Characterization of sucrose nonfermenting-1-related protein kinase 2 (SnRK2) gene family in Haynaldia villosa demonstrated SnRK2.9-V enhances drought and salt stress tolerance of common wheat

Background The sucrose nonfermenting-1-related protein kinase 2 (SnRK2) plays a crucial role in responses to diverse biotic/abiotic stresses. Currently, there are reports on these genes in Haynaldia villosa, a diploid wild relative of wheat. Results To understand the evolution of SnRK2-V family genes and their roles in various stress conditions, we performed genome-wide identification of the SnRK2-V gene family in H. villosa. Ten SnRK2-V genes were identified and characterized for their structures, functions and spatial expressions. Analysis of gene exon/intron structure further revealed the presence of evolutionary paths and replication events of SnRK2-V gene family in the H. villosa. In addition, the features of gene structure, the chromosomal location, subcellular localization of the gene family were investigated and the phylogenetic relationship were determined using computational approaches. Analysis of cis-regulatory elements of SnRK2-V gene members revealed their close correlation with different phytohormone signals. The expression profiling revealed that ten SnRK2-V genes expressed at least one tissue (leave, stem, root, or grain), or in response to at least one of the biotic (stripe rust or powdery mildew) or abiotic (drought or salt) stresses. Moreover, SnRK2.9-V was up-regulated in H. villosa under the drought and salt stress and overexpressing of SnRK2.9-V in wheat enhanced drought and salt tolerances via enhancing the genes expression of antioxidant enzymes, revealing a potential value of SnRK2.9-V in wheat improvement for salt tolerance. Conclusion Our present study provides a basic genome-wide overview of SnRK2-V genes in H. villosa and demonstrates the potential use of SnRK2.9-V in enhancing the drought and salt tolerances in common wheat. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-024-10114-7.


Background
Wheat (Triticum aestivum L.) is an important food crop providing essential nutrients to human beings [1], and its production is crucial to ensuring global food supply security [2,3].However, the abiotic stresses, such as drought and salt, threatened wheat production by reducing its grain yield and quality [3][4][5].Therefore, identification of genes regulating abiotic stress tolerances and the elucidation of their regulatory mechanisms are of great significance for the improvement of wheat tolerances to various abiotic stresses via a molecular approach.
Under drought-or high salt-stress conditions, reactive oxygen species (ROS) accumulated in plants and brought about oxidative damage and programmed cell death [27].Compelling molecular evidence revealed that the increased transcriptions of SnRK2s can increment the transcripts of antioxidant enzymes genes, such as superoxide dismutase (SODs), ascorbate peroxidase (APXs), catalase (CATs), peroxidase (PODs), glutathione (GSHs) and/or corresponding protein levels, which are involved in scavenging ROS.Consequently, they relieved oxidative stress to decrease malondialdehyde (MDA) and kept normal growth and development of plant under different stresses [28,29].Under high salinity tolerance, AtSnRK2. 4 and AtSnRK2.10 regulated the expression of CATs and APXs that responsible for ROS homeostasis [28,29].Conversely, overexpression of OsSAPK1 and OsSAPK2 promoted the production of ROS scavenger (ascorbic acid) and increased the protein levels of SOD and CAT, leading to the improved ROS detoxification [30,31].Of note, Ossapk2 mutants were more sensitive to drought stress, with the remarkable increased transcript levels of OsCAT , OsCu/Zn-SOD1, OsCu/Zn-SOD2 and OsAPX2 [30,31].Recently, it has been demonstrated that heterologous overexpression of TaSnRK2.9 in tobacco enhanced drought and high salt tolerances via reducing H 2 O 2 content by SOD, CAT, POD and GSH [24].More recently, overexpressing CsSnRK2.5 in Arabidopsis has been reported to enhance drought tolerance by decreasing accumulation of ROS and MDA [32].
Haynaldia villosa L. (2n = 2x = 14, VV), a diploid wild relative of wheat, is a valuable genetic resource harboring many elite traits, such as resistance to tolerance to various abiotic stresses [33][34][35].Simultaneously, SnRK2 gene family plays an important role in different abiotic stresses, however, there is still a sustained lack of research on understanding the functional import of the SnRK2 genes in H. villosa.Herein, we aimed to elucidate the evolution and diversification of SnRK2-V genes and potential roles of SnRK2.9-V in drought and high salinity stresses.First, different members of SnRK2-V genes were identified in H. villosa, and phylogenetic tree and evolutionary relationship of SnRK2-V genes were analyzed.Secondly, chromosome distribution and gene structure were further studied to gain a better understanding of SnRK2-V genes, in which ten genes were cloned from H. villosa and their potential functions were elucidated by quantitative RT-PCR (qRT-PCR).Finally, SnRK2.9-V was transformed into common wheat to analyze its role upon salt and drought stresses.In a word, our results may provide new candidate genes for wheat to improve the tolerance of drought and soil salinization.

