A TaSnRK1α Modulates TaPAP6L‐Mediated Wheat Cold Tolerance through Regulating Endogenous Jasmonic Acid

Abstract Here, a sucrose non‐fermenting‐1‐related protein kinase alpha subunit (TaSnRK1α‐1A) is identified as associated with cold stress through integration of genome‐wide association study, bulked segregant RNA sequencing, and virus‐induced gene silencing. It is confirmed that TaSnRK1α positively regulates cold tolerance by transgenes and ethyl methanesulfonate (EMS) mutants. A plastid‐lipid‐associated protein 6, chloroplastic‐like (TaPAP6L‐2B) strongly interacting with TaSnRK1α‐1A is screened. Molecular chaperone DJ‐1 family protein (TaDJ‐1‐7B) possibly bridged the interaction of TaSnRK1α‐1A and TaPAP6L‐2B. It is further revealed that TaSnRK1α‐1A phosphorylated TaPAP6L‐2B. Subsequently, a superior haplotype TaPAP6L‐2B30S /38S is identified and confirmed that both R30S and G38S are important phosphorylation sites that influence TaPAP6L‐2B in cold tolerance. Overexpression (OE) and EMS‐mutant lines verified TaPAP6L positively modulating cold tolerance. Furthermore, transcriptome sequencing revealed that TaPAP6L‐2B‐OE lines significantly increased jasmonic acid (JA) content, possibly by improving precursor α‐linolenic acid contributing to JA synthesis and by repressing JAR1 degrading JA. Exogenous JA significantly improved the cold tolerance of wheat plants. In summary, TaSnRK1α profoundly regulated cold stress, possibly through phosphorylating TaPAP6L to increase endogenous JA content of wheat plants.


Introduction
Wheat is one of the most widely cultivated major food crops.However, cold stress is one of the key abiotic stresses that damage wheat growth and cause severe yield loss in common wheat. [1,2]OI: 10.1002/advs.202303478   Numerous studies have demonstrated that the probability and potential risk of extreme cold events are gradually increasing as a result of global warming.The discovery of genes modulating cold stress for the development of cold-tolerant wheat varieties is an effective method to prevent cold injury in wheat.
Previous studies have identified some important cold stress genes in plants, including transcription factors (CBF, MYB, ZIPI, and DREB), protein kinases (MPK, OST1), and cold response protein chillingtolerance divergence 1 (COLD1), etc.The CBF/DREB-mediated transcriptional regulatory cascade has been shown to be the major cold signaling pathway, and this cascade is essential to activate a set of cold responsive genes. [3,4]The MPK3/6-ICE1 and ICE-CBF-COR models are also widely reported to regulate plant hypothermia response. [5,6][9] Plant U-box E3 ligases PUB25 and PUB26 dynamically modulate ICE1 stability via differential ubiquitination during cold stress in Arabidopsis. [10]COLD1 interacts with the G-protein  subunit to activate the Ca 2+ channel for sensing low temperature and to accelerate G-protein GTPase activity. [11]The Ca 2+ transporter ANNEXIN1 was found to mediate cold-induced calcium signaling and freezing tolerance in plants. [12]A recent study indicated that loss-of-function mutations of COLD1 caused reduced rice chilling tolerance, and natural variation of codon repeats in COLD11 endows rice with chilling resilience. [13][16] The plastid-lipid-associated protein 6 (PAP6)silenced wheat plants showed significantly decreased cold tolerance. [17]Silencing of glycine-rich RNA-binding protein 2, CBS domain-containing protein chloroplastic-like, and cold acclimation protein Wcor410c reduced cold tolerance in common wheat. [18]Overexpression and silencing of the gene encoding this DHN 13 in Arabidopsis and wheat led to increased tolerance and sensitivity to cold stress, respectively. [19]hytohormones are important regulators of plant growth and development, as well as abiotic stress response signal networks, suggesting that plant hormones interact with signals for plant growth and environmental stresses. [14]Phytohormone regulation in wheat cold stress is another strategy to study wheat cold tolerance.Exogenous abscisic acid (ABA) enhances cold tolerance, possibly by increasing the activity of antioxidant enzymes. [20,21]nitiation of melatonin during grain filling of mother plants may improve the cold tolerance of wheat offspring at the seedling stage. [22]During the recovery period of wheat after cold stress, foliar spraying of melatonin enhances the cold tolerance of wheat plants. [23][26][27] JA positively regulates cold tolerance by promoting ABA biosynthesis in tomato. [28]Fibrillins (FBNs), generally described as plastid lipid-associated proteins (PAPs), are the most abundant plastidial proteins. [29]Some evidences suggest that FBNs are involved in phytohormone signaling, especially JA signaling. [30,31]Arabidopsis fibrins FBN1a, FBN1b, and FBN2 regulate JA synthesis induced by light or cold stress. [32][35] Plants with JA pretreatment increased significantly the activity of antioxidant enzymes, and thereby improved the cold tolerance. [36,37]A was also reported to positively regulate downstream cold response genes in the CBF transcription pathway and ultimately enhanced cold tolerance. [38]n this study, we found that the expression of sucrose nonfermenting-1-related protein kinase alpha subunit (TaSnRK1) alleviates the damage of cold stress in wheat plants.We further found that TaSnRK1 interacts with PAP6, chloroplastic-like (TaPAP6L) and promotes its accumulation by phosphorylation.Moreover, our results indicate that TaSnRK1 and TaPAP6L increase the content of plant hormone JA, thereby regulating plants to prevent the damage from cold stress.Consequently, our study reveals the important role of TaSnRK1 in regulating wheat resistance to cold stress.

