Global crotonylatome and GWAS revealed a TaSRT1-TaPGK model regulating wheat cold tolerance through mediating pyruvate

Abstract

Here, we reported the complete profiling of the crotonylation proteome in common wheat. Through a combination of crotonylation and multi-omics analysis, we identified a TaPGK associated with wheat cold stress. Then, we confirmed the positive role of TaPGK-modulating wheat cold tolerance. Meanwhile, we found that cold stress induced lysine crotonylation of TaPGK. Moreover, we screened a lysine decrotonylase TaSRT1 interacting with TaPGK and found that TaSRT1 negatively regulated wheat cold tolerance. We subsequently demonstrated TaSRT1 inhibiting the accumulation of TaPGK protein, and this inhibition was possibly resulted from decrotonylation of TaPGK by TaSRT1. Transcriptome sequencing indicated that overexpression of TaPGK activated glycolytic key genes and thereby increased pyruvate content. Moreover, we found that exogenous application of pyruvate sharply enhanced wheat cold tolerance. These findings suggest that the TaSRT1-TaPGK model regulating wheat cold tolerance is possibly through mediating pyruvate. This study provided two valuable cold tolerance genes and dissected diverse mechanism of glycolytic pathway involving in wheat cold stress.

A global wheat crotonylation profiling was constructed and TaPGK was identified to regulate cold tolerance by mediating pyruvate.

INTRODUCTION

Low temperature is one of the most seriously abiotic stresses that adversely affects wheat production (1). Cold stress frequently occurred in many wheat regions worldwide and caused a serious yield loss (2). Cold stress results in the accumulation of reactive oxygen species, leading to oxidative damage of proteins, nucleic acids, and lipids in plants (3). Plants adopt multiple strategies to cope with this adverse condition, such as raising the level of antioxidants and chaperones, maintaining osmotic balance by altering membrane structure, and producing more energy by activation of primary metabolisms (4). Wheat cold tolerance is a complex trait as it could be affected by genetic factors and environment. It is well known that the ICE-CBF-COR signaling pathway is essential for plant cold tolerance (5). In wheat, 37 C-repeat binding factor (CBF) genes have been characterized (6). In addition, a number of genes (e.g., TaDREB3, TaCBF5L, WCS120, and Wrab19) were identified in response to cold stress (7, 8). Previous studies revealed two major QTL of Fr-1 (frost resistance-1) and Fr-2 for wheat cold tolerance on chromosome 5A (2, 9). Fr-1 is assumed to be a pleiotropic effect of Vrn-1 due to their physically close positions, and Fr-2 is possibly a copy number variation of a CBF gene (9–11). Because restricted important genes regulating cold stress were reported in wheat, the identification of more genes is urgent for the improvement of wheat cold tolerance and is valuable for further dissection of the regulatory mechanisms of wheat cold stress.

Protein posttranslational modifications (PTMs) represent fundamental regulatory events that integrate signaling and gene expression with cellular metabolic networks (12). Emerging evidences indicate that PTMs are also important for plant cold responses. In Arabidopsis, cold-activated protein kinase open stomata 1 phosphorylates the inducer of CBF expression 1 (ICE1) to promote its protein stability, thereby enhancing freezing tolerance (13). ICE1 is subjected to sumoylation and polyubiquitylation and subsequent proteasomal degradation, mediated by the SUMO E3 ligase SIZ1 and ubiquitin E3 ligase osmotically responsive gene 1 (HOS1), respectively (14). In rice, mitogen-activated protein kinase 3 (OsMAPK3) phosphorylates OsICE1 and inhibits its ubiquitination to activate trehalose-6-phosphate phosphatase (OsTPP1) and enhances chilling tolerance (15). As a novel lysine PTM, crotonylation (Kcr) was also found to be related to cold stress in plants (16). In chrysanthemum, Kcr of a temperature-induced lipocalin-1-like gene (DgTIL1) promoted the cold tolerance by inhibiting a nonspecific lipid transfer protein (DgnsLTP) degradation (17). Kcr was first identified to be mainly associated with active chromatin in human beings in 2011 (18). From then on, there was a growing interest in Kcr because it has emerged as a powerful novel epigenetic marker. Histone Kcr has been associated with DNA damage and repair, transcriptional silencing and repair, carcinogenesis, kidney injury, inflammation, the building of some sperm super-enhancers, endoderm differentiation (19, 20), and depressive behaviors. In addition to histone lysine residues, there are global profiles of the Kcr proteome in humans, animals, microorganisms, and plants (21, 22). The rice crotonylome provided evidence for histone Kcr in transcriptional regulation in plants (23) and sharply affected plant stress adaptation (24). These studies indicated that Kcr possibly plays vital roles in regulating cellular metabolism and protein function.

To date, several regulatory enzymes (writers and erasers) of histones have been identified in mammal cells, yeast cells, or in vitro, e.g., histone acetyltransferases [HATs; p300/CBP (CREB-binding protein) , the catalytic subunit of nucleosome acetyltransferase of H4 complex Esa1, and Males absent on the first (MOF)], histone deacetylases (HDAC1, HDAC2, and HDAC3), and sirtuins (SIRT1, SIRT2, and SIRT3) (19, 25). The NAD⁺-dependent deacetylase CobB and the putative GCN5-family acetyl-transferase Kct1 were found as “erasers” in Streptomyces roseosporus, but they primarily functioned in different developmental stages (26). OsSRT2 was found to be associated with the erasure of histone Kcr in rice, and its decrotonylase activity was also verified in vitro (24). Recently, human HDAC6 was reported to decrotonylate lamin A and was down-regulated in response to hypoxia (27).

The energy defensing kinds of abiotic stresses in plants is mainly produced in the glycolytic pathway. Glycolysis is a central feature of metabolism, and its regulation plays important roles in cold responses (12). Some crucial glycolytic genes are directly related to cold stress. In Machilus macrocarpa, the up-regulation of glycolytic genes, including glyceraldehyde 3-phosphate dehydrogenase (GAPC), phosphofructokinase (PFK), phosphoglycerate mutase (PGM), and pyruvate kinase (PK), could provide energy to enhance cold stress (28). In orange, glycolytic genes fructose-bisphosphate aldolase (FBA) and enolase (ENO) were strongly induced by cold stress (29). In rice, a PGM gene (TCM12) plays an important role in regulating cold response (30). In Saussurea involucrata, the SiFBA5 positively regulates plant cold stress (31). Recent advances in proteomics and mass spectrometry (MS) have documented extensive PTMs of most glycolytic enzymes in plants (12). Phosphorylation, redox-sensitive cysteine PTMs, and polyubiquitination, and mono-ubiquitination were reported to be involved in plant glycolytic control (12).

The large genome size of hexaploid wheat is a big barrier to clone key genes. Therefore, previous identification of abiotic stress response genes mainly relies on reverse genetic strategy, especially proteome and transcriptome sequencing. Genome-wide association studies (GWAS) have been widely used to identify important genetic loci associated with complex traits in multiple plants (32). Therefore, integration of multi-omics and GWAS is an efficient strategy to identify key genes in hexaploid wheat. It has been proven that Kcr widely exists in multiple plants and potentially affects plant development and defense (21, 24). In this work, we performed a global profile of the Kcr proteome of common wheat and subsequently combined multi-omics and Kcr proteome to identify a glycolytic gene phosphoglycerate kinase (PGK) (TaPGK) regulating wheat cold stress. We further revealed TaSRT1 negatively modulating wheat cold tolerance through inhibiting the accumulation of TaPGK protein by erasing Kcr. Last, we demonstrated the TaSRT1-TaPGK model regulating wheat cold stress, possibly by promoting pyruvate accumulation.

RESULTS

Evolutionarily conservatism of wheat protein crotonylation

To illustrate whether Kcr commonly exists in wheat, we performed Western blot using pan anti-Kcr antibody and found that Kcr is widely distributed and is mainly enriched in the range of 15 to 130 kDa in hexaploid wheat (AABBDD) and its progenitors Triticum urartu (AA), Aegilops speltoides (SS), Aegilops tauschii (DD), and Triticum durum Langdon (AABB) (Fig. 1A). Hexaploid wheat exhibited the highest Kcr level compared with its progenitors. In addition, the Kcr level of the T. urartu was relatively lower than those of the tetraploid, A. speltoides and A. tauschii. It suggests that Kcr is an evolutionarily conserved marker widely distributed in wheat and its progenitors. Furthermore, Western blot indicated that Kcr level changed irregularly with seedling growth but was the highest at the two-leaf stage (fig. S1A). Kcr also widely occurred in various types of proteins in wheat germ, root, stem, and seed (fig. S1B). The detection of dynamic Kcr under different abiotic stresses indicated that the Kcr levels significantly changed under the multiple stress treatments (Fig. 1B). The Kcr level decreased significantly under high temperature but increased mainly in the 25 to 130 kDa range under low temperature, suggesting that the Kcr level is potentially associated with cold stresses.