Identification of SnRK2-V gene family in H. villosa and analysis of phylogenetic relationship
Out of 98 SnRK2 genes from seven Triticeae species, thirty SnRK2 members were characterized in T. aestivum, eight in T. urartu, ten in Aegilops speltoides, ten in Ae. tauschii, twenty in T. dicoccoides, ten in H. vulgare, and ten in H. villosa genome sequence.These genes were named from SnRK2.1-V to SnRK2.10-V according to the phylogenetic relationship to wheat SnRK2 genes (Fig. 1).

Gene structure and conserved motif analysis of SnRK2-V genes
Crystal structure analysis of SnRK2-V demonstrated that there were significant differences in the structures of SnRK2-V genes among three subgroups or within individual subgroup.Firstly, the number of introns varied significantly in Group I. Maximal number of intron was eight in SnRK2.4-V,SnRK2.6-V and SnRK2.7-V,which were the same as their orthologues in other Triticeae species and rice, e.g.SAPK4, SAPK6 and SAPK7 (Fig. 4).Conversely, at least two introns were detected in SnRK2.5-Vgene, and a similar result was observed in TaSnRK2.5-A,TaSnRK2.5-B and TaSnRK2.5-Dgenes (Fig. 4).Simultaneously, except SnRK2.1-Vcontaining six introns, eight introns were detected in SnRK2.2-V and SnRK2.3-Vgenes in Group II, and a similar result was observed in SnRK2.8-V and SnRK2.9-Vgenes in Group III (Fig. 4).
Secondly, the length of every intron varied dramatically and the gene length of each SnRK2-V gene was significantly different.The longest intron of the SnRK2.8-V was eight kb, whereas the shortest intron of the SnRK2.5-Vgene was only three kb and the rest SnRK2-V genes varied from four kb to seven kb.Of note, the coding DNA sequence (CDS) length of SnRK2-V genes did not change remarkably, varying from 1,026 bp (SnRK2.3-V) to 1,179 bp (SnRK2.5-V)(Table S1).

Subcellular localization and expression profiling of SnRK2-V genes
Investigating the subcellular localization of a protein may provide clues towards the elucidation of its function.To verify where the SnRK2-V proteins were localized in vivo, the Agrobacterium method was performed to observe the transient expression of GFP (green fluorescent protein)tagged fusion proteins in the leaves of Nicotiana Benthamiana.The GFP signals of the fusion proteins could be detected for all ten cloned SnRK2-V proteins, with the signals deferentially localized (Fig. 8).Compared with an even distribution of GFP fluorescence in the control group (Fig. 8a), all ten identified SnRK2-V proteins localized on the plasma membrane (PM) (Fig. 8).Notably, some SnRK2-V proteins also had obvious GFP fluorescence in the cytoplasm and nucleus, such as SnRK2.1-V,SnRK2.2-V,SnRK2.3-V,SnRK2.6-V,SnRK2.7-V,SnRK2.8-V,SnRK2.9-V and SnRK2.10-V(Fig. 8b, c, d, g,  h, i, j, k).To further predict the functions of the identified SnRK2-V genes, we investigated their transcription levels in different tissues or in responses to various biotic or abiotic stresses.The transcription patterns of SnRK2-V genes in different tissues of H. villosa (e.g., roots, stems, leaves, spikes, and grains) under two abiotic stresses (e.g., drought and salt) or two biotic stresses for Blumeria graminis f. sp.Tritici (Bgt) isolate E26 and Puccinia sriiformis f. sp.Tritici (Pst) isolate CYR32 were investigated by qRT-PCR.A three-fold change of transcription level was arbitrarily considered to be positive expression induction.Moreover, the tissue-specific expression analysis displayed that there were dramatic differences in the transcription levels of SnRK2-V genes.A relatively highest transcription level of SnRK2.2-Vgene was detected in roots, SnRK2.4-Vgene was mainly in stems and spikes, SnRK2.9-Vgene was in leaves, and SnRK2.8-Vgene was in grains (Figure S1).Meanwhile, the lowest transcription level of SnRK2.3-Vgene was detected in grains, and the transcription level of SnRK2.5-Vgene was in stems and grains.Contrarily, the lowest transcription level of SnRK2.1-V was detected in all tested tissues (Figure S1).
To further verify the transcription levels of SnRK2-V genes induced by exogenous ABA, seedlings of H. villosa were transferred with ABA and then the samples were separately collected at 0, 6, 12, 24, and 48 h for qRT-PCR analysis.The data showed that, after ABA treatment, the transcript level of SnRK2.8-Vgene was induced significantly at 6 h, and reached a maximum of five-fold change at 24 h.Compared with SnRK2.8-V, the transcription level of SnRK2.9-Vincreased obviously at 12 h, and peaked with a maximum of ten-fold change at 24 h (Figure S2).In contrary, the transcription levels of other SnRK2-V genes were not induced markedly by exogenous ABA treatment (Figure S2).