Identification of Candidate Genes Associated with Wheat Cold Stress by GWAS
Genome-wide association analysis (GWAS) was conducted for cold tolerance index (CTI) in an association panel consisting of 243 wheat accessions previously genotyped using Wheat 660K SNP array. [39]GWAS identified an important genetic locus gCTI-1A significantly associated with cold stress on chromosome 1A.To further map gCTI-1A, we re-run GWAS using only SNPs on 1A genome, and found that the gCTI-1A containing five significant SNPs was in a 9-Mb (532-541 Mb) interval containing 28 annotation genes (Figure 1A).BSR-Seq (bulked segregant RNA sequencing) analysis in a Recombinant Inbred Lines (RIL) (Shanghai3/Catbird×Naxos, SC) population indicated that 13 of the 28 genes were significantly induced at both 24 h and 48 h after cold stress, and 3 (TraesCS1A02G350500, TraesCS1A02G360300, and TraesCS1A02G360400) of these 13 genes showed differential expression between cold-tolerant and cold-sensitive pools at both 24 h and 48 h after cold treatment (Figure 1B; Table S1, Supporting Information).

VIGS Revealed the Role of TaSnRK1𝜶 Regulating Wheat Cold Stress
To confirm the gCTI-1A gene, we silenced three above-mentioned candidate genes in wheat plants by virus-induced gene silencing (VIGS) using a Barley Stripe Mosaic Virus (BSMV) vector.After inoculation for 14 days, the relative expression levels were identified by qRT-PCR, and the silenced-plants were subjected to cold stress at −4 °C for 12 h.Results indicated that only TraesCS1A02G350500-silenced plants among three candidates showed obvious drooping and wilting (Figure 1C-E; Figure S1, Supporting Information).The TraesCS1A02G350500 encodes a sucrose non-fermenting-1-related protein kinase alpha subunit, hereafter designated as TaSnRK1−1A on 1A, was selected as candidate for further study.

Function Confirmation of TaSnRK1𝜶 by EMS Mutants, Overexpression, and CRISPR/Cas9
To further functional verification of TaSnRK1, we screened Ethyl methanesulfonate (EMS)-mutagenized tetraploid wheat Kronos library, and identified three mutants aaBB1 (K331), aaBB2 (K4218), and AAbb (K4220) of TaSnRK1.We backcrossed these three mutants twice with wild type to obtain the BC 2 lines, respectively.After cold stress (−4 °C, 4 h) for BC 2 lines, all three mutants showed relatively serious drooping and dehydration, and possessed significantly increased relative electrolyte leakage rates compared with wild type (Figure 1G).
To determine whether the expression of TaSnRK1 contributes to cold tolerance in hexaploid wheat, we generated TaSnRK1−1A-overexpression (TaSnRK1−1A-OE) lines by transferring full-length cDNA of TaSnRK1−1A from cold tolerance parent Shanghai3/Catbird into hexaploid wheat cultivar Fielder.Three positively high-expression lines detected by qRT-PCR were self-crossed into T 3 generation and three T 3 lines with high expression levels were selected for cold stress treatment.After cold stress (−4 °C, 10 h), TaSnRK1−1A-OE lines showed significantly stronger resistance to drooping and dehydration and possessed significantly decreased relative electrolyte leakage rate compared with wild type (Figure 1H).
To validate the effect of TaSnRK1 mutants in hexaploid wheat, we used CRISPR/Cas9-mediated gene editing technique to knock out TaSnRK1 gene in Fielder.Mutation sites were confirmed through sequencing by Hi-TOM and were further verified through Sanger sequencing (Table S2, Supporting Information). [16]After cold stress (−4 °C, 6 h), TaSnRK1-edited wheat mutant lines showed more serious drooping and dehydration and possessed significantly increased relative electrolyte leakage rate compared with non-edited wild type (Figure 1I).These results suggested that TaSnRK1 positively modulated wheat cold tolerance.

TaSnRK1𝜶 Interacted with TaPAP6L
To dissect the regulatory mechanism of TaSnRK1 in cold tolerance, we conducted a yeast two-hybrid (Y2-H) to screen TaSnRK1−1A-interacting proteins using TaSnRK1−1A as a bait in a wheat cDNA library.Among several interacting proteins, a client TaPAP6L-2B (TraesCS2B02G171300, plastid-lipidassociated protein 6, chloroplastic-like) on 2B was selected for further analysis because TaPAP6L is a member of the PAP/ FBNs protein family and it has been reported that this family was involved in plant response to temperature. [17,40]We next confirmed the interaction of TaSnRK1−1A and TaPAP6L-2B proteins in the yeast cells by Y2-H (Figure 2A) and in the tobacco leaves by Luciferase Reporter systems, respectively (Figure 2B).We then performed an in vitro pull-down, and results indicated that GST-TaPAP6L specifically pulled down TaSnRK1-His (Figure 2C).Together, these results indicated that TaSnRK1−1A interacted with TaPAP6L-2B in vivo and in vitro.