Fig. 1. Proteome-wide identification and properties of lysine crotonylation sites in wheat.

(A) Immunoblots of Kcr (crotonylation) proteins in wheat and its donors (SS: A. speltoides; AA: T. urartu; AABB: Triticum durum; DD: A. tauschii; AABBDD: Triticum aestivum L.). (B) Immunoblots of Kcr proteins after wheat exposed to different stresses [2°C, 42°C, 20% polyethylene glycol, molecular weight 6000 (PEG-600), and 100 mM NaCl]. (C) Experimental procedures used in this study. (D) Venn diagram of the number of Kcr sites among three biological replicates. (E) Pie chart illustrating the number and percentage of Kcr sites per protein. (F) Conservation and variance of Kcr among protein triplets. (G and H) Venn diagram of Kcr proteins (G) and sites (H) from Kac (acetylome), Ksu (succinylome), Kcr, and Kma (malonylome). (I) Heatmap showed the frequency of different types of amino acids around Kcr sites. Red indicates high enrichment and green indicates depletion. (J) Probabilities of Kcr in different protein secondary structures and predicted surface accessibility of Kcr sites.

Global landscape of the lysine crotonylome in wheat

To identify Kcr characteristics in wheat, we determined Kcr proteome of wheat leaves by combining the affinity enrichment and liquid chromatography–tandem MS (LC-MS/MS) (Fig. 1C). In total, we identified 12,066 distinct Kcr sites in 3458 proteins (Fig. 1D and table S2), and 4696 Kcr sites from 1726 proteins overlapped in three replicates (Fig. 1D and table S3). Most of the mass errors were less than 5 parts per million (ppm), confirming the high accuracy of our MS data (fig. S2, A to C). The Kcr peptide length ranged from 7 to 40 amino acids and 91% of them showed 7 to 20 amino acids (fig. S2D). Of the 1726 Kcr proteins, more than 800 wheat proteins were found to have multiple Kcr sites (Fig. 1E and table S3). Analysis of Kcr conservation in 22,097 gene traids previously reported (http://wheat.cau.edu.cn/TGT/download.html) showed that single subgenome had relatively more Kcr proteins (Fig. 1F). Only 75 proteins (2%) were identified to have Kcr in protein traids (expressed homologs proteins from three subgenomes) of common wheat (Fig. 1F and table S4).

A comparison showed many proteins or sites had multiple PTMs (Fig. 1, G and H), but only three proteins were overlapped in Kcr, Kac (33), Ksuc (34), and Kma (35) proteins previously determined in wheat. Analysis in different species indicated that 680 Kcr proteins were orthologous among wheat, tobacco, and rice (23, 36, 37), and 171 were simultaneously found in the three species (fig. S3, A and B). In addition, 1046 Kcr proteins were exclusively found in the wheat lysine crotonylome. Motif analysis results indicated that 21 conserved sequences around the Kcr sites were identified in wheat (fig. S4), and three (KcrD, EKcr, and DKcr) of them simultaneously existed in wheat, tobacco (36), and rice (23). Both heatmap (Fig. 1I) and motif-x (fig. S4) results showed the preference of several amino acid residues for Kcr in wheat. Protein secondary structures indicated that 27.4, 6.5, and 66% of the Kcr sites were located in α helices, β strands, and disordered coils, respectively (Fig. 1J). The distribution pattern of Kcr indicated that there was structural preference in the β strand regions compared with unmodified lysine residues. The average surface accessibility of protein Kcr was significantly lower than that of unmodified protein lysine residues (Fig. 1J), implying that Kcr is preferably located within protein structures.

The Gene Ontology (GO) analysis of the identified 1726 Kcr proteins showed that a total of 30 nonredundant terms were statistically enriched (fig. S5A), and the largest proportion was assigned to the chloroplast (50%), followed by the cytoplasm (23%), nucleus (13%), and mitochondria 96 (6%) (fig. S5B). The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that Kcr proteins were related to a variety of metabolic pathways, such as photosynthesis, oxidative phosphorylation, and carbon metabolism (fig. S6A). Protein domains enrichment showed that the nicotinamide adenine dinucleotide phosphate–binding domain, chlorophyll a/b binding protein domain, and thioredoxin domain were significantly enriched in Kcr proteins (fig. S6B), which is consistent with the GO and KEGG results.

Identification of cold response candidate genes modified by lysine crotonylation

PTMs have been proven to play important roles in plant abiotic stresses (13, 15, 38). Because the Kcr level increased significantly under cold stress as mentioned above, we assumed that Kcr potentially affected wheat cold tolerance. Subsequently, we performed bulked segregation transcriptome [bulked segregant transcriptome using RNA sequencing (BSR-seq)] and proteomics [bulked segregant proteome using tandem mass tag (BSP-TMT)] sequencing in the extreme phenotype lines of RIL (recombinant inbred line) population Shanghai3/Catbird × Naxos (RIL-SC) (SC-1: Shanghai3/Catbird; SC-2: Naxos) after investigation of cold tolerance index (CTI). Results showed that a total of 6781 (table S5 and fig. S7) and 444 (table S6 and fig. S8) genes/proteins were identified as differentially expressed genes (DEGs) and proteins (DEPs) between cold-tolerant and cold-sensitive pools. Furthermore, molecular weight of 300 DEPs identified by BSP-TMT ranged from 25 to 130 kDa (table S7), and 45 of them were modified by Kcr (Fig. 2A). Combination analysis with BSR-seq indicated that 12 of the 45 genes were differentially expressed at the transcriptome level (Fig. 2A and table S8), and thereby, they were selected to be potential candidates with Kcr modification.

Fig. 2. Identification of TaPGK through a combination of multi-omics, GWAS, and geographical distribution.

(A) Venn diagram of the number of bulked segregant transcriptome and proteome (25 to 130 kDa) and the Kcr proteome. (B) Distribution of the CTI in Chinese wheat cultivars in three environments. (C and D) Regional Manhattan plots for cold resistance for chromosomes 6B and regional Manhattan plots. (E) Gene structure and haplotype analysis of TaPGK and comparison of the CTI among two haplotypes in multiple environments. SNP1, single-nucleotide polymorphism 1. (F) Geographic distribution of accessions carrying TaPGK-hap1. Values are presented as means ± SE. **P < 0.01, as determined by paired Student’s t tests. E1: 2018_Yuanyang; E2: 2020_Yuanyang; E3: 2020_Zhengzhou.

Association of TaPGK with wheat cold tolerance by GWAS

To further uncover the candidate genes associated with cold tolerance, we investigated CTI for 2 years in the 406 wheat accessions containing two panels that have been previously genotyped using wheat 660K and 90K single-nucleotide polymorphism (SNP) arrays (39, 40), respectively. Phenotypic distribution showed that 120, 240, and 46 of the tested wheat accessions were highly sensitive (2 < CTI ≤3), moderately sensitive (1 < CTI ≤ 2) and poorly sensitive (0 ≤ CTI <1) to cold stress (Fig. 2B). Sequencing the above-mentioned 12 candidate genes generated a total of 126 variants (table S9) in all 406 accessions. Running GWAS using wheat 660K and 90K SNP arrays after adding the 126 variants showed that 425 and 865 SNPs were significant in panels I and II across 2 years, respectively (table S10), and they were mainly distributed on 1B, 2D, and 5A (fig. S9A and table S10). Moreover, 10 variants associated with 3 (TraesCS7A02G010300, TraesCS7D02G091500, and TraesCS6B02G187500) of the above 12 genes were significant in multiple environments (table S11). However, only TraesCS6B02G187500 (TaPGK) of the three genes was highly expressed in wheat roots and leaves (fig. S9B; http://202.194.139.32/). In addition, variants of TaPGK were simultaneously significant in two panels (Fig. 2, C and D, and table S11). Sequencing results showed that TaPGK had seven variants between two parents of RIL-SC (Fig. 2E). Wheat varieties with Hap2 (SC-1) exhibited significantly stronger cold tolerance (CTI = 0.25) than those with Hap1 (SC-2) (CTI = 1.69) (Fig. 2E). Quantitative real-time polymerase chain reaction (qRT-PCR) showed that TaPGK was significantly up-regulated under cold stress (fig. S10, A and B). These results suggested that TaPGK is intimately associated with wheat cold tolerance.