Overexpression of SnRK2.9-V in wheat enhances drought and salt tolerances
Previous studies have demonstrated that SnRK2 proteins were involved in drought and salt stresses [6,7].To further confirm the positive function of SnRK2.9-V in drought and salt stresses, we generated three independent 2 × 35S:SnRK2.9-Vtransgenic OE lines (OESnRK2.9-V#1,OESnRK2.9-V#5 and OESnRK2.9-V#6) in the wheat cv.Fielder, which carried the full CDS of SnRK2.9-V.The transgenic plants were confirmed by PCR using specific primers to amplify a 308 bp DNA segment in T 2 generations.Compared with negative transgenic plant (#2) in which no amplification was detected (Fig. 10a).The results indicated that transcript levels of SnRK2.9-V in the leaves of three OE lines increased prominently compared with WT and #2 as was shown in Fig. 10b.

Overexpression of SnRK2.9-V enhances the transcription levels of antioxidase genes upon drought and salt stresses in wheat
Stresses are perpetually associated with the generation of ROS, such as H 2 O 2 , and ROS accumulation, leads to lipid peroxidation, and results in the production of MDA [20].MDA is a stress-specific molecular marker that is indicative of the extent of membrane injury and cell and tissue damage.To determine whether overexpression of SnRK2.9-V can induce the ROS generation, we measured H 2 O 2 and MDA contents in positive transgenic lines (OESnRK2.9-V#1,OESnRK2.9-V#5 and OESnRK2.9-V#6),Fielder and negative transgenic line #2 under different conditions.Our data verified that H 2 O 2 and MDA contents decreased in the positive transgenic lines under PEG6000 and NaCl conditions.In contrast, increases of H 2 O 2 and MDA contents were observed in Fielder and negative transgenic line #2 after PEG6000 and NaCl treatment, indicating that overexpression of SnRK2.9-V can reduce oxidative damage via decreasing ROS content under PEG6000 and NaCl conditions (Fig. 12).
To elucidate the possible molecular mechanisms underlying the SnRK2.9-Vgene in stress responses, the transcription levels of drought-and salt-responsive genes of antioxidant system including TaAPX, TaSOD, TaCAT and TaPOD in leaves (Fig. 13a) and roots (Fig. 13b) were separately investigated in positive transgenic lines, Fielder and negative transgenic line.A three-fold change of transcription level was arbitrarily considered to be represent positive expression induction.qRT-PCR analysis revealed no significant differences in the transcription levels of TaAPX, TaSOD, TaCAT and TaPOD genes between the positive lines and negative lines under normal conditions (ddH 2 O).Under PEG6000 and NaCl conditions, the elevated transcription levels of TaSOD, TaCAT and TaPOD were much higher in roots than those in leaves (Fig. 13).The transcription levels of TaSOD, TaCAT and TaPOD genes increased 10.00-fold, 6.50-fold and 12.00fold in the roots of OESnRK2.9-Vlines, and 4.50-fold, 3.50-fold and 3.50-fold in the corresponding leaves after PEG6000 treatment.However, the transcription levels of these genes were not markedly changed in the Fielder and #2 plants.In response to high salt condition, the transcription levels of TaSOD, TaCAT and TaPOD genes increased by 8.00-fold, 5.50-fold and 7.50-fold, respectively in the roots of OESnRK2.9-Vlines, along with the increased by 3.50-fold, 3.00-fold and 3.50-fold in the Fig. 10 PCR identification and transcription profiling of SnRK2.9-V in common wheat transgenic plants.a PCR identification of SnRK2.9-V in common wheat transgenic plants.Lanes 1-8 represented Marker, plasmid, ddH 2 O, Fielder, negative control and three OESnRK2.9-Vtransgenic wheat, respectively.The red arrow represented the target strip.b Related transcription levels of SnRK2.9-V in transgenic wheat.Significant differences are indicated as **P < 0.01, *P < 0.05.Data are mean ± SE (n ≥ 3) corresponding leaves.However, no significantly changes were detected in these transcription levels in Fielder and #2 plants as well as those under PEG6000 condition.Intriguingly, the maximal transcription level of TaAPX gene was detected in OESnRK2.9-Vlines, with 8.00-fold increase in the leaves compared with 6.00-fold in the roots under PEG6000 condition.Conversely, under high salt condition, the transcription level of TaAPX increased more than 5.00-fold in the leaves of OESnRK2.9-Vlines in comparison with 6.00-fold in the corresponding roots.