TaDJ-1 as a Molecular Chaperone Possibly Bridged the Interaction of TaSnRK1𝜶 and TaPAP6L
To identify locations of TaSnRK1−1A and TaPAP6L-2B in cells, we performed subcellular localization in wheat protoplasts by transiently transforming them with Ubi::GFP-TaSnRK1 or Ubi::GFP-TaPAP6L, respectively.Results showed that TaSnRK1−1A was mainly localized in the nucleus and cytoplasm, but TaPAP6L-2B was in chloroplast (Figure 2D).We subsequently extracted wheat protoplast protein and chloroplast protein and imprinted them with anti-GFP antibody, respectively.Results showed that both TaSnRK1−1A and TaPAP6L-2B could be detected in wheat protoplast protein, but only TaPAP6L-2B could be detected in chloroplast protein, implying that TaSnRK1−1A was not expressed in chloroplasts (Figure 2E).Bimolecular Fluorescent Complementary (BiFC) in wheat mesophyll protoplasts showed that TaSnRK1−1A interacted with TaPAP6L-2B on the chloroplast (Figure 2F).
in the yeast cell by Y2-H (Figure 3A) and Luciferase Reporter systems, respectively (Figure 3B,C).Subcellular localization in wheat protoplasts showed that Ubi::GFP-TaDJ-1 was obviously observed in almost the whole cell (Figure 3D).Therefore, we assumed that TaDJ-1, as a molecular chaperone, accompanied with TaSnRK1−1A to the chloroplast by interaction.To verify this hypothesis, we silenced TaDJ-1 in wheat plants by VIGS and performed BiFC assay for verifying whether the interaction of TaSnRK1-His with TaPAP6L-2B occurs using protoplasts from BSMV TaDJ-1 and BSMV 0 plants.Results showed that YFP florescence signal was significantly weaker in BSMV TaDJ-1 plants than in BSMV 0 plants (Figure 3E).Moreover, we extracted chloroplast proteins from protoplasts of BSMV 0 and BSMV TaDJ-1 plants and performed western blotting with anti-His.Results indicated that TaSnRK1-His was only detected in chloroplast proteins from BSMV 0 plants (Figure 3F), implying that silencing of TaDJ-1 possibly hindered the bridge for moving TaSnRK1−1A to the chloroplast.Therefore, we hypothesized that TaSnRK1−1A, with the assistance of molecular chaperone TaDJ-1, moved to the chloroplast to interact with TaPAP6L-2B.
To further illustrate whether TaSnRK1−1A could affect the protein level of TaPAP6L-2B, we detected proteins in tobacco leaf cells transfected with 35S::GFP-TaSnRK1-His and 35S::GFP-TaPAP6L-Flag by immunoblots with anti-Flag antibody.Results revealed that the addition of TaSnRK1−1A significantly improved the protein abundance of TaPAP6L-2B (Figure 4B).These TaDJ-1 as a molecular chaperone is involved in the interaction of TaSnRK1 and TaPAP6L.A) Yeast two-hybrid (Y2-H) assays to verify the interaction of TaDJ-1-7B with TaSnRK1−1A and TaPAP6L-2B.AD-T+BD-53 group was used as positive control, while AD-T+BD-Lam group was used as negative control.B) Firefly luciferase complementation imaging assay of the interaction between TaDJ-1-7B and TaPAP6L-2B in tobacco leaves.The empty vector groups were as negative controls.C) Firefly luciferase complementation imaging assay of the interaction between TaDJ-1-7B and TaSnRK1−1A in tobacco leaves.The empty vector groups were considered as negative controls.D) Subcellular localization of the TaDJ-1-7B protein in wheat protoplast.GFP fusion proteins were as controls.E) Bimolecular fluorescence complementation (BiFC) analysis of the interaction between TaSnRK1-His and TaPAP6L-2B in the protoplasts of BSMV TaDJ-1 and BSMV 0 plants.F) The expression of TaSnRK1-His protein in chloroplast proteins from protoplasts of BSMV 0 and BSMV TaDJ-1 plants, respectively.
Blast analysis in the genome of Chinese Spring showed that TaPAP6L has three homologous genes TaPAP6L-2A (TraesCS2A02G145900) on 2A, TaPAP6L-2B (TraesCS2B02G171300) on 2B, and TaPAP6L-2D (TraesCS2D02G150500) on 2D.To identify which one plays a more important role, we cloned sequences of the three homologous genes from Chinese Spring and purified the corresponding proteins TaPAP6L-2A, TaPAP6L-2B, and TaPAP6L-2D by prokaryotic expression.In vitro phosphorylation results showed that TaPAP6L-2B had the significantly highest phosphorylation level among the three proteins (Figure 4D).Therefore, we assumed that TaPAP6L-2B possibly played a relatively more important role in regulating wheat cold stress among three homologous proteins.
Next, we transiently overexpressed TaPAP6L-2B and TaPAP6L-2B R30S/G38S in protoplasts of wheat leaves before and after cold treatment.TaPAP6L-2B proteins from wheat protoplasts were immunoprecipitated with anti-Flag antibody and were detected by immunoblots.Results showed that cold stress increased the phosphorylation level of TaPAP6L, and visibly affected the phosphorylation level of TaPAP6L R30S/G38S (Figure 4G), implying that R30S and G38S as keys phosphorylation site of TaPAP6L-2B is response to cold stress.

EMS Mutants and Overexpression Verified TaPAP6L Positively Modulating Wheat Cold Stress
To demonstrate the role of TaPAP6L in regulating wheat cold stress, we screened two mutants, aaBB (K2368) and AAbb (K4490) of TaPAP6L from the EMS-mutagenized tetraploid wheat Kronos library.We then backcrossed them twice with wildtype Kronos to get BC 2 lines, respectively, and also crossed aaBB and AAbb mutants to obtain aabb double mutants.The BC 2 lines and aabb mutants of TaPAP6L were used for cold treatment.After cold stress (−4 °C, 4 h), all three mutants (aaBB, AAbb, and aabb) showed significantly decreased cold tolerance, and possessed significantly increased relative electrolyte leakage rates compared with wild type (Figure 5A).
To further verify TaPAP6L regulating cold stress by overexpression, we constructed an overexpression vector LGY-OE3 containing TaPAP6L-2B and then transferred it into Fielder for overexpression.We detected expression levels of positive TaPAP6L-2Boverexpression (TaPAP6L-2B-OE) lines by qRT-PCR.Three positive lines with high expression levels were further self-crossed into T 3 generation and then were further detected for positive and high expression level.The T 3 non-segregating plants of three lines with high expression levels were selected for cold stress.After cold treatment (−4 °C, 10 h), TaPAP6L-2B-OE transgenic wheat plants showed significantly enhanced cold tolerance and possessed significantly decreased relative electrolyte leakage rates compared with wild type (Figure 5B).These results demonstrated that TaPAP6L positively regulated cold tolerance in common wheat.

TaSnRK1𝜶−1A Modulated Cold Tolerance Possibly through the Interaction with TaPAP6L-2B Mediating JA Content
Phytohormones as plant growth regulators modulate the adaptation process of plants to the environment.][54] To determine whether TaSnRK1 was associated with phytohormones, we measured four common endogenous phytohormones (abscisic acid, ABA; 3-Indoleacetic acid, IAA; jasmonic acid, JA; salicylic acid, SA) in overexpression and mutant lines of TaSnRK1.Results found that JA content was significantly increased in TaSnRK1−1A-OE wheat plants but was significantly decreased in TaSnRK1 EMS mutants compared with their wild types (Figure 5C; Figure S2, Supporting Information).
As a member of fibrillins, TaPAP6L was possibly involved in biosynthesis JA. [55] To detect the influence of TaPAP6L on JA, we measured JA content in overexpression and mutant lines of TaPAP6L.The TaPAP6L-2B-OE lines had significantly higher JA, whereas TaPAP6L EMS mutants (aaBB, AAbb, and aabb) had lower JA compared with their wild types (Figure 5D).To illustrate possible mechanism of TaPAP6L regulating JA, we sequenced the transcriptomes of TaPAP6L-2B-OE and wild-type plants.Results showed that 27 significantly up-regulated differentially expressed genes (DEGs) were involved in the synthesis of -Linolenic acid which is the precursor of JA biosynthesis, whereas three DEGs (Jasmonic acid-amino synthetases, JAR1s) responsible for JA degradation into jasmonoyl-isoleucine (JA-Ile) were significantly down-regulated (Figure 5E; Table S3, Supporting Information). [56,57]These results suggested that TaPAP6L-2B-OE increased the JA content, most likely by promoting the accumulation of -Linolenic acid and inhibiting the degradation of JA.

