of a cluster of key genes in addition to hsps, that play a significant role (although Hsps might regulate the expression of the gene cluster – an independent study for each gene is required) in imparting heat stress tolerance during grain filling without affecting grain yield significantly. An individual overview of the cluster of genes that were identified in this study is provided below.

3.3. Functional annotation From the annotation of genes at 14-dpa for three genotypes inde­ pendently between control and stress, the top five annotated genes for BP of Banks and EGA Gregory (non-tolerant) were enriched with “catabolic process” while in the tolerant genotype Fang-60 it is “biosynthetic process” (Supp 3–5). Comparison between tolerant and non-tolerant genotypes during stress, highlighted the role of “carbohy­ drate metabolic process” differentially in Fang-60 relative to the other two indicating their potential crucial role in sustained grain filling and withstanding heat stress. Enrichment of genes with “transcription factor activity, DNA bind­ ing” activity for MF uniquely in Fang-60 during stress, might play a key role in heat stress tolerance; when the genotypes under stress are compared with respect to their controls (Supp 3–5). Whereas, compar­ ison between the tolerant and non-tolerant genotypes under stress revealed the importance of translation events through the gene enrich­ ment with “translation factor activity, RNA binding” activity. Enrichment of CC annotation with “endoplasmic reticulum” (Supp 3–5) specifically in Fang-60 between control and stress and “ribosome” in comparison between Fang-60 and non-tolerant genotypes reflects the importance of translation for sustenance even at high temperature stress. Functional annotation analyses at 30dpa identified “biosynthetic

4.1. 6-Phosphogluconate dehydrogenase The chloroplast localized 6-phosphogluconate dehydrogenase (pgd3) gene involved in the oxidative pentose phosphate pathway has been reported to affect starch biosynthesis significantly (Zhang et al., 2016). The pgd and zwf (glucose-6-phosphate dehydrogenase) genes are known to play a balancing role between the oxidative pentose phosphate pathway (OPP), Calvin-Benson (C3) cycle, and Entner-Doudoroff (ED) pathway. The present study (Table 2; Supp 1, and Supp 2.xlsx) reports the first evidence for the importance of expression of the pgd3 gene for 6

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Journal of Cereal Science 91 (2020) 102895

sustained grain filling during heat stress. This result suggests the possible involvement of pgd3 in diverting the glycolytic route to the OPP thereby increasing the threshold level of ribulose-5-phosphate towards the C3 cycle for starch accumulation.

involvement of FtsH protease in heat stress tolerance (Table 2; Supp 1, and Supp 2.xlsx).

4.2. S6 RPS6-2 ribosomal protein

Activation of RuBisCO in plants is performed through an enzymatic action of RuBisCO activase (RCA) with the highest activity for RuBisCO at >50 � C while that of RCA was reported to be at only 42 � C indicating the thermal sensitivity of RCA (Crafts-Brandner and Salvucci, 2000). Although RuBisCO has the highest activity at higher temperatures; during heat stress two things happen, one, RCA fails to activate RuBisCO and secondly, the RuBisCO that is active has an altered response to oxygenation requiring higher concentrations of CO2 to function. Tem­ perature and CO2 concentration play a crucial role in balancing the action of RuBisCO between the C3 cycle and the photorespiration pathway. Chloroplast HSPs are reported to protect RCA and photosyn­ thesis during heat stress (Salvucci, 2008). Our study indicates the dif­ ferential expression of RCA (B isoform) during heat stress might reduce their deactivation and thereby sustain photosynthesis under stressed conditions in the tolerant genotype Fang-60. Based on the understanding obtained through this study, we could group the set of significantly differentially expressed genes into three categories: 1) genes involved in imparting heat stress tolerance; 2) the ones that are stress responsive; and 3) genes associated with heat sus­ ceptibility during stress. Although the present findings were an outcome from a few genotypes (one tolerant and two susceptible), extending the study to a larger set of genotypes might help validating the identified genes and their role in heat stress tolerance. Also, our focus was to study the impact of heat stress during grain filling and hence chose only two stages (11-14dpa and 27-30dpa). However, extending heat stress ex­ periments of different durations, intensities and developmental stages might give a better perspective on the impact of heat stress in wheat and might be useful in choosing appropriate strategies fo breeding for wheat with resilience to high temperature.