TaPGK positively modulating wheat cold tolerance

To verify the function of TaPGK regulating wheat cold stress, we screened two mutants (K3282, a splice acceptor variant; K167, a premature stop codon) of TaPGK-B1 from the ethyl methanesulfonate (EMS)–mutagenized Kronos library and backcrossed them into BC₂ lines with wild type (WT) for two times (Fig. 3, A and B). We determined sharply decreased PGK activities in TaPGK mutants compared with control (Fig. 3C). After cold treatment, leaves of two TaPGK mutants displayed extreme dehydration and drooping (Fig. 3B), and their relative electrolyte leakage rate (REC), hydrogen peroxide (H₂O₂), and malondialdehyde (MD) contents were sharply increased (fig. S11, A to C) compared with the control.

Fig. 3. TaPGK was verified to regulate cold stress through mutants and overexpressed plants.

(A to D) Mutation sites of TaPGK (A) in Kronos EMS mutants and a comparison of phenotype, PGK activity, and pyruvate content of TaPGK EMS mutants and the WT (n = 6 plants per replicate) under cold stress (B to D). (E to H) Relative expression of TaPGK in TaPGK-overexpressed lines (E) and a comparison of phenotype, PGK activity, and pyruvate content of TaPGK-overexpressed lines and the WT (n = 6) under cold stress (F to H). The β-actin gene was served as the endogenous control. (I to L) Mutation sites of TaPGK (I) in gene edited (GE) Fielder mutants using CRISPER-Cas9 and comparison of phenotype, PGK activity, and pyruvate content of TaPGK-GE mutants and the WT (n = 6) under cold stress (J to L). gRNA, guide RNA. Experiments were conducted with three biological replicates. Values are presented as mean ± SE. **P < 0.01, as determined by paired Student’s t tests. KO, gene knockout. KO is the same to GE.

To identify the influence of TaPGK overexpression on cold stress, we cloned the cDNA of TaPGK from cold tolerance parent SC-1 and constructed the wheat LGY-OE3 vector containing TaPGK. Moreover, we transferred the LGY-OE3-TaPGK into cold-sensitive cultivar Fielder for overexpression. After positive detection by specific primers, three TaPGK-overexpressed lines with relatively high expression by qRT-PCR were self-crossed into T₂ generation (Fig. 3, E and F, and fig. S12A). PGK activity was sharply increased in TaPGK-overexpressed T₂ plants (Fig. 3G). After cold treatment (−4°C, 36 hours), TaPGK-overexpressed plants showed significantly stronger cold tolerance (Fig. 3F) and lower contents of REC, H₂O₂, and MDA than the controls (fig. S11, D to F). In addition, TaPGK-overexpressed lines showed slightly preferred change of agronomic traits in the field (table S12).

To further verify the mutation of TaPGK affecting cold stress in hexaploid wheat, we used CRISPR-Cas9 to create TaPGK-edited mutants in Fielder. After mutation sites were confirmed through sequencing by high-throughput tracking of mutations (Hi-TOM) (table S13) (41), we selected four TaPGK-edited lines to self-cross them into T₂ generation. After further verification of mutation sites through Sanger sequencing (Fig. 3, I and J, and fig. S13), we selected four TaPGK-edited T₂ lines for further analysis, i.e., TaPGK_GE#1 (aaBBdd), TaPGK_GE#3 (AABBdd), TaPGK_GE#8 (AAbbDD), and TaPGK_GE#17 (aabbDD). PGK activities were sharply reduced in the four TaPGK-edited lines (Fig. 3K). After cold treatment (−4°C, 18 hours), all four TaPGK-edited lines were severely dehydrated and drooped (Fig. 3J) and had a significant increase of REC, H₂O₂, and MDA (fig. S11, G to I). Together, these results confirmed that TaPGK positively regulated wheat cold tolerance.

Lysine crotonylation of TaPGK induced by cold stress

To illustrate the relationship of cold stress and Kcr level of TaPGK, we identified eight Kcr sites in TaPGK (table S8 and fig. S14) by LC-MS/MS. TaPGK proteins from Fielder before and after cold treatment were immunopreciptated with anti-TaPGK antibody recognizing plant cytosolic PGK and subsequently were detected by immunoblots. Results indicated that the Kcr level of TaPGK was significantly increased in Fielder after cold treatment (Fig. 4A), indicating that cold stress positively induced the Kcr of TaPGK. Moreover, we extracted the total proteins of TaSRT1-overexpressed wheat plants before and after cold treatment. Western blot results by immunoblots indicated that cold stress increased the Kcr level and protein accumulation of TaPGK (Fig. 4, B and C). These results revealed intimate association of cold stress with the Kcr level of TaPGK.

Fig. 4. Cold stress induced lysine crotonylation of TaPGK.

(A) Immunoblots detected increased Kcr level of TaPGK in Fielder after cold treatment. IP, immunoprecipitation. (B) Immunoblots detected increased Kcr level of TaPGK in TaPGK-overexpressed wheat after cold treatment. (C) Immunoblots detected increased expression of TaPGK in TaPGK-overexpressed wheat after cold treatment. (D) Immunoblots detected decreased Kcr level of TaPGK^K206R but not TaPGK^K35R and TaPGK^K366R in transient expression tobacco. (E) In vivo detection results showed that TaPGK^K206R but not TaPGK^K35R and TaPGK^K366R had decreased enzymatic activity in transient expression tobacco. (F) Immunoblots detected decreased protein abundance of TaPGK^K206R in transient expression tobacco. (G) Immunoblots detected sharply increased Kcr level of TaPGK and but no visible change of Kcr level of TaPGK^206R in transient expression wheat leaf protoplasts after cold stress. Relative Kcr/TaPGK signals in the different genotypes are measured. The bands were quantified with ImageJ software. Bars are means ± SE from three measures of each of the two repeats. *P < 0.05 and **P < 0.01, as determined by paired Student’s t tests.

Integration of LC-MS/MS and previous reported results showed that TaPGK was modified by multi-PTMs but three (K35, K206, and K366) of the eight key sites in TaPGK were modified only by Kcr (fig. S14). To illustrate the association of Kcr sites of TaPGK with cold stress, we mutated lysine (K) in these three sites into arginine (R) to generate crotonyl-dead mutants, respectively. Subsequently, we transiently overexpressed TaPGK and its three mutants (TaPGK^K35R, TaPGK^K206R, and TaPGK^K366R) in tobacco. Results showed that the Kcr level and enzyme activity of TaPGK^K206R were substantially decreased compared with WT (Fig. 4, D and E). Moreover, TaPGK^K206R showed significantly decreased protein abundance compared with WT (Fig. 4F). These results suggested that K206 is a key Kcr site of TaPGK. Next, we transiently overexpressed TaPGK and TaPGK^K206R in protoplasts of wheat leaves before and after cold treatment. TaPGK proteins from wheat protoplasts were immunopreciptated with anti-TaPGK antibody and were detected by immunoblots. Results showed that cold stress sharply increased the Kcr level of TaPGK but did not visibly affect the Kcr level of the TaPGK^K206R (Fig. 4G), implying that K206 as a key Kcr site of TaPGK is response to cold stress.

Interaction of TaPGK with TaSRT1

To dissect the mechanism of TaPGK regulating wheat cold tolerance, we used TaPGK as a bait to screen interaction proteins in a wheat cDNA library by yeast two-hybrid (Y2-H), and a sirtuin-like gene (TaSRT1, TraesCS2B02G092700) was selected because decrotonylase activity was previously reported in sirtuins of mammals (42) and OsSRT2 of rice (24). We then confirmed that TaSRT1 interacted with TaPGK in the yeast cell by Y2-H (Fig. 5A). A firefly luciferase complementation imaging (LCI) assay in tobacco cells showed that the co-infiltration of TaPGK-cLUC and TaSRT1-nLUC generated luminescence by the complemented luciferase, implying that TaPGK interacted with TaSRT1 in vivo (Fig. 5B). In vitro pull-down assay showed that the TaSRT1–glutathione S-transferase (GST) pulled down TaPGK-His after a GST-tag protein purification resin, indicating that TaSRT1-GST interacted with TaPGK-His (Fig. 5C). These results suggested that TaPGK strongly interacted with TaSRT1 in vivo and in vitro.