Exogenous JA can Enhance Cold Tolerance of Wheat
To further strengthen the relationship between JA and cold stress in wheat plants, we sprayed exogenous JA on wild-type Fielder and TaSnRK1-edited plants.Results showed that wildtype Fielder plants with exogenous JA significantly improved cold tolerance and possessed significantly increased relative water content and significantly decreased relative electrolyte leakage rate after cold stress (−4 °C, 8 h) compared with controls (Figure 5F).On the other hand, TaSnRK1-edited wheat plants with exogenous JA obviously restored cold tolerance and possessed significantly increased relative water content and significantly decreased relative electrolyte leakage rate after cold stress (−4 °C, 4 h) compared with controls (Figure 5G).In addition, we sprayed exogenous JA on TaPAP6L EMS mutants and TaPAP6L-2B-OE plants.Results showed that TaPAP6L EMS mutants with exogenous JA significantly increased cold tolerance after cold stress (−4 °C, 4 h) compared with controls (Figure S3, Supporting Information), and TaPAP6L-2B-OE plants with exogenous JA showed obviously enhanced cold tolerance after cold stress (−4 °C, 14 h) compared with controls (Figure S4, Supporting Information).These results suggested that exogenous application of JA could significantly enhance the cold tolerance of wheat.
In conclusion, cold stress resulted in the enrichment of TaSnRK1 at the chloroplast under the guidance of TaDJ-1, where it facilitated the accumulation of TaPAP6L protein through phosphorylation.TaPAP6L increased JA content by promoting linolenic acid synthesis and inhibiting JA degradation, thereby enhancing cold tolerance in wheat plants (Figure 6).

Discussion
As one of the most important food crops, wheat is widely planted all over the world.However, cold stress seriously threatens wheat growth and yield.Frost events led to $360 million of yield losses annually in the Australian wheat production. [58]Cold stress can significantly reduce the live leaf area and soluble carbohydrate accumulation and ultimately negatively affect yield. [59]In the vegetative stage of wheat, cold stress can lead to leaf wilt, which is not conducive to wheat growth and development. [60]Developing cold-tolerant wheat germplasm for wheat breeding by pyramiding multiple superior genes is an effective strategy to alleviate the damage of cold stress in wheat production.In the present study, we identified two important cold stress genes, TaSnRK1 and TaPAP6L, that could be potentially used for pyramiding breeding to improve cold tolerance in wheat breeding program.
in plants. [61,62]SnRK1 is a heterotrimeric complex containing  subunit with catalytic activity,  subunit with regulatory function and  subunit with anchoring function, and its function is mainly determined by the  subunit. [63,64][67] In S. lycopersicum, SnRK1 is involved in the immunity of biviridae. [68]In rice, OsSnRK1a positively modulated salicylic acid and enhanced JA-mediated defense responses after inoculation with M. grisea. [69]The SnRK1 kinases control metabolism, growth, and development, as well as stress tolerance, by direct phosphorylation of metabolic enzymes and regulatory proteins and by extensive transcriptional regulation. [70]In Arabidopsis, SnRK1 phosphorylation of FUSCA3 positively regulates embryogenesis, seed yield, and plant growth at high temperature. [71]In rice, the OsNAC23-Tre6P-SnRK1a feed-forward loop regulates sugar homeostasis and grain yield. [47]SnRK1 phosphorylation of AL2 delays cabbage leaf curl virus infection, [72] and SnRK1 regulates chromatin-associated OXS3 family proteins localization through phosphorylation. [73]nRK1-mediated phosphorylation of a cytosolic ATPase positively regulates rice innate immunity and is inhibited by U. virens effector SCRE1. [48]Compared with many plant species (i.e., Arabidopsis and rice etc.), hexaploid wheat possessed a higher number of SnRK genes.Genome-wide identification and expression analysis of SnRK gene in wheat showed that the SnRK gene family plays an important role in the regulation of abiotic stress and different pathways. [74]In wheat, SnRK1 was reported to be involved in seed germination and fusarium head blight resistance. [4,75]Up to date, study of SnRK1 in wheat has mainly focused on growth and development as well as disease resistance, while the related research on SnRK1 on abiotic stress has been less reported.In this study, the potential of TaSnRK1 to regulate wheat cold tolerance was successfully explored through multiple molecular techniques.We revealed that TaSnRK1 phosphorylated TaPAP6L and mediated endogenous JA content to regulate cold tolerance, and this regulation was involved in molecular chaperone TaDJ-1 due to different sub-cellular localizations of TaSnRK1 and TaPAP6L.Therefore, we brought forth a new insight into dissecting molecular mechanism of TaSnRK1 modulating wheat cold tolerance.
FBNs (Fibrillins) are a large protein family in photosynthetic organisms. [76]Members of the FBN family in the thylakoid and stroma have been reported to be associated with multiple environment stresses through involving in the storage, transport, and synthesis of lipid molecules. [77]As a member of FBN family, PAP6 is a homologue of FBN4 in Arabidopsis, [78] and was reported to regulate the expression of genes involved in cytokinins synthesis in Arabidopsis. [79]A recent study indicated that TaPAP6L modulated JA-mediated grain size in wheat, [80] and may play an important role in wheat cold tolerance by VIGS. [17]n this study, we investigated a significant increase of JA content in TaSnRK1−1A-OE transgenic wheat lines and screened an interaction protein TaPAP6L-2B that was possibly involved in JA synthesis.We further verified TaSnRK1−1A interacting and phosphorylating TaPAP6L-2B and revealed important phosphorylation sites and superior haplotypes of TaPAP6L-2B.Therefore, our study further elucidates the regulatory mechanism of TaPAP6L as a member of the FBN family in cold tolerance.
Jasmonic acid (JA), a group of oxidized phospholipid compounds ubiquitous in the plant kingdom, is a key signal regulating various plant processes.The lipid-derived phytohormone JA regulates plant growth, development, secondary metabolism, resistance to insect attack and pathogen infection, tolerance to abiotic stresses, etc. [81] MYC2/JIN1 protein is involved in JA signal transduction and plant adaptation to salt stress, and JA impairs salt tolerance in Arabidopsis seedlings through MYC2mediated inhibition of Catalase 2 () expression. [82]Recently, JA is reported to be involved in the regulation of root development under drought stress by promoting xylem differentiation from procambial cells in roots of Arabidopsis. [83]The regulatory effect of JA under salt stress is correlated with the increase in JA level, thus improving ion transport, osmotic regulation, and antioxidant defense. [84]JA was also reported to be involved in tolerance to cold stress.JA positively regulates the transcription pathway of CBF to up-regulate downstream cold response genes, and ultimately improve plant cold tolerance; meanwhile, JA interacts with other hormone signaling pathways (such as auxin, ethylene, and gibberellic acid) to regulate leaf aging and cold stress. [38]In this study, TaPAP6L-2B significantly up-regulated 27 differentially DEGs involved in the synthesis of -Linolenic acid but down-regulated 3 DEGs that were responsible for JA degradation.We further revealed that upregulation of TaPAP6L-2B significantly increased JA content in overexpression lines under cold stress.Moreover, exogenous JA application significantly enhanced wheat cold tolerance.These results indicated that TaSnRK1 modulates TaPAP6L-mediated wheat cold tolerance possibly through regulating endogenous JA contents.
In summary, we found that TaSnRK1-edited lines and EMS mutants showed significantly reduced cold tolerance and JA content in wheat plants but TaSnRK1−1A−OE lines showed significantly increased cold tolerance and JA content.Therefore, we assumed that TaSnRK1 regulated wheat cold tolerance by mediating endogenous JA content.Exogenous JA significantly enhanced cold tolerance in TaSnRK1-edited wheat lines and wild type.Subsequently, we found that overexpression of TaPAP6L-2B which is a phosphorylation substrate of TaSnRK1−1A also significantly increased cold tolerance and JA content in wheat plants.These results suggested that TaSnRK1−1A altered endogenous JA content, possibly through phosphorylating TaPAP6L-2B, and thereby enhanced wheat cold tolerance (Figure 6).Therefore, the relationship among TaSnRK1, TaPAP6L, and JA could be used to construct a regulatory network between phytohormones and cold stress for potential application of wheat cold tolerance breeding.