Discussion
SnRK2 is a family of highly conserved protein kinases, which play important roles in plant adaptation to various environmental signals.With the completion of whole genome and high-quality genome assembly in H. villosa, understanding the whole gene family of SnRK2-V is helpful for researchers to explore the functions of specific SnRK2-V genes in various stress responses.

Evolutionary feature of SnRK2 gene family
In plants, the SnRK2 gene family has been well investigated.Until now, 108 SnRK2 genes have been isolated from plants, including ten SnRK2 genes in A. thaliana [8], ten in O. sativa [9], thirty in T. aestivum [16], eight in T. urartu, ten in Ae. speltoides, ten in Ae. tauschii, twenty in T. dicoccoides, and ten in H. vulgare [15].Our study revealed that there were ten SnRK2-V genes in H. villosa, a diploid wild relative of wheat.It was similar to rice, Arabidopsis and other diploid Triticeae species, in which seven chromosome pairs possessed ten SnRK2 genes.All these data indicate that the SnRK2-V gene family is evolutionarily conserved in Triticeae species in the context of gene number [15,16].The widely accepted classification system helps to categorize structures of SnRK2-V genes into three subgroups (three in Group I, four in Group II and three in Group III), which are well defined in wheat and other plants, as well as similar members proportion of each subgroup between H. villosa and other diploid species, suggesting that the classification of SnRK2-V Fig. 11 SnRK2.9-Voverexpression improved drought and salt stresses in common wheat.a Growth performance of seeding stages under different treatments in control and OESnRK2.9-V-T 2 plants.Scale bar = 5 cm.b Scan analysis of root in seeding stages under different treatments in control and OESnRK2.9-V-T 2 plants.Scale bar = 5 cm.c Shoot length, total root length and total root area in seeding stages under different treatments in control and OESnRK2.9-V-T 2 plants.Significant differences are indicated as **P < 0.01, *P < 0.05.Data are mean ± SE (n ≥ 3) genes is also conserved.Moreover, analysis of chromosome location indicated that the SnRK2-V genes were distributed on the chromosome 1, 2, 3, 4 and 5.This indicates that SnRK2-V genes were formed before the differentiation of Triticeae species [36][37][38][39].