Fig. 5. TaSRT1 inhibited the protein level of TaPGK through de-crotonylation.

(A to C) Interaction of TaPGK and TaSRT1 was verified on the basis of Y2-H assay (A), a firefly LCI assay in tobacco (B), and pull down (C). (D) TaSRT1 reduces accumulation of TaPGK protein. Immunoblots detected transient expression of 35S::TaPGK-GFP-Flag with (+) or without (−) 35S::TaSRT1-GFP in tobacco leaves cells; immunoblots detected transient expression of 35S:TaPGK-GFP in WT and TaSRT1-overexpressed wheat leaf protoplasts. (E) Immunoblots detected that Kcr level of wheat endogenous TaPGK is increased in TaSRT1 mutant K3494 and is decreased in TaSRT1-overexpressed plants using anti-TaPGK antibody and pan anti-Kcr antibody. (F) TaSRT1 reduces Kcr level of TaPGK. Immunoblots detected Kcr levels of TaPGK together with (+) or without (−) TaSRT1 in tobacco leaves cells; immunoblots detected Kcr levels of WT and TaSRT1-overexpressed wheat leaf protoplasts transfected with 35S::TaPGK-GFP. (G) In vivo enzymatic activities of TaPGK with (+) or without (−) TaSRT1. (H) TaPGK can be decrotonylated by TaSRT1 in vitro. The Kcr status of TaPGK together with (+) or without (−) TaSRT1 in E. coli cells were determined by immunoblots. (I) In vitro enzymatic activities of PGK with (+) or without (−) TaSRT1. The bands were quantified with ImageJ software. Bars are means ± SE from three measures of each of the two repeats. **P < 0.01, as determined by paired Student’s t tests. AD, pGADT7; BD, pGBKT7; GST, glutathione S-transferase.

TaSRT1 negatively regulating wheat cold tolerance

As TaSRT1 physically interacted with TaPGK, we speculated that expression of TaSRT1 possibly affected wheat cold tolerance. qRT-PCR results indicated that TaSRT1 expression was significantly induced by cold temperature (fig. S10, C and D). To identify the function of TaSRT1, we screened mutants (K872, a splice acceptor variant; K3494, a splice acceptor variant) from the EMS-mutagenized Kronos library and backcrossed them into BC₂ lines with WT for two times (Fig. 6, A and B). Compared with control, two mutants of TaSRT1 showed sharply increased PGK activity (Fig. 6C). After cold treatment (−3°C, 12 hours), the two mutants of TaSRT1 showed stronger cold tolerance (Fig. 6B) and exhibited a significant decrease of REC, H₂O₂, and MDA contents (fig. S11, J to L).

Fig. 6. TaSRT1 was verified to regulate cold stress through mutants and overexpressed plants.

(A to D) Mutation sites of TaSRT1 (A) in Kronos EMS mutants and comparison of phenotype, PGK activity, and pyruvate content of TaSRT1 EMS mutants and the WT (n = 6) under cold stress (B to D). (E to H) Relative expression of TaSRT1 in TaSRT1-overexpressed lines (E) and a comparison of the phenotype, PGK activity, and pyruvate content of TaSRT1-overexpressed lines and the WT (n = 6) under cold stress (F to H). The β-actin gene was served as the endogenous control. The experiments were conducted with three biological replicates. Values are presented as means ± SE. **P < 0.01, as determined by paired Student’s t tests.

To identify the influence of TaSRT1 overexpression on cold stress, we construct the LGY-OE3 vector containing the cDNA of TaSRT1 and transferred it into Fielder for overexpression. After positive detection by specific primers, three TaSRT1-overexpressed lines with relatively high expression by qRT-PCR were self-crossed into T₂ generation (Fig. 6, E and F, and fig. S12B). Compared with control, TaSRT1-overexpressed T₂ plants showed sharply decreased PGK activity (Fig. 6G), implying that TaSRT1 possibly affect PGK protein level in wheat plants. After cold treatment (−4°C, 18 hours), the TaSRT1-overexpressed T₂ lines showed serious drooping and dehydration (Fig. 6F) and had significantly increase of REC, H₂O₂, and MDA contents (fig. S11, M to O). These results suggested that TaSRT1 negatively modulated wheat cold tolerance.

Inhibition of TaSRT1 on the protein level of TaPGK through decrotonylation

To illustrate whether TaSRT1 affects the protein level of TaPGK, we transiently overexpressed vectors 35S::TaPGK-GFP-Flag + 35S::GFP and 35S::TaPGK-GFP-Flag + 35S::TaSRT1-GFP in tobacco leaf cells, respectively. Western blot results by immunoblots using anti-flag antibody revealed that the addition of TaSRT1 significantly reduced the protein abundance of TaPGK (Fig. 5D). Next, we transiently overexpressed 35S::TaPGK-GFP in protoplasts from leaves of TaSRT1-overexpressed and WT wheat plants, respectively. Western blot results by immunoblots using anti–green fluorescent protein (GFP) antibody revealed that the accumulation of TaPGK protein was significantly decreased in the TaSRT1-overexpressed plants (Fig. 5D). These results suggested that TaSRT1 could inhibit the accumulation of TaPGK protein in tobacco and wheat protoplast.

As TaSRT1 had a potential role of decrotonylation, we used the BC₂ K3494 of TaSRT1 mutant for immunoblots with the pan anti-Kcr antibody. Western blot showed that K3494 exhibited a significant increase in Kcr levels, but three mutant lines of other genes (K2082: HDA6, Kronos423: HDA9, and K4387: HDA15) showed no clear difference in Kcr levels compared with the control (fig. S15A). Moreover, TaSRT1-overexpressed wheat lines showed a significant decrease in Kcr levels (fig. S15B). These results suggested that TaSRT1 is an important decrotonylase in wheat.

To check whether wheat endogenous TaPGK is affected by TaSRT1, we precipitated leaf protein extracts of TaSRT1-EMS mutants, TaSRT1-overexpressed lines, and their controls with anti-TaPGK antibody. Western blot results by immunoblots indicated that TaPGK exhibited relatively higher Kcr level in TaSRT1-EMS mutants but showed relatively lower Kcr level in TaSRT1-overexpressed lines compared their controls (Fig. 5E). To explore whether TaSRT1 could decrotonylate TaPGK, we transiently overexpressed proteins vectors 35S::TaPGK-GFP-Flag + 35S::GFP and 35S::TaPGK-GFP-Flag + 35S::TaSRT1-GFP into tobacco leaf cells, respectively. Western blot results by immunoblots indicated that adding TaSRT1 significantly reduced the Kcr level (Fig. 5F) and enzyme activity (Fig. 5G) and of TaPGK. Furthermore, we introduced the 35S::TaPGK-GFP into protoplasts of TaSRT1-overexpressed and WT wheat leaves, respectively. TaPGK-GFP fusion proteins were immunopreciptated with anti-GFP antibody and were detected by immunoblots. Results showed that Kcr level (Fig. 5F) and enzyme activity (Fig. 5G) of PGK were significantly decreased in the TaSRT1-overexpressed plants compared to the WT. These results suggested that TaSRT1 could decrotonylate TaPGK in both tobacco and wheat. To test whether TaSRT1 could decrotonylate TaPGK in vitro, we transformed the Escherichia coli strain containing TaPGK-His + GST and TaPGK-His + TaSRT1-GST constructs, respectively. Compared with cells producing TaPGK-His, the Kcr level and enzyme activity of TaPGK-His were significantly decreased in the cells producing TaPGK-His and TaSRT1-GST (Fig. 5, H and I). These results demonstrated that TaPGK could be decrotonylated by TaSRT1 both in vivo and in vitro. In addition, the substitution mutant of TaPGK indicated that K206 is a major site decrotonylated by TaSRT1 (Fig. 4, D to F). TaSRT1-mediated inhibition of TaPGK protein is likely through decrotonylation. This hypothesis was further supported by reduced accumulation of the substitution mutant of TaPGK.