Experimental Section
Growth Conditions and Cold Stress: Wheat seedlings were grown in an illuminated incubator at 23/18 °C day/night temperatures under 16/8 h light/dark photoperiod and 500 mol m −2 s −1 light intensity.The cold treatments were performed according to the method as previously reported with minor modification. [17]After 3-4 weeks, the seedlings were treated with cold stress.All plants with Fielder background were exposed to cold stress at −4 °C from 4 to 14 h.All plants with Kronos background were exposed to cold stress at −4 °C for 4 h.All plants with Zhengmai9023 background were exposed to cold stress at −4 °C for 12 h.Jasmonic acid (JA) pretreatment was performed according to methods described previously with minor modification. [85]Wheat seedlings at three-leaf stage of Fielder, CRISPR-Cas9-mediated lines of TaSnRK1 (TraesCS1A02G350500, sucrose non-fermenting-1-related protein kinase alpha subunit), EMS (Ethyl methanesulfonate) mutants, and overexpressed (TaPAP6L-2B-OE) lines of TaPAP6L (TraesCS2B02G171300, plastid-lipid-associated protein 6, chloroplastic-like), were spray with either 100 μm JA or an equal amount of distilled water (control) for 5 consecutive days, respectively.All plants with Fielder background were exposed to cold stress at −4 °C from 4 to 14 h.All plants with Kronos background were exposed to cold stress at −4 °C for 4 h.
Population Materials and Cold Tolerance Index: The wheat association panel, consisting of 243 wheat accessions, as we previously reported was planted at Yuanyang (35.04°N, 113.94°E) in 2017-2018 and 2019-2020 cropping seasons, and at Zhengzhou (34.87°N, 113.60°E) in 2017-2018 cropping season. [80,86]The F 10 RIL population (SC) containing 166 lines, developed from the cross of Shanghai3/Catbird×Naxos, was planted at Zhengzhou in 2015-2016 cropping season and at Yuanyang in 2017-2018 cropping season.No drought stresses occurred in field.The cold tolerance index of all surveyed accessions was investigated in February and March of each year and classified into four ranks (3, 2, 1, 0) from high to low according to the standards of the Wheat Cultivar Approval Committee of the Yellow and Huang wheat regions (i.e., sensitive, moderately sensitive, moderately tolerant, and cold tolerant, respectively) as previously described. [17]enome-Wide Association Study (GWAS): All 243 wheat accessions were genotyped using the Wheat 660K SNP array as we described previously. [80,86]The PCA analysis of the Panel was showed in our previous study. [87]After quality control, only SNPs with minor allele frequency (MAF) > 0.05 and missing data < 20% in the association panel were kept for GWAS analysis using PLINK software. [88]The threshold (1.0E-3) for Pvalue was calculated using a modified Bonferroni correction method. [89]aplotype analysis was performed using Haploview v4.2.
Bulked Segregant RNA-Seq Analysis in the F10 RIL Population: The F 10 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.The leaves of 3 cold-tolerant pools (CTP) (cold stress for 0 h and 24 h, respectively) and 3 cold-sensitive pools (CSP) were collected under cold stress for 0 h, 24 h, and 48 h at the three-leaf stage in the greenhouse, respectively.Each cold-tolerant or cold-sensitive pool was composed of an equivalent mixture of leaves from 10 lines of the RIL population with level 0 or 3 under the 4 environments.Sampled leaves were rapidly frozen in liquid nitrogen and stored at −80 °C for bulked segregant RNA sequencing (BSR-Seq).In addition, TaPAP6L-2B-OE lines and Fielder were sampled for transcriptome analysis.
The RNA concentration was 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.Approximately 11G clean reads were generated for each sample.Bowtie2v2.2.3 was used to map clean reads to the genome of Chinese Spring (IWGS Ref Seqv1.0) to obtain the location information of these genes.The difference expression genes (DEGs) were detected using the DESeq1.18.0 R package functions estimate SizeFactors and nbinomTest.The threshold for significantly differentially expressed genes was set as P value < 0.05 and fold change > 2. Results of bulked segregant RNA-seq (BSR) analysis of the F 10 RIL population (SC) in a 9-Mb (532-541 Mb) interval on 1A are shown in Table S1 (Supporting Information).
Virus-Induced Gene Silencing (VIGS): The cold sensitive wheat cultivar Zhengmai 9023 was used for virus-induced gene silencing (VIGS) using infection of barley stripe mosaic virus (BSMV) vector.Primers (Table S4, Supporting Information) of candidate genes were designed using Primer 3.0, and 237-bp, 194-bp, and 181-bp fragments were generated for TraesCS1A02G350500, TraesCS1A02G360300, and TraesCS1A02G360400, respectively.The , , and  RNAs of the BSMV genome were synthesized from linearized plasmids, using Ribo MAXTM Large Scale RNA Production System-T7 (Promega, Madison).The original BSMV 0 was constructed from , , and  RNAs derived from the original empty pSL038-1 vector and served as the viral control.The plants inoculated with BSMV:PDS (phytoene desaturase) were used as the positive control.BSMV TaPDS (Gen-Bank: FJ517553.1),as mentioned by Zhang et al. (2016) was used to monitor the time course of VIGS as a positive control.Transcripts of each vector (, , , or recombinant -gene) were mixed in a 1:1:1 ratio to prepare for inoculating wheat plants.The mixture RNA virus was added to the FES buffer to inoculate onto the second leaves of the silenced seedlings, and after darkness for 24 h, all plants were grown at a condition of 23-25 °C with 60-80% relative humidity.After two weeks, the third and fourth leaf tissues were collected to measure the physiological index.qRT-PCR was performed to determine the efficiency of silencing using primers in Table S4 (Supporting Information).
qRT-PCR Analysis: Total RNA was extracted from different materials using total RNA kits (TaKaRa, Dalian, China).Two-step PrimeScript TM RT reagent kit and gDNA Eraser were used for RT reaction by the Bio-Rad CFX96 Real-Time PCR system.The -actin gene (No.AB181991) was an endogenous control.Relative expression levels were evaluated according to the relative quantification method (2 −ΔΔCT ). [90]MS Mutants of TaSnRK1 and TaPAP6L Genes: Seeds of the tetraploid wheat Kronos were mutated by the team of Jorge Dubcovsky from the University of California, Davis, using the chemical mutagen methanesulfonic acid (EMS).Two TaSnRK1a-1A (from 1A chromosome) mutants Kronos331 (G/A, stop gained) and Kronos4218 (G/A, splice_acceptor_variant), TaSnRK1a-1B (from 1B chromosome) mutant Kronos4220 (C/T, missense variant); TaPAP6L-2A (from 2A chromosome) mutant Kronos2368 (G/A, stop gained), and TaPAP6L-2B (from 2B chromosome) mutant Kronos4490 (G/A, splice_donor_variant) were used to backcross twice with Kronos to generate BC 2 lines, respectively.We crossed Kronos2368 and Kronos4490 to obtain aabb (TaPAP6L) double mutants.