4.7. RuBisCO activase B

Ribosomal proteins, especially S6, play a crucial role in regulating cellular metabolism, transcription of rRNA genes, and especially trans­ lation process (Kim et al., 2014). In this study, various ribosomal pro­ teins viz., S6, S8, L28, L30, and L9 were found to be differentially regulated during heat stress in the tolerant genotype (Table 2; Supp 1, and Supp 2.xlsx) and might play a crucial role in the synthesis of mo­ lecular chaperones and other proteins involved in heat stress tolerance. Generally, transcripts containing terminal oligopyrimidine tracts (TOP) like ribosomal proteins (RPS6) and elongation factors are energy demanding and hence inhibited during stress through various regulatory roles of untranslated regions (UTRs) both 50 and 3’ (Sajjanar et al., 2017). 4.3. Peptidylprolyl isomerases Peptidylprolyl isomerases are the family of proteins altering the 3D structure of the proteins through the catalytic activity of rotating the covalent bond preceding the proline amino acid in a protein resulting in various functional implications like chaperone or signal transduction activity through interaction with HSP. The role of such isomerases as intracellular receptors and during heat stress has been well documented (Sykes et al., 1993). 4.4. Plasma membrane proton ATPase These are primary transporters that generate proton motive force and thereby regulate pH and the potential difference across the plasma membrane that primarily enables various secondary transport activity. Other reports indicate the regulation of multiple physiological activities during development, and during abiotic stress conditions (Jung et al., 2017) in plants. The key role played by the plasma membrane proton ATPase in determining heat stress tolerance and its interaction with HSP30 (Braley and Piper, 1997) has been well established in microbial systems.

5. Conclusion The regulated expression of a cluster of genes, especially, 6-phos­ phogluconate dehydrogenase (pgd3), S6 RPS6-2 Ribosomal protein, Pepti­ dylprolyl isomerases, Plasma membrane proton ATPase, heat shock cognate70, FtsH protease and RuBisCO activase B was identified as playing a crucial role in imparting heat stress tolerance in addition to known HSPs. The importance of the sustained expression of the starch biosynthesis regulatory gene pgd3, during heat stress for tolerance is suggested here for the first time. In addition to the expression of genes required for heat stress tolerance; the up- (e.g: β-glucanase (1,3 & 1,4)) or down-regulation (e.g: glucose-1-phosphate adenylyltransferase) of genes uniquely in nontolerant genotypes during heat stress is also a crucial observation requiring attention. Heat stress is one abiotic factor that cannot be overcome by protective or preventive measures and requires tolerant genotypes for sustained productivity under climate change (Henry et al., 2016). The use of the cluster of genes identified or the inclusion of Fang-60 as apparent in a breeding program for introgression of these genes/locus using marker assisted breeding or through modern genetic tools, might help develop high yielding wheat cultivars with heat stress tolerance.

4.5. Heat shock cognate-70 The heat shock cognate-70 protein categorized under the HSP70 family is basically a constitutively produced chaperone reported with multitude functionalities: protein folding, translocation across mem­ branes, and preventing protein aggregation during stress conditions. Heat shock cognate (HSC-70) in combination with HSR1 and eEF1A play a crucial role in activation of the monomeric heat shock factor (HSF1) to trimer which in turn regulates transcription of various genes encoding HSPs and other stress proteins (Åkerfelt et al., 2010) leading to heat stress tolerance. 4.6. FtsH protease An inner-membrane bound protease with AAA (ATPase associated with various cellular activities) and metalloprotease domain – fila­ mentation temperature sensitive (FtsH) protease – was first reported in a bacterial system and found to be linked to temperature sensitivity and later identified in animals and plants (Wagner et al., 2012). The FtsH protease degrades short-lived proteins with a high rate of turnover and misassembled proteins. The core reaction centre of Photosystem II (PS2) consists of the heterodimer proteins D1 and D2. The protein D1 has a high rate of turnover with a half-life of 2 h due to a photo-inhibitory effect resulting in repair cycle functionalized through dephosphoryla­ tion events (Rintam€ aki et al., 1996). Our study clearly indicates the