Expression of TaPGK enhancing wheat cold tolerance possibly through promoting pyruvate accumulation

In plants, PGK is a highly conserved reversible key enzyme in glycolysis (43). Glycolysis plays an important role in plant cold response (28–30). To explain why TaPGK affects wheat cold tolerance, we sequenced the transcriptome of TaPGK-overexpressed plants and WT before and after the cold stress. The results indicated that 1384 and 3351 of all identified 134,939 genes were DEGs before cold stress and after stress, respectively, in the TaPGK-overexpressed plants compared with WT (fig. S16, A and B). Further analysis showed that 42 of 362 glycolytic genes were DEGs, and 7 of them (Fig. 7A) were key genes involved in 10 enzymatic steps of glycolysis pathway (12), including hexokinase (TaHK), phosphofructokinase l (TaPFK1), Fructose-bisphosphate aldolase (TaFBA), glyceraldehyde 3-phosphate dehydrogenase (TaGAPC), phosphoglycerate mutase (TaPGM), enolase (TaENO), and pyruvate kinase (TaPK). We further selected four key genes (TaGAPC, TaPGM, TaENO, and TaPK) adjacent to TaPGK in glycolysis pathway to determine the expression level by qRT-PCR. Compared with controls, these four genes showed sharply decreased expression in the TaPGK EMS mutants and TaPGK-edited lines (Fig. 7, B and C) but showed significantly increased expression in the TaPGK-overexpressed plants (Fig. 7D and fig. S12, C to E) after cold stress, indicating that they were induced by TaPGK.

Fig. 7. TaPGK enhanced wheat cold tolerance possibly through promoting the accumulation of pyruvate content.

(A) The expression of 7 key glycolytic genes identified by transcriptome sequencing (n = 10). (B-D) Relative transcript levels of 4 glycolytic genes (TaGAPC, TaPGM, TaENO, and TaPK) in the mutants, overexpressed plants, and gene-edited (GE) lines of TaPGK and their corresponding controls after cold stress. (E) Exogenous pyruvate (10 mM) significantly enhanced wheat cold tolerance in TaPGK EMS mutants and TaPGK-edited lines (n = 6) after cold stress. (F) Putative model of TaSRT1-TaPGK in response to cold stress in wheat. The experiments were conducted with three biological replicates. Values are presented as mean ± SE. **P < 0.01, as determined by paired Student’s t tests. TaHK, hexokinase; TaPHI, phosphohexose isomerase; TaPFK1, phosphofructokinase l; TaFBA, fructose-bisphosphate aldolase; TaTPI, triose phosphate isomerase; TaGAPC, glyceraldehyde 3-phosphate dehydrogenase; TaPGM, phosphoglycerate mutase; TaENO, enolase; TaPK, pyruvate kinase; G-6-P, glucose-6-phosphate; F-6-P, fructose-6-phosphate; F-1,6-P₂, fructose-1,6- diphosphate; DHAP, dihydroxyacetone phosphate; GA-3-P, glyceraldehyde 3-phosphate; 1,3-BisPGAP, 1,3-bisphosphoglycerate; 3-PGA, 3-phosphoglycerate; 2-PGA, 2-phosphoglycerate; PEP, phosphoenolpyruvate.

Glycolysis is a catabolic process to convert glucose into pyruvate (12). Therefore, pyruvate is the end product of glycolysis. To observe whether pyruvate affects wheat cold tolerance, we determined pyruvate contents of overexpression and mutant lines of TaPGK and TaSRT1, respectively. Results indicated that pyruvate contents were significantly decreased in the TaPGK EMS mutants and TaPGK-edited lines but were significantly increased in the TaPGK-overexpressed lines (Fig. 3, D, H, and L); however, pyruvate contents were significantly increased in the TaSRT1 EMS mutants but were significantly decreased in the TaSRT1-overexpressed lines (Fig. 6, D and H). Therefore, we concluded that TaPGK and TaSRT1 regulated wheat clod tolerance, possibly through the change of the pyruvate content. Furthermore, we found that exogenous application of pyruvate sharply enhanced wheat cold tolerance in Fielder, TaPGK EMS mutants, and TaPGK-edited lines (Fig. 7E and fig. S17, A to D). These results demonstrated that the TaPGK-TaSRT1 model regulated cold tolerance, possibly by modulating pyruvate content in wheat plants.

A big potential of TaPGK on improving wheat cold tolerance worldwide

To illustrate the geographical distribution of the cold-tolerant allele, we investigated haplotypes of TaPGK in 1168 worldwide wheat accessions (table S14) (44–49). Results showed that the cold tolerance allele TaPGK-hap2 were widely distributed worldwide but showed relatively low percentages (Fig. 2F), i.e., 1.7% in Oceania, 2.3% in Asia, and 2.4% in Africa; however, the percentage reached 4.7% in East Europe and 8.1% in North America. In addition, the percentage of TaPGK-Hap2 was 0.8% in Chinese modern cultivars and 4.6% in Chinese historical cultivars. These results revealed the big potential of the TaPGK for the utilization of improving cold tolerance in a worldwide wheat breeding program.

DISCUSSION

Cold damage frequently occurs in many wheat production areas worldwide and has resulted in millions of tons of grain yield losses (2). The ICE-CBF-COR cold signaling pathway is universally well known to be related to cold stress in plants (5). In wheat, some genes involved in the ICE-CBF-COR pathway have been reported to be associated with cold stress (7, 50, 51). In addition, other kinds of genes in response to cold stress have been identified in wheat, e.g., PAP6-like, Hsp90, REP14, and WCS19, (52–54) etc. In this study, through an integration of Kcr proteome, GWAS, BSR-seq, and BSP-TMT, we functionally identified a TaPGK gene positively regulating wheat cold tolerance. In plants, there are two isoforms of PGKs, i.e., the cytosolic PGK (PGKc) and the plastidial/chloroplast PGK (PGKp) (55, 56). The TaPGK we cloned was PGKc. In addition, we also identified a TaSRT1 gene negatively regulating wheat cold tolerance. Therefore, identification of TaPGK and TaSRT1 provides valuable gene resources for pyramiding breeding in view of improvement of cold tolerance in wheat breeding programs.

PTMs play important roles in plant cold response (38). Many proteins for PTMs were reported to be involved in ICE-CBF-COR cold signaling pathway in Arabidopsis (5). Phosphorylation, ubiquitination, and sumoylation of ICE1 have been proven to function in regulating freezing tolerance of Arabidopsis (57). Kcr is a recently identified PTM and has been found in diverse organisms, including animals, microorganisms, and plants (21, 22). Kcr proteins in response to cold stress have been reported in chrysanthemum (17). In this study, we found that Kcr was an evolutionarily conserved PTM in wheat and its progenitors. Different conserved motifs for the Kcr have been reported in plants (21). For instance, Carica papaya (37), Dendranthema grandiforum (16), and Arachis hypogaea (58) have 8, 14, and 6 motifs, respectively. Our results indicated that wheat containing 21 motifs has the highest number of conserved motifs among the reported plants so far. Considerable acidic amino acids, glutamic acid (E) and aspartic acid (D), were enriched at the −1 or +1 position. The EKcr motif is present in all reported plants, while the KcrE is conserved in plants but not rice, and KcrD motifs is conserved in plants but not papaya (21). Detection of specific Kcr motifs is beneficial to understand the biological significance of Kcr in plants.

In this study, TaPGK could be crotonylated and positively modulated wheat cold tolerance possibly through mediating Kcr levels. On the basis of interaction assays, we cloned a TaSRT1 that is possibly a homolog to human SIRT7. We further verified that TaSRT1 could decrotonylate TaPGK, and K206 is a major site decrotonylated by TaSRT1. A previous study indicated that AtSRT1 deacetylates histone proteins (e.g., H3K9ac) and nonhistone proteins (e.g., AtMBP-1) to control stress tolerance as a chromatin regulator in Arabidopsis (59). OsSRT1 in the nucleus is involved in epigenetic regulation of the gene expression in diverse pathways including stress responses (60–62). OsSRT2 was associated with the erasure of histone Kcr in rice, and its decrotonylase activity was also verified in vitro (24). In the present study, we confirmed that TaSRT1 negatively modulating wheat cold tolerance, possibly because TaSRT1 inhibited the abundance of TaPGK protein through decrotonylation.

PGK is a highly conserved reversible enzyme that catalyzes the conversion of 1,3-bisphosphoglycerate and adenosine diphosphate to 3-phosphoglycerate and adenosine triphosphate in glycolysis and mediates the reverse reaction in gluconeogenesis and the Calvin-Benson cycle (43). Glycolysis is intimately related with cold tress (28–30), and PGK as a key enzyme plays crucial roles in glycolysis (43). Previous studies indicated that glycolytic genes (e.g., GAPC, PFK, PGM, ENO, and PK) could be induced by cold stress (28–30). In this study, we found that TaPGK up-regulated the expression of TaGAPC, TaPGM, TaENO, and TaPK. We further revealed that up-regulation of TaPGK significantly increased pyruvate (the end product of glycolysis) content, but up-regulation of TaSRT1 significantly decreased pyruvate content in overexpression lines under cold stress. Moreover, exogenous application of pyruvate significantly enhanced wheat cold tolerance. These results indicated that the TaSRT1-TaPGK model regulated wheat cold tolerance, possibly via promoting pyruvate accumulation. In summary, we propose that cold stress down-regulates the expression of TaSRT1 and thus increase the expression of TaPGK by relieving the decrotonylation limitation from TaSRT1, thereby promotes pyruvate accumulation to minimize cold damage (Fig. 7F) in wheat plants. These results provide a different regulatory mechanism of wheat cold stress. In addition, this study implies that pyruvate could be used as a potentially important substance to prevent wheat cold damage.