Overexpression of the TaSnRK1−1A Gene and TaPAP6L-2B Gene: To produce TaSnRK1−1A-OE and TaPAP6L-2B-OE plants, the entire coding sequences (CDSs) of TaSnRK1−1A and TaPAP6L-2B were cloned and linked into the LGY-OE3 vector with the Ubi promoter, respectively.These vectors containing targeted genes were transformed by Agrobacteriummediated infection into immature embryos of hexaploid wheat cultivar Fielder to obtain the TaSnRK1−1A-OE and TaPAP6L-2B-OE transgenic lines.Positively T 0 transgenic plants detected by PCR were self-pollinated into T 3 generations.Three independent lines of T 3 plants with high expression levels by qRT-PCR were used for further analysis.
CRISPR/Cas9 Gene Editing of TaSnRK1: The target sequences were designed and selected using CRISPRdirect (http://crispr.dbcls.jp/)and CRISPOR (http://crispor.tefor.net/) to minimize off-target effects.Two sgRNAs of TaSnRK1 were designed based on homology searches against the genome of Chinese Spring.The recombinant plasmid was generated by introducing the guide sgRNA 1 and sgRNA 2 into the binary vector pBUE411 that is modified with the wheat TaU3 promoter and guides the RNA scaffold.The constructed vector was introduced into the A. tumefaciens strain EHA105 and then transferred into Fielder for genetic transformation.TaSnRK1-edited plants were self-pollinated into T 2 generations.TaSnRK1-edited plants were sequenced to detect the targeted mutation by Hi-Tom and Sanger sequencing (Table S2, Supporting Information). [16]easurement of Phytohormones and Physiological Indexes: TaSnRK1−1A-OE lines and EMS mutants were used to determine the phytohormones, including Abscisic acid (ABA), 3-Indoleacetic acid (IAA), Jasmonic acid (JA) and Salicylic acid (SA) based on the liquid chromatography analysis method according to previous reports. [91]esides, the JA content in TaPAP6L-2B-OE lines and EMS mutants were also determined.In order to assess the effect of cold stress, relative water content and relative electrolyte leakage rate were measured using fresh wheat seedlings.
Yeast Two-Hybrid (Y2-H) Assays: The CDS sequence of TaSnRK1−1A was recombined to the vector pGBKT7.The cold-stressed wheat seedling of Ak58 was used to construct a cDNA library for screening interaction proteins using yeast two-hybrid (Y2-H) assay following the manufacturer's protocol (Clontech, USA).The yeast colonies that grew on SD/-Trp-Leu-His-Ade and showed -galactosidase activity were selected as putative interaction colonies.After sequencing, the client genes were identified.Then the cDNAs of TaPAP6L-2B and TaDJ-1-7B (TraesCS7A02G446200, DJ-1 family protein) were amplified and cloned into the pGADT7 vector.The bait and prey vectors were co-transformed into yeast strain AH109 and screened on SD/-Leu-Trp and SD/-Trp-Leu-His-Ade to verify their interactions with TaSnRK1−1A.
Firefly Complementation Assay: To investigate the interaction between TaSnRK1−1A and TaPAP6L-2B, the CDS of TaSnRK1−1A was inserted into pCAMBIA1300-cLUC vector, while the CDS of TaPAP6L-2B was inserted into pCAMBIA1300-nLUC vector.The recombinant vectors were transformed into A. strain GV3101 and then different vector combinations were co-transfected into lower epidermis of the N. benthamiana leaves.TaPAP6L-nLUC and cLUC, nLUC and TaSnRK1-cLUC, cLUC and nLUC were used as negative controls.After 24 h dark and 24 h light, the coinfiltrated leaves were observed for luciferase activity using a plant living imaging system (Berthold, Night Shade LB 985).
Pull-Down Assay: TaSnRK1-His and TaPAP6L-GST or GST protein was expressed in Escherichia coli (E.coli).TaPAP6L-GST or GST proteins were purified with buffer (50 mm Tris, 150 mm NaCl, 10 mm GSH, pH8.0) using beyoGold TM GST-tag Purification Resin according to the instruction of a GST-tag Protein Purification Kit (Beyotime, China, Catalog No. P2262), then the pulled down proteins mixed with the SDS sample buffer.The samples were detected by immunoblot using an anti-GST antibody (Abmart, M20007) and anti-His antibody (Abmart, M20001).
Protoplast Isolation and Transformation: Protoplast isolation and transactivation assays were performed as previously described by Fujii et al. (2009).Two-week old wheat seedling (23/20 °C day/night, 10/14 h light/dark, and 500 mol m −2 s −1 light intensity) were used for protoplast isolation.Strips of young rosette leaves were treated with enzyme solution containing Cellulase R-10 (Yakult Pharmaceutical Industry) and Macerozyme R-10 (Yakult Pharmaceutical Industry) in the dark.After being diluted with equal volume of W5 solution (2 mm MES, pH 5.7, 154 mm NaCl, 125 mm CaCl 2 , and 5 mm KCl), the protoplasts were filtered through a nylon mesh and pelleted at 100 g for 2 min.Protoplasts were resuspended in W5 solution and kept for 30 min.Protoplasts (100 μL) in MMG solution (4 mm MES, pH 5.7, 0.4 m mannitol, and 15 mm MgCl 2 ) were mixed with the plasmid mix, and incubated for 5 min with 110 μL PEG solution (40% w/v PEG-4000, 0.2 m mannitol, and 100 mm CaCl 2 ).The protoplasts were washed twice with 1 mL W5 solution, and then were cultured at room temperature for 12 h for further observation.
Chloroplast Protein Extraction of Wheat Protoplast: An appropriate amount of chloroplast extraction buffer (0.33 m Sorbitol, 50 mm HEPES-KOH pH 8.0, 2.0 mm EDTA-Na 2 , 2.0 mm MgCl 2 , 2.0 mm MnCl 2, 2.0 mm Ascorbic acid, 0.1% BSA) was added to the cultured protoplasts, resuspension protoplasts.Supernatant was taken and centrifuged at 4 °C at 1500 g for 10 min.The resulting precipitates were crude extracted protoplasts.The precipitates were suspended with 2 mL chloroplast extraction buffer solution for further use.Taking a 15 mL centrifuge tube, the bottom contains 80% Percoll (v/v) gradient solution (0.33 m Sorbitol, 50 mm HEPES-KOH pH 8.0, 2.0 mm EDTA-Na 2 ), the upper part contains a gradient liquid covering of 30% Percoll (v/v).The prepared crude chloroplast extract was suspended on 30% Percoll and centrifuged at 7000 g for 1 h.The chloroplasts were washed two times with clean solution (0.33 m Sorbitol, 50 mm HEPES-KOH pH 8.0, 2.0 mm EDTA-Na 2 , 1.0 mm MgCl 2 ) and were then suspended in the buffer solution.The chloroplast proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoreses (SDS-PAGE) with coomassie blue staining and western blotting using anti-GFP antibody (Abmart, M20004).