SnRK2-V family members undertake diverse roles
The complete genome sequences for conserved domains, cis-elements and gene expressions in H. villosa offer an opportunity to learn more about functional characterization of the individual SnRK2-V genes.At present study, ten SnRK2-V genes contained highly conserved domains at the N-terminal kinase domains regions with an ATP binding site, alpha C helix, the serine/threonine protein kinase active-site and activation loop, which is highly consistent with rice and Arabidopsis [8,9].These demonstrated that the N-terminal kinase of SnRK2-V genes was evolutionarily conserved in Triticeae species [15,16].Otherwise, domain II with osmotic stress-mediated activation existed in all the promotor regions of SnRK2-V genes, while only SnRK2.8-V,SnRK2.9-V and SnRK2.10-Vexisted ABA-responsive kinases in the domain II.Accumulating evidence has demonstrated that, under exogenous ABA treatment, the transcription level of SnRK2.9-Vincreased obviously by ten-fold, with five markedly induced SnRK2 genes (AtSnRK2.2,AtSnRK2.3,AtSnRK2.6,AtSnRK2.7 and AtSnRK2.8) in Arabidopsis [8], strongly induced OsSAPK8-OsSAPK10 in rice [9] and prominently induced TaSnRK2.8-TaSnRK2.10 in Wheat [16].Additionally, the most frequently present types of CAREs were ABREs, and TGACG-motifs, TCA-elements and LTRs, which are separately present in the promoters of SnRK2.4,SnRK2.6 and SnRK2.8 genes [40].Conversely, SnRK2.9-V had the maximum ABREs numbers, and TCrich repeats were only located in SnRK2.9-V.Moreover, tissue-specific expression analysis displayed the different transcription patterns of SnRK2 genes.These evidences show that different members of SnRK2-V family have different functions [41].However, their specific functions need to be further validated by transgenic approach.
Expression pattern analysis can provide an opportunity to estimation of gene functions, and the transcription levels of SnRK2 genes can be obviously induced in various types of abiotic stresses [42].Among them, AtSnRK2.4 and AtSnRK2.10 were strongly induced under salt treatment [28].Under high salt and PEG conditions, rice OsSAPK1 and OsSAPK2 genes [30] and potato (Solanum tuberosum) StSnRK2.4 gene [43] were rapidly activated.Similarly, GhSnRK2.3/2.7/2.8/2.9/2.10 genes in cotton (Gossypium hirsutum) were notably induced under salt and PEG conditions [44], and TaS-nRK2.4,TaSnRK2.7, and TaSnRK2.8 genes in wheat were up-regulated in response to drought and high salt [16].In our current study, we analyzed the expression profiles of SnRK2-V genes under abiotic (drought and salt) stress and verified that ten SnRK2-V genes were shown to respond to drought and salt stresses, emphasizing that these genes may be positive regulators in responses to drought and salt stresses.Simultaneously, we also detected the transcription levels of SnRK2-V genes under biotic (Bgt and Pst) stress and demonstrated that SnRK2.1-V and SnRK2.3-Vdid not respond to Bgt infection.Notably, six SnRK2-V genes including SnRK2.2-V,SnRK2.4-V,SnRK2.5-V,SnRK2.7-V,SnRK2.8-V and SnRK2.9-V were dramatically induced by Bgt and Pst infection.Therefore, these six SnRK2-V genes are worthy to be further studied for their function in disease resistance for the demonstrated cross-link between abiotic and biotic stress regulation [45,46].