Now, the application of cold tolerance genes is still limited in wheat-breeding programs. Although global temperature is increasing, extreme and abnormal cold weather still occurred frequently in many wheat regions worldwide during the most recent years, which sharply increased the risk of cold damage to wheat production. In this study, the proportion of the cold-tolerant haplotype TaPGK-hap2 ranged from 0.8 to 8.1% in worldwide wheat production regions, and TaPGK-overexpressed lines did not show significant change of field agronomic traits, suggesting that TaPGK_hap2 has a big potential to improve cold tolerance through utilization in wheat breeding programs, especially in major wheat regions that frequently suffer from cold damage.

MATERIALS AND METHODS

Plant materials and cold treatment

Seedling preparation for crotonylation experiments

Wheat donor progenitors, including T. urartu (PI428185) with an AA genome, A. speltoides (PI542245) with an SS genome, A. tauschii var. strangulate (AS2393) with a DD genome, tetraploid T. durum Langdon (AABB), and hexaploid wheat Aikang 58 (Ak58) were growth at 25°/15°C day/night under a 16-hour light/8-hour dark photoperiod in an illuminated incubator (RTOP-1000D, China). In addition, multiple different stresses were used to treat wheat seedlings of Ak58, including low temperature (2°C), high temperature (42°C), osmotic stress (20% polyethylene glycol, molecular weight 6000, −0.49 MPa), and salt stress (100 mM NaCl), and wheat was grown under normal conditions as the control (CK). Positions of plants in the growth chamber were randomly shuffled during the plant growth to minimize the micro environmental effect. Plants grown normally and consistently were sampled for each experiment. Seedling leaf tissues from two-leaf stage of above-mentioned samples and one-leaf to five-leaf stages of Ak58 as well as roots, stems, and seeds of Ak58 were freshly collected and were quickly frozen in liquid nitrogen for subsequent protein extraction.

Cold treatments

Cold treatment was performed according to the method from our previous report with minor modification (52). Wheat seedlings at two-leaf stage of Fielder exposed to low temperature (2°C) for 0, 3, 6, 12, and 24 hours, respectively, then the leaf tissues were collected for qRT-PCR. Wheat seedlings at three-leaf stage of Fielder and TaPGK-overexpressed (OE)#1 plants were exposed to low temperature (2°C) for 24 hours, and the leaf tissues of all plants including controls were collected for protein extraction. Wheat seedlings at three-leaf stage of mutants and transgenes of TaPGK and TaSRT1 were subjected to cold stress. To evaluate phenotype change of wheat plant after cold treatment, all plants with Fielder background were exposed to cold stress at −4°C from 18 to 36 hours, and all plants with Kronos background were exposed to cold stress at −3°C for 8 hours. The leaf tissues of all plants were collected for determination of physiological index.

Pyruvate treatment

Pyruvate treatment was performed as following: Wheat seedlings at three-leaf stage of Fielder, EMS mutants, and CRISPR-Cas9–mediated lines of TaPGK were irrigated with either fresh pyruvate (10 mM) or an equal amount of distilled water (control) for three consecutive days. TaPGK-edited plants and controls with Fielder background were exposed to cold stress at −4°C from 18 to 36 hours. All plants with Kronos background were exposed to cold stress at −3°C for 8 hours. All leaf tissue after cold treatment was collected and stored at −80°C.

RIL population and association panels

The F₁₀ RIL population SC containing 166 lines from the cross of Shanghai3/Catbird(SHA3/CBRD)✕Naxos was planted at the Scientific Research and Education Center of Henan agricultural University at Zhengzhou (34.87°N, 113.60°E) during the 2015–2016 cropping season and Yuanyang (35.04°N, 113.94°E) during the 2017–2018 cropping season. Two wheat association panels were used for GWAS in this study. Panel I consisted of 243 wheat cultivars or advanced lines developed after 2014, representing current breeding situations in the Huanghuai valley (39). Panel II consisted of 163 historical wheat cultivars from different periods in China (40). These accessions were planted at the Scientific Research and Education Center of Henan agricultural University at Yuanyang during the 2017–2018 and 2019–2020 cropping seasons and Zhengzhou during the 2017–2018 cropping season. All of the accessions surveyed were planted in three land parcels in each location. No drought stresses occurred in field. The CTI of all surveyed accessions was investigated in February and March of each year and was classified into four ranks (3, 2, 1, and 0) from high to low according to the standards of the wheat cultivar approval committee of the Yellow and Huang wheat region (i.e., sensitive, moderately sensitive, moderately tolerant, and cold tolerant, respectively) as we previously described (52).

Crotonylation proteomics analysis

Protein extract

The leaf tissue with three independent biological replicates from two-leaf periods of Ak58 wheat seedlings was used to extract proteins in lysis buffer [8 M urea, 1% Triton X-100, 10 mM dithiothreitol (DTT), and 1% protease inhibitor cocktail, 3 μM trichostatin (TSA), and 50 mM nicotinamid (NAM)]. A bicinchoninic acid kit (Beyotime Institute of Biotechnology, Nantong, China) was used to check the protein concentration. Trypsin was used to digest the extracted proteins. For digestion, the protein solution was reduced with 5 mM DTT for 30 min at 56°C and alkylated with 11 mM DTT for 15 min at room temperature in darkness. The protein sample was then diluted by adding 100 mM NH₄HCO₃ to urea concentration less than 2 M. Last, trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4-hour digestion.

High-performance liquid chromatography fractionation

The tryptic peptides were fractionated into fractions by high pH reverse-phase high-performance liquid chromatography (HPLC) using Thermo BetaSil C18 column (5-μm particles, 10 mm in inside diameter (ID), and 250 mm in length). Briefly, peptides were first separated with a gradient of 8 to 32% acetonitrile (pH 9.0) over 60 min into 60 fractions. Then, the peptides were combined into fractions and dried by vacuum centrifuging.

Affinity enrichment

To enrich crotonylation (Kcr) modified peptides, prewashed pan anti-crotonyllysine antibody agarose-conjugated antibody beads (PTM Biolabs, lot number 503) was used to incubate the tryptic peptides [dissolved in NETN buffer containing 100 mM NaCl, 1 mM EDTA, 50 mM tris-HCl, and 0.5% NP-40 (pH 8.0)] at 4°C overnight with gentle shaking. Then, we used NETN buffer and deionized water to wash the beads four times and twice, respectively. Trifluoroacetic acid (0.1%) was used to elute the bound peptides from the beads. Last, the eluted fractions were combined and vacuum-dried. C18 ZipTips (Millipore) was used to clean the resulting peptides according to the manufacturer’s instructions.

LC-MS/MS analysis

Three parallel analyses for each fraction were performed. The tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto a home-made reversed-phase analytical column (15 cm in length and 75 μm in ID). The gradient was composed of an increase from 6 to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23 to 35% in 8 min, and climbing to 80% in 3 min and then holding at 80% for the last 3 min, all at a constant flow rate of 400 nl/min on an EASY-nLC 1000 ultra performance LC (UPLC) system.

The peptides were subjected to NSI source followed by MS/MS in Q Exactive Plus (Thermo Fisher Scientific) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The mass/charge ratio (m/z) scan range was 350 to 1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using various normalized collision energies (NCE) setting as 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0-s dynamic exclusion. Automatic gain control (AGC) was set at 5 × 10⁴. For MS scans, the m/z scan range was 350 to 1800.

Database search

The resulting MS/MS data were processed using MaxQuant search engine (v.1.5.2.8). Tandem mass spectra were searched against Uniprot_Triticum_aestivum_20170318.fasta (released at 18 March 2017, 136884 sequences) concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme, allowing up to four missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in first search and 5 ppm in main search, and the mass tolerance for fragment ions was set as 0.02 Da. The minimum peptide length was set at 7. Carbamidomethyl on Cys was specified as fixed modification and crotonylation on lysine, deamidation (NQ), acetylation on protein N-terminal, and oxidation on Met were specified as variable modifications. The false discovery rate was adjusted to <1%, and the minimum score for modified peptides was set to >40.