Funding The research reported here was jointly funded by DBT, Govt. of India in the form of an Indo-Australian Career Boosting Gold Fellowship to RP and QAAFI, UQ, Australia. ICAR-NBPGR supported P.R. Data deposition and accession number The transcriptome sequence (in the form of raw reads) for 36 samples 7

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Journal of Cereal Science 91 (2020) 102895

reported in this paper has been submitted to the Arrayexpress database (with the accession number E-MTAB-6140).

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Authors contributions P.R. and R.H. conceived the project; P.R., A.F. and R.H. planned the experiment; A.F. and R.H. supervised the experiment; P.R. per­ formed the experiments, analysed data and wrote the article with con­ tributions of all the authors. All authors have read and approved the manuscript Availability of data and material The transcriptome datasets reported in this study are available in a raw read format under the accession number E-MTAB-6140 for down­ load through ArrayExpress (https://www.ebi.ac.uk/arrayexpress/expe riments/E-MTAB-6140). Declaration of competing interest The authors declare that they have no competing interests. The research funders had no influence on the conduct of the research, writing of the manuscript or decision to submit. Acknowledgements We thank, Sally Norton, Australian Grains Genebank, for the supply of germplasm reported in this study and Ken Hayes for facilitating glasshouse services. The research reported here was jointly funded by DBT, Govt. of India in the form of an Indo-Australian Career Boosting Gold Fellowship to RP and QAAFI, UQ, Australia. RP thanks the Direc­ tor, ICAR-NBPGR for institutional support. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.jcs.2019.102895. References Åkerfelt, M., Morimoto, R.I., Sistonen, L., 2010. Heat shock factors: integrators of cell stress, development and lifespan. Nat. Rev. Mol. Cell Biol. 11, 545–555. Blumenthal, C., Bekes, F., Gras, P.W., Barlow, E.W.R., Wrigley, C.W., 1995. Identification of wheat genotypes tolerant to the effects of heat stress on grain quality. Cereal Chem. 72, 539–544. Braley, R., Piper, P., 1997. The C-terminus of yeast plasma membrane Hþ-ATPase is essential for the regulation of this enzyme by heat shock protein Hsp30, but not for stress activation. FEBS Lett. 418, 123–126. Chen, X., Schreiber, K., Appel, J., Makowka, A., F€ ahnrich, B., Roettger, M., Hajirezaei, M. R., S€ onnichsen, F.D., Sch€ onheit, P., Martin, W.F., 2016. The Entner-Doudoroff pathway is an overlooked glycolytic route in cyanobacteria and plants. Proc. Natl. Acad. Sci. 113, 5441–5446. Crafts-Brandner, S.J., Salvucci, M.E., 2000. Rubisco activase constrains the photosynthetic potential of leaves at hgh temperature and CO2. Proc. Natl. Acad. Sci. 97, 13430–13435. Furtado, A., 2014. DNA extraction from vegetative tissue for next-generation sequencing. In: Henry, R., Furtado, A. (Eds.), Cereal Genomics: Methods and Protocols. Humana Press, New York, pp. 1–5. Furtado, A., Bundock, P.C., Banks, P., Fox, G., Yin, X., Henry, R., 2015. A novel highly differentially expressed gene in wheat endosperm associated with bread quality. Sci. Rep. 5, 10446.

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