SnRK2.9-V acts as the positive regulator of drought and salt tolerances via antioxidant system
Several studies have demonstrated the positive roles of SnRK2 genes in numerous defenses against harsh environments (e.g., drought and salinity) [46][47][48][49][50]. Overexpression of SnRK2 member OsSAPK4 or OsSAPK6 improved SnRK2 transgenic rice drought tolerance [51], and overexpression of B. distachyon SnRK2.9 dramatically improved drought and high salt resistance [12].Besides, heterologous overexpressing TaSnRK2.9 in N. tabacum significantly enhanced tolerances of transgenic plants to drought and salt stresses [24], and heterologous overexpressing TaSnRK2.4 in Arabidopsis improved salt resistance [16].Similarly, heterologous overexpressing Fig. 13 SnRK2.9-Vregulates the transcription of antioxidant enzyme genes in transgenic wheat.a Transcription profiling of TaAPX, TaSOD, TaCAT and TaPOD in response to drought and salt treatments in the leaves of control and OESnRK2.9-V-T 2 plants.b Transcription profiling of TaAPX, TaSOD, TaCAT and TaPOD in the root of control and OESnRK2.9-V-T 2 plants under drought and salt treatments.Significant differences were indicated as **P < 0.01, *P < 0.05.Data were mean ± SE (n ≥ 3) TaSnRK2.3 in Arabidopsis enhanced tolerance to drought by more developed roots [49].In our study, SnRK2.8-V,SnRK2.9-V and SnRK2.10-Vshowed sensible responses to drought and high salt condition.It closely resembled the results from the orthologous genes AtSnRK2.2, AtSnRK2.3 and AtSnRK2.6 in regulating ABA synthesis and relevant defense-signaling under drought [8].However, defense/stress-responsiveness (TC-rich repeats) only existed in the promoter sequence of SnRK2.9-V,indicating a conservative function of SnRK2.9-V in stress resistance.Consistently, our research demonstrated that there were significantly enhanced tolerance to drought and high salt in SnRK2.9-Vtransgenic lines, implying that ectopic expression of SnRK2.9-V can improve the tolerance of wheat to drought and salt stresses.Collectively, our present study and others indicate that SnRK2.9-V may represent a gene resource for improving wheat resistance to drought and soil salinization stresses.
To survive biotic and abiotic stresses, plants have developed elaborate mechanisms to adaptation by modulating the expression of genes.As we know, the most important functions of SnRK2 proteins against abiotic stresses are their phosphorylation modifications.AtSnRK2.6 phosphorylated CHYR1 (E3 ubiquitin ligase) and bZIP transcription factors ABF1/2/3/4 to enhance drought stress tolerance [52], and it also phosphorylated the transcription factor ICE1, activated the transcription of CBF genes, and enhanced low-temperature tolerance [53].Additionally, overexpression of rice OsSAPK6 enhanced phosphorylation level of OsABF transcription factors and increased salt tolerance [54].Moreover, OsSAPK9 positively enhanced drought stress through the potentiality of transactivation of the OsbZIP transcription factors [20].ZmSnRK2.11effectively alleviated the damage caused by high salt intake and drought stresses via phosphorylating ZmABI1/ABI2/DREB2A/P5CS1 [55].In response to drought, αC-helix in the N-terminal lobe is required for correct folding of the catalytic centre and kinase activation loop, which is stabilized by the SnRK2 box [42].In our search, ten SnRK2-Vs were highly conserved at the N-terminal containing the serine/threonine protein kinase active-site and activation loop, but their C-terminal varied significantly, therefore their features are identical to other Snf1 kinase domains [20].They also displayed the well-ordered characteristic SnRK2 box with a single α-helix in the N-terminal lobe packed parallel against the αC helix.In the activation loop, the predicted phosphorylation site S176 and T177 of SnRK2.9-Vcorresponded to the same positions of OsSAPK9, which had both autophosphorylation and transphosphorylation activities in vitro [20].Taken together, SnRK2.9-V has a characteristic kinase fold structure and the recombinant protein might possess autophosphorylation and transphosphorylation activities.However, the phosphorylation function of SnRK2.9-Vrequires more biochemical experiments to further verify.
It is well known that APX, SOD, CAT and POD are key enzymes that play a decisive role in scavenging ROS and relieving oxidative stress to keep normal growth and development of plant [56,57], and corresponding genes have been reported to be up-regulated upon various stresses [27,[58][59][60][61]. Several studies have demonstrated that overexpression of SnRK2s can promote an increase in activities or transcription levels of antioxidant enzymes, such as SOD, APX, CAT and POD, to scavenge ROS and maintain ROS homeostasis, thus relieving oxidative stress to keep the normal growth and development of plants under osmotic stresses [41,45,46].In our research, heterologous overexpression of SnRK2.9-Vincreased the transcriptions of ROS scavenging enzymerelated genes, such as TaAPX, TaSOD, TaCAT and TaPOD in leaves and roots under high salt and drought stresses.Conversely, the orthologous gene TaSnRK2.9 also activated SOD, CAT, and POD antioxidant system genes to reduce the H 2 O 2 accumulation in transgenic tobacco under drought or salt stresses [24].Likewise, its orthologous gene BdSnRK2.9transgenic plants exhibited lower levels of H 2 O 2 under drought and high salt conditions [12].Moreover, overexpressing SAPK9 in planta enhanced the drought tolerance phenotype of transgenic lines through ROS detoxification, consequently reducing membrane damage [20].Besides, overexpression of CsS-nRK2.5 improved its drought resistance through reducing the accumulation of ROS [32].Our results of SnRK2.9-Vsupported its involvement in drought and salt stresses through expanding the transcription levels of antioxidant enzymes genes.Nevertheless, more biochemical experiments are needed to confirm the role of SnRK2.9-V in regulating antioxidant enzymes genes directly.

Conclusions
Collectively, our study provides comprehensive insights into the SnRK2 gene family in H. villosa.Classification of all SnRK2-V genes from the whole genome survey further contributes to the fundamental researches for the better understanding of key SnRK2-V genes against various biotic and abiotic stresses.Meanwhile, the preliminary verification of SnRK2.9-V in enhancing common wheat resistance to drought and salt stresses might provide a useful gene resource for improving wheat resistance in breeding.