Protein structure prediction and annotation

To specify the sequence model proximal to the Kcr residues, we examined the relative frequencies of 10 amino acids in upstream and downstream of Kcr sites by motif-x. All protein sequences in the database were treated as background controls. The minimum number of occurrences was set to 20, P value < 0.000001. Other parameters were set to default. NetSurfP was used to predict the secondary structures of the proteins. The GO annotation proteome was derived from www.ebi.ac.uk/GOA. WoLFPSORT (www.genscript.com/wolf-psort.html) was used to predict subcellular localization. The KEGG database was used to annotate protein pathways (https://www.kegg.jp/). InterPro (www.ebi.ac.uk/interpro/) was used to annotate the protein domain functional description.

Crotonylation analysis of the A, B, and D subgenome homologs across triads

The analysis focused exclusively on the gene triads, which had a 1:1:1 correspondence across the three wheat homologous subgenomes, including 22,097 syntenic triads (total of 66,291 genes) previously reported (http://wheat.cau.edu.cn/TGT/download.html). We defined protein triads that A, B, and D subgenome homologous were identified at the protein level. At least one subgenome homologs modified by Kcr was analyzed.

Transcriptome and proteome analysis in an RIL population

Sample preparation

The F₁₀ RIL population SC displayed clear segregation of cold tolerance based on the investigation of classification for cold tolerance (from 0 to 3) in the field. Leaves from three cold-tolerant pools (CTPs; T1_H) (cold stress for 24 h) and three cold-sensitive pools (CSPs; S1_H) (cold stress for 24 h) were collected in the greenhouse. Each CTP or CSP with three independent biological replicates was composed of an equivalent mixture of leaves from 10 lines of the RIL population with level 0 or 3 under the four environments. Sampled leaves were rapidly frozen in liquid nitrogen and stored at −80°C for BSR-seq and TMT (BSP-TMT). In addition, seedlings of TaPGK-OE#1 line and Fielder before and after cold stress were sampled for transcriptome analysis.

Transcriptome sequencing

RNA concentration and purity were measured using NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v4-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generated. Approximately 11-gauge clean reads were generated for each sample. Bowtie2 v2.2.3 was used to map the clean reads to the Wheat Chinese Spring genome published by the International Wheat Genome Sequencing Consortium (IWGS, RefSeq. v1.0) to obtain the location information of the gene and the specific sequence characteristic information of the sequencing sample. The reads were reassembled using StringTie 1.3.3b. Quantification of gene expression levels was estimated by fragments per kilobase of transcript per million fragments mapped. Differential expression analysis of two conditions/groups was performed using the DESeq2. The resulting P values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P value < 0.05 found by DESeq2 and fold change > 2 are assigned as differentially expressed.

Proteome analysis

Experimental procedure of protein extraction and trypsin digestion were conducted as above. After trypsin digestion, peptide was desalted by Strata-X-C18 SPE column (Phenomenex) and vacuum-dried. Peptide was reconstituted in 0.5 M Tetraethylammonium bromide (TEAB) and processed according to the manufacturer’s protocol for TMT kit. Briefly, one unit of TMT reagent was thawed and reconstituted in acetonitrile. The peptide mixtures were then incubated for 2 hours at room temperature and pooled, desalted, and dried by vacuum centrifugation. In HPLC fractionation, LC-MS/MS analysis were conducted as above. The resulting MS/MS data were processed using MaxQuant search engine (v.1.5.2.8). Tandem mass spectra were searched against Z9415_Triticum_aestivum_4565_IWGSC_20190910 (133346), concatenated with reverse decoy database. Other procedure was conducted as above. Two sample t test was used to identify significant (P < 0.05) differences in means between samples were defined on the basis of thresholds of >1.5- or <0.67 fold change ratios.

Genetic analysis of candidate genes associated with cold tolerance

Genotyping and GWAS

The association panel I composed of 243 wheat accessions and panel II composed of 163 wheat accessions were genotyped using the wheat 660K SNP arrays and 90K SNP arrays, respectively. The principal components analysis of panel I and panel II were showed in our previous study (39, 40). The uniform suggestive threshold of the 660K assay (P value = 1.0 × 10³) and 90K assay (P value = 1.0 × 10²) was presented to identify significance. Significant SNPs with minor allele frequency > 0.05 were used for further analysis. Haplotype analysis was performed using Haploview 4.2 software.

Identification of candidate genes associated with cold tolerance

The candidate genes identified by combination analysis of BSR-seq, BSP-TMT, and the Kcr proteome were sequenced for their allelic variation in two association panels, and all polymorphic markers associated with these genes were added to rerun GWAS. The genes with significant SNPs were chosen as key candidate genes for further study.

RNA isolation and qRT-PCR

TRIzol isolation reagent (Invitrogen) was used to isolate total RNA, and reverse transcription reactions were performed using the two-step PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa) by following the protocol. The β-actin gene (GenBank accession no. AB181991) and glyceraldehyde 3-phosphate dehydrogenase (GenBank accession no. EF592180) gene were served as the endogenous controls (table S1). The specific primers of candidate genes for qRT-PCR were shown in table S1.

Y2-H assay

The entire coding sequence (CDS) of phosphoglycerate kinase (TaPGK, TraesCS6B02G187500) gene were cloned into the bait vector pGBKT7. The cold-stressed wheat seedling of Ak58 was used to construct a cDNA library for screening interaction proteins using Y2-H assay following the manufacturer’s protocol (Clontech, USA). Yeast colonies were amplified by PCR and sequenced using T7 specific primers for the cDNA library plasmid (table S1). Furthermore, sequences were blasted for homologous genes in the wheat genome (http://plants.ensembl.org/Triticum_aestivum/Tools/Blast). To identify proteins that interacted with the bait in the Y2-H screening, the CDS of a nicotinamide adenine dinucleotide–dependent histone deacetylase (sirtuin-like gene, TaSRT1) was amplified and fused to the pGADT7 vector to create the prey (pGADT7-TaSRT1) vector (table S1). Then, the recombinant bait and prey plasmids were used for Y2-H assays.

Firefly LCI assay

The LCI assay for the interaction between TaPGK and TaSRT1 was performed in Nicotiana benthamiana leaves. The CDSs of TaPGK and TaSRT1 were inserted into pCAMBIA1300-nLUC and pCAMBIA1300-nLUC to form TaSRT1-nLUC and TaPGK-cLUC, respectively. Four-week-old N. benthamiana leaves co-infiltrated with Agrobacterium-expressing TaSRT1-nLUC and TaPGK-cLUC. TaSRT1-nLUC and cLUC, nLUC and TaPGK-cLUC, cLUC, and nLUC were used as negative controls. The luminescence images were captured using a plant living imaging system (Berthold, NightShade LB 985).

Pull-down protein interaction assay

TaPGK-His and TaSRT1-GST or GST protein were expressed in E. coli. TaSRT1-GST or GST proteins were purified with buffer [50 mM tris, 150 mM NaCl, 10 mM glutathione (pH 8.0)] using BeyoGold GST-tag Purification Resin according to the instruction of the GST-tag Protein Purification Kit (Beyotime, China, catalog no. P2262), then the pulled-down proteins were mixed with the SDS sample buffer. The samples were detected by immunoblot using anti-GST antibody (PTM Biolabs, PTM 5046) and anti-His antibody (Abmart, M30111).

Antibody production and immunoprecipitation

The full-length protein of TaPGK was used as the antigen for generating polyclonal antibodies in rabbits by Abclonal (Wuhan, China). Total leaf (0.5 g) proteins from Fielder and TaPGK-overexpressed wheat plants before and after cold treatment (2°C, 24 hours) were lysed in lysis buffer [20 mM tris-HCl (pH 8.0), 2 mM DTT, 1% Triton X-100, 800 μM phenylmethylsulfonyl fluoride, and 250 mM sucrose] containing protease inhibitor cocktail, respectively. Immunoprecipitation was carried out by incubation with or without 25 μl of the anti-TaPGK antibody; 30 μl of Protein A/G Plus Agarose (Santa Cruz Biotechnology, lot number sc-2003) was added, and the solution was incubated for 4 to 6 hours on ice with gentle shaking. After incubation, the bound proteins were washed four times with phosphate-buffered saline (PBS) buffer [137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄ (pH 7.4), and 0.2 mM DTT] and were then suspended in 40 μl of PBS buffer. The proteins in the buffer were analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie blue staining and Western blotting by immunoblot using anti-TaPGK antibody and pan anti-Kcr antibody (PTM Biolabs, lot number 502). To examine whether cold stress could induce the expression of TaPGK in wheat, protein extracts of leaf from TaPGK-overexpressed wheat before and after cold treatment (2°C, 24 hours) were analyzed by immunoblots using anti-TaPGK–specific antibody. An anti-actin (Abbkine, lot number A01050) antibody was used as loading control.