Figure 1 .
Figure 1.TaSnRK1−1A was identified by GWAS, BSR-seq and VIGS; TaSnRK1−1A positively regulated wheat cold tolerance.A) GWAS results revealed an important genetic locus gCTI-1A (532-541 Mb) regulating cold tolerance on chromosome 1A in two environments (2018 and 2020 in Yuanyang).B) Expressions of 28 annotation genes in a 9-Mb (532-541 Mb) interval between cold-tolerant pools (CTPs) and cold-sensitive pools (CSPs) based on transcriptome analysis."+" indicated differentially expressed genes (DEGs); "-" indicated none DEG.S Cold : cold-sensitive pools under cold stress for 24 h or 48 h.S CK : cold-sensitive pools without cold stress.T Cold : cold-tolerant pools under cold stress for 24 h or 48 h.C-E) VIGS (virus-induced gene silencing) experiment verified the function of TaSnRK1−1A gene.C,D) Phenotypes of wild type (WT), BSMV 0 , and BSMV TaSnRK1 plants (n = 5 plants per replicate) before and after cold stress; E) the relative expression levels (n = 3).CK represents wheat plants without cold stress; NS represents unsilenced plants.G) Mutation sites of TaSnRK1 in Kronos EMS mutants and a comparison of phenotype and physiological indices of mutants and the controls (n = 5) under cold stress.H) The relative expression of TaSnRK1−1A in TaSnRK1−1A-OE lines (n = 3) and a comparison of phenotype and physiological indices of TaSnRK1−1A-OE lines and the controls (n = 5) under cold stress.I) The editing sites in TaSnRK1-edited lines and a comparison of phenotype and physiological indices of TaSnRK1-edited lines and the controls (n = 5) under cold stress.Values are presented as mean ± SE.Statistical significance was determined by a two-sided t-test (* P < 0.05, ** P < 0.01).