Plant materials
H. villosa (genome VV, accession no.91C43) was obtained from Cambridge Botanical Garden in the UK and maintained by the Cytogenetic Institute, Nanjing Agricultural University (CINAU) in the 1970s [36].Sun et al. used cytogenetics methods to identify H.villosa in our laboratory in 2018 [62].Zhang et al. used this plant material for genome sequencing in our laboratory in 2023 [36].And this plant material was used for gene cloning and expression analysis in this study [63].Wheat varieties Fielder was maintained in Cytogenetics Institute of Nanjing Agricultural University (CINAU) and it was performed as described by Fan et al. [64].And Fielder was used as the recipient cultivar for genetic transformation.N. benthamiana (NC89) plants were maintained in Cytogenetics Institute of Nanjing Agricultural University (CINAU) and it was performed as described by Zhang et al. [65].And it used for subcellular localization analysis.All plants were grown in the greenhouse and the growth conditions were as follows: 14/10 h day/night cycle, 24/20 °C day/night temperature, 8,000 lx light intensity, and 70% relative humidity [63,65].

Phylogenetic analysis and gene characteristics of SnRK2 gene family in Triticeae
Multiple sequence alignment was conducted by ClustalW which was integrated in MEGA 7.0 [72].Phylogenetic analysis was performed through online software PhyML 3.0 [73] using maximum-likelihood method with default parameter [74].To understand the phylogenetic relationship of SnRK2 genes, the unrooted phylogenetic tree was built using MEGA 7.0 via the Neighbor Joining (NJ) method.

Chromosomal distribution and exon-intron structure of SnRK2 gene family in Triticeae
Chromosomal information of predicted SnRK2 genes was obtained from each species and their chromosomal locations were determined after using cDNA sequences as the query sequences blasted to the genomic sequences [63,65].Then we drew their locations onto the physical map of each chromosome using MapInspect tool (http:// mapin spect.softw are.infor mer.com/) [63,65].MCScanX tool kit was used to investigate gene duplication events within species and sequence similarity between SnRK2 genes in wheat and other plant species.

Cis-acting regulatory elements (CAREs) analysis
To analyze putative cis-acting elements in the promoter region, 1.5-kb promoter regions were selected and screened against the Plant CARE database (http:// bioin forma tics.psb.ugent.be/ webto ols/ plant care/ html) [15].Thereafter, the number of occurrences for each CARE motif was counted for SnRK2 genes, and the most commonly occurring CAREs were used to produce figures in TBtools.

Cloning of SnRK2-V genes from H. villosa
Prediction of SnRK2-V genes in H. villosa was carried out by comparing the genomic data of H. villosa [36], and the predicted proteins were processed according to the above method.According to the sequences, the primers (Table S3) were designed to clone the full-length cDNA of SnRK2-V genes from H. villosa using online software Primer3 (v.0.4.0,University of California, Oakland, CA, USA) [65].cDNA of H. villosa tissues (root, stem, leaf and grain) was served as a template for the isolation, and the specific primers for SnRK2-V genes of H. villosa were used for cloning [63].This was performed at 95 °C for 30 s, followed by 32 cycles of 95 °C for 30 s, 56 °C for 45 s, and 72 °C for 1 min, and then 5 min at 72 °C in Phanta Max Super-Fidelity DNA polymerase (Vazyme, Nanjing, China) [63].

Plant treatments
Plant treatments were performed as described by Zhao et al. [63] and Zhang et al. [75] with some modifications.Sterilized seeds were germinated and cultured with water in a climate chamber under a 12 h light/12 h dark cycle at 22 °C.The seedlings of H. villosa were grown in aqueous solution with 1/2 Murashige and Skoog (MS) medium or soil [63,75].Three-leaf stage H. villosa seedlings were in a plethora of multiple abiotic and biotic stresses.For Bgt infection, the plants were inoculated with Bgt virulent race E26 and the leaf tissues were sampled at 0, 6, 24, 48 and 72 h after inoculation [63,75].For Pst infection, the plants were inoculated with Pst virulent race CYR32 and the leaf tissues were sampled at 0, 24, 48, 72 and 120 h after inoculation.For drought stress, the plants were transferred to 1/2 MS medium with 15% PEG 6000, all leaf tissues were collected 0, 6, 12, 24 and 48 h [63,75] For high salinity treatment, the plants were transferred to 1/2 MS medium with 150 mM NaCl, all leaf tissues were collected 0, 6, 12, 24 and 48 h [63,75].For phytohormones treatments, the plants were sprayed with 0.2 mmol abscisic acid and all leaf tissues were collected at 0, 6, 12, 24 and 48 h after spraying.All the samples were rapidly frozen in liquid nitrogen, then stored in an ultra-freezer (-80 °C) before use [63,75].
For the analysis of high salinity and drought tolerances, seeds of SnRK2.9-Vtransgenic plants and the receptor variety Fielder were sterilized with 75% ethanol for 10 min and 12% sodium hypochlorite for 10 min respectively, and then washed five times with sterile water [65].Wild type and SnRK2.9-Vtransgenic plants were cultured in 1/2 MS medium for two weeks.For the high salt and drought stresses, seedlings at 14 days old stage were cultured with hydroponics for seven days with 1/2 MS medium supplemented with the 150 mM NaCl solution and 15% PEG6000 solution.Therefore, the shoot lengths, total root lengths and total root areas of transgenic lines and the receptor variety were measured and analyzed.At the one-week drought and salt stress treatment stage, leaves and roots from each line were collected to measure.All the samples were rapidly frozen in liquid nitrogen, then stored in an ultra-freezer (-80 °C) before use.The averages of data were computed based on the means of three independent experiments, and each based on at least 10 independent plants [65].

RNA isolation and transcription analysis of ROS-related genes
Total RNA was extracted using a Trizol Reagent kit (Invitrogen, CA, USA) according to the manufacturers' instructions [63].Three microliters RNA was used in agarose gel electrophoresis to check the quality and integrity of the obtained RNA samples.The first-strand cDNA was synthesized with random oligonucleotides using the HiScript ® II Reverse Transcriptase system (Vazyme, Nanjing, China).

Determination of H2O2 contents
Determination of H 2 O 2 contents were performed according to the method of Malondialdehyde Microplate Assay Kit (Absin, Shanghai, China).Briefly, wild type and SnRK2.9-Vtransgenic plants were cultured in 1/2 MS medium for two weeks.For the high salt and drought treatments, seedlings at 14-day old stage were cultured with hydroponics supplemented with 150 mM NaCl solution or 15% PEG6000 solution for seven days.Then, 0.1 g fresh tissue of leaves or roots were weighed and 1 mL pre-cooled acetone was added for ice bath homogenization.The sample was transferred to an EP tube and diluted to 1 mL with acetone for centrifugation at 12,000 rpm at 4 °C for 10 min.Thereafter, 0.5 mL supernatant was taken and mixed well with Substrate and Reaction buffer on ice for centrifugation at 12,000 rpm at 25 °C for 10 min, the supernatant was removed and mixed with Dissolution Buffer well.Finally, 200 μL reaction liquid was added into a microplate to measure the absorbance at 415 nm.H 2 O 2 content (μmol/g) = 100 × (O D Sample -OD Black )/(OD Standard -OD Black )/W.

Determination of malondialdehyde (MDA) contents
MDA contents were measured according to the method of Hydrogen Peroxide Microplate Assay Kit (Absin, Shanghai, China).Wild type and SnRK2.9-Vtransgenic plants and seedlings with high salt and drought stresses were cultured as above mentioned.Then 0.1 g fresh tissue of leaves or rootswere taken and 1 mL Assay Buffer was added for ice bath homogenization.After centrifugation at 8,000 g at 4 °C for 10 min, the supernatant was taken and placed on ice for detection.Briefly, 10 μL sample was added into a 1.5 mL centrifuge tube and mixed with 0.6 mL Reaction Buffer (100 μL Reaction Buffer I and 10 μL Reaction Buffer II) gently.After keeping warm in a 90 °C-water bath for 30 min (cover tightly to prevent moisture loss), the tube was cooled in an ice bath and then centrifuged at 10,000 g for 10 min at 25 °C.Thereafter, 200 μL reaction liquid was added into a microplate to measure the absorbance at 532 nm.MDA content (μmoL /g) = (OD Sample -OD Black )/(OD Standard -OD Black )/W.

Subcellular localization assay
Subcellular localization was performed as described by Zhao [63] with the following modifications.The ORFs of SnRK2-V genes (without stop codon) were amplified from the pTOPO-Blunt