To examine whether TaSRT1 affect the Kcr level of wheat endogenous TaPGK, the proteins of Kronos mutant SRT1-2BS (K3494) and TaSRT1-overexpressed wheat and their controls were immunoprecipitated by anti-TaPGK–specific antibody, respectively. Then, eluted proteins were analyzed by immunoblots with anti-TaPGK–specific antibody and pan anti-Kcr antibody (PTM Biolabs, lot number 502), respectively.

Transient expression assays

35S::TaPGK-GFP-Flag and 35S::TaSRT1-GFP or 35S::GFP were introduced into 4-week-old tobacco (N. benthamiana) leaf cells by injection, and total proteins were extracted. TaPGK-GFP proteins from the above extracts were immunoprecipitated with anti-Flag antibody (Abcam, lot number AF0036) and Protein A/G Plus Agarose (Santa Cruz Biotechnology, lot number sc-2003) after a series of washing steps. Eluted proteins were detected by immunoblotting with anti-Flag (Abcam, lot number AF0036) and pan anti-Kcr (PTM Biolabs, lot number 502) antibodies using the ECL SuperSignal system. Furthermore, protoplasts of WT and TaSRT1-overexpressed wheat leaves were transiently transfected with 35S::TaPGK-GFP. Proteins of transfected protoplast were immunoprecipitated by anti-GFP and were analyzed by immunoblots with anti-GFP antibody (Abcam, lot number ab13970) and pan anti-Kcr antibody (PTM Biolabs, lot number 502), respectively.

To examine whether TaSRT1 could regulate the expression of TaPGK, protein extracts of tobacco leaves of transient expression with 35S::TaPGK-GFP-Flag together with 35S::TaSRT1-GFP or 35S::GFP were analyzed by immunoblots using anti-Flag (Abcam, lot number AF0036). Moreover, protein extracts of WT and TaSRT1-overexpressed wheat leaf protoplasts transfected with 35S::TaPGK-GFP were analyzed by immunoblots using anti-GFP antibody (Abcam, lot number ab13970). An anti-actin (Abbkine, lot number A01050) antibody was used as loading controls.

Prokaryotic expression

TaPGK was cloned into the prokaryotic expression vector PET28a using Bam HI and Not I as restriction enzyme cutting sites (table S1). E. coli BL21 (DE3) was used to express recombinant TaPGK-His. The elution buffer [137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄ (pH 7.4), and 0.2 mM DTT] was used to elute the proteins using BeyoGold His-tag Purification Resin according to the instruction of the His-tag Protein Purification Kit (Beyotime, China, catalog no. P2226), then the collected TaPGK-His recombinant proteins were analyzed by SDS-PAGE and immunoblot by using anti-His antibody (Abmart, M30111) and pan anti-Kcr antibody (PTM Biolabs, lot number 502), respectively.

In vitro lysine decrotonylase activity assay

To test whether TaSRT1 can decrotonylate TaPGK in vitro, TaPGK-His and TaSRT1-GST or GST vector were transformed in E. coli. We purified TaPGK-His recombinant protein from E. coli. The purified proteins were detected by immunoblot using anti-His antibody (Abmart, M30111) and pan anti-Kcr antibody (PTM Biolabs, lot number 502).

Site-directed mutagenesis and expression

We performed site-directed mutagenesis of K35R, 206R, and K366R (AAA-AGA) within the 35S::TaPGK-GFP-Flag plasmid using the Fast Site-Directed Mutagenesis Kit (Tiangen, China, catalog no. KM101), respectively. PCR was conducted using site-specific primers (table S1). Mutant sites were verified by DNA sequencing. We transiently overexpressed recombinant WT and mutants of 35S::TaPGK-GFP-Flag in 4-week-old tobacco (N. benthamiana) leaf cells, respectively. Then, the proteins of transfected tobacco leaf were immunoprecipitated by anti-TaPGK antibody and were analyzed by immunoblots with anti-TaPGK antibody and pan anti-Kcr antibody (PTM Biolabs, lot number 502), respectively. In addition, protein extracts of tobacco leaf of transient overexpression with 35S::TaPGK-GFP-Flag and mutants of 35S::TaPGK-GFP-Flag were analyzed by immunoblots using anti-TaPGK antibody. An anti-actin (Abbkine, lot number A01050) antibody was used as loading control.

Furthermore, we transiently overexpressed 35S::TaPGK-GFP and 35S::TaPGK ^K206-GFP in protoplasts of wheat leaves (Fielder) before and after cold stress (6°C, 4 hours). Proteins of transfected protoplast were immunoprecipitated by anti-PGK antibody and were analyzed by immunoblots with anti-PGK antibody and pan anti-Kcr antibody (PTM Biolabs, lot number 502), respectively.

Ethyl methanesulfonate mutants and transgenic plants

Seeds of the tetraploid wheat cultivar Kronos were mutagenized by the group of Jorge Dubcovsky from UC Davis using the chemical mutagen EMS. All of the EMS mutant lines of Kronos were sequenced using exome capture and next-generation sequencing (63). The BC₂ mutant lines PGK-6BL (Kronos3272: C/T, mutation effect = splice_acceptor_variant), PGK-6BL (Kronos167: C/T, mutation effect = stop gained), SRT1-2AS (K872: G/A, mutation effect = splice_acceptor_variant), and SRT1-2BS (K3494: G/A, mutation effect = splice_acceptor_variant), HDA6-6AS (Kronos2082: C/T, mutation effect = splice acceptor variant), HDA9-2AL (Kronos4236: G/A, mutation effect = stop gained), and HDA15-5BS (Kronos4387: G/A, mutation effect = splice acceptor variant) (table S1) were screened to use for functional verification.

To produce TaPGK-OE and TaSRT1-OE plants, the CDSs of TaPGK (TraesCS6B02G187500) and TaSRT1 (TraesCS2B02G092700) were cloned into the LGY-OE3 vector with the ubiquitin promoter. These vectors containing targeted genes were transformed by Agrobacterium-mediated infection into immature embryos of Fielder to obtain the TaPGK-OE and TaSRT1-OE lines. T₀ transgenic plants detected to be positive by PCR using specific primers (table S1) were further self-pollinated. The T₂ nonsegregating plants of three lines with high expression levels were selected for further analysis.

The target sequences were designed and selected using CRISPRdirect (http://crispr.dbcls.jp/) and CRISPOR (http://37.187.154.234/) to minimize off-target effects. Two signal guide RNAs (sgRNAs) of TaPGK were designed on the basis of homology searches against the genome of Chinese Spring. The recombinant plasmid was generated by introducing the guide sgRNA1 and sgRNA2 into the binary vector pBUE411, which is modified with the wheat TaU3 promoter and guides the RNA scaffold. The constructed vector was introduced into the Agrobacterium tumefaciens strain EHA105. Fielder was used as explants for genetic transformation. Transgenic wheat plants were identified with the Enviologix QuickStix kit for Bar protein (EnviroLogix, Portland, ME, USA), and the leaf DNA of these transformants was extracted as a template for PCR amplification using a primer flanking the target site (table S1). The PCR products were sequenced to detect the targeted mutation by Hi-TOM (table S1). Two 20-nucleotide (nt) sequences in the TaPGK gene were chosen as the target site for Cas9 cleavage, and multiple mutant transgenic lines were generated mainly in the second 20-nt sequence.

Enzymatic assays and measurement of physiological indexes

To assess the stress effects, the REC was measured using fresh wheat seedlings (64). The H₂O₂ contents and lipid peroxidation were measured by estimating the MDA contents as described previously (65). The enzyme activity of PGK and pyruvate content were measured in triplicate using commercial assay kits (Comin Biotechnology, Suzhou, China) according to the manufacturer’s instructions, respectively.

Supplementary Materials

This PDF file includes:

Figs. S1 to S17

Other Supplementary Material for this manuscript includes the following:

Tables S1 to S14

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