Figure 2 .
Figure 2. TaSnRK1 strongly interacted with TaPAP6L.A) Yeast two-hybrid (Y2-H) assays verified the interaction of TaSnRK1−1A and TaPAP6L-2B.AD-T+BD-53 group was used as positive control, while AD-T+BD-Lam group was used as negative controls.B) Firefly luciferase complementation imaging assay of the interaction between TaSnRK1−1A and TaPAP6L-2B in tobacco leaves.The empty vector groups were as negative controls.C) Pulldown assay confirmed the interaction between GST-TaSnRK1 and His-TaPAP6L.D) Subcellular localization of the GFP-TaSnRK1 and GFP-TaPAP6L fusion protein in wheat protoplast.GFP fusion protein acts as the controls.E) Western blotting detected transient expression of Ubi::GFP-TaSnRK1 or Ubi::GFP-TaPAP6L in wheat protoplasts and chloroplasts, respectively.F) Bimolecular fluorescence complementary (BiFC) confirm the interaction between TaSnRK1 and TaPAP6L.cEYFP+nEYFP, cEYFP+nEYFP-TaSnRK1, cEYFP-TaPAP6L+nEYFP were used as controls.

Figure 3 .
Figure 3.TaDJ-1 as a molecular chaperone is involved in the interaction of TaSnRK1 and TaPAP6L.A) Yeast two-hybrid (Y2-H) assays to verify the interaction of TaDJ-1-7B with TaSnRK1−1A and TaPAP6L-2B.AD-T+BD-53 group was used as positive control, while AD-T+BD-Lam group was used as negative control.B) Firefly luciferase complementation imaging assay of the interaction between TaDJ-1-7B and TaPAP6L-2B in tobacco leaves.The empty vector groups were as negative controls.C) Firefly luciferase complementation imaging assay of the interaction between TaDJ-1-7B and TaSnRK1−1A in tobacco leaves.The empty vector groups were considered as negative controls.D) Subcellular localization of the TaDJ-1-7B protein in wheat protoplast.GFP fusion proteins were as controls.E) Bimolecular fluorescence complementation (BiFC) analysis of the interaction between TaSnRK1-His and TaPAP6L-2B in the protoplasts of BSMV TaDJ-1 and BSMV 0 plants.F) The expression of TaSnRK1-His protein in chloroplast proteins from protoplasts of BSMV 0 and BSMV TaDJ-1 plants, respectively.

Figure 4 .
Figure 4. TaSnRK1 phosphorylated TaPAP6L in vivo and in vitro.A) TaSnRK1−1A significantly improved the phosphorylation level of TaPAP6L-2B.Immunoblots detected phosphorylation levels of TaPAP6L-2B together with (+) or without (-) TaSnRK1−1A in tobacco leaves cells.B) TaSnRK1−1A significantly improved the protein abundance of TaPAP6L-2B in tobacco leaves cells.Immunoblots detected transient expression of TaSnRK1−1A together with (+) or without (-) TaPAP6L-2B in tobacco leaves cells.Anti-actin was used for loading controls.C) TaSnRK1−1A phosphorylated TaPAP6L-2B in E. coli cells.The phosphorylation status of E. coli-produced TaPAP6L-2B incubated with E coli-produced TaSnRK1−1A or/and TaGRIK1 or alone were determined by immunoblots, respectively.D) TaPAP6L-2B had the highest phosphorylation level than its homologous proteins TaPAP6L-2B and TaPAP6L-2D.The phosphorylation status of E. coli-produced TaPAP6L-2B homologous proteins incubated with E. coli-produced TaSnRK1−1A and TaGRIK1 were determined by immunoblots.E) Gene structure and haplotype analysis of TaPAP6L-2B, and comparison of the cold tolerance index among 8 haplotypes in multiple environments.I: Mar.2018, Yuanyang; II: Feb. 2020, Yuanyang and III: Mar.2020, Yuanyang.Statistical significance was determined by a two-sided t-test (* P < 0.05).F) Changes of R30S and G38S significantly enhanced the phosphorylation of TaPAP6L-2B mediated by TaSnRK1−1A in E. coli cells.The phosphorylation status of E. coli-produced wild type and mutants of TaPAP6L-2B incubated with E coli-produced TaSnRK1−1A and TaGRIK1 were determined by immunoblots.G) Changes in R30S and G38S significantly enhanced TaPAP6L-2B phosphorylation in wheat protoplasts affected by low temperature.

Figure 5 .
Figure 5. TaPAP6L positively modulated wheat cold stress possibly due to the accumulation of JA.A) Mutation sites of TaPAP6L in Kronos EMS mutants and a comparison of phenotype and physiological indices of mutants and the controls (n = 5) under cold stress.B) The relative expressions of TaPAP6L-2B in TaPAP6L-2B-OE lines (n = 3) and a comparison of phenotype and physiological indices of TaPAP6L-2B-OE lines and control (n = 5) under cold stress.C) JA (Jasmonic acid) was measured in TaSnRK1−1A-OE lines and EMS mutants (n = 5); D) JA was measured in TaPAP6L-2B-OE lines and EMS mutants (n = 5).E) A total of 27 DEGs were involved in the synthesis of -linolenic acid identified by transcriptome sequencing.F) Exogenous JA (100 μm) significantly enhanced wheat (n = 5) cold tolerance.The relative electrolyte leakage rate and relative water content (n = 5) were determined.G) Exogenous JA (100 μm) significantly enhanced wheat (n = 5) cold tolerance in TaSnRK1-edited lines.The relative electrolyte leakage rate and relative water content (n = 5) were determined.Values are presented as mean ± SE.Statistical significance was determined by a two-sided t-test (* P < 0.05, ** P < 0.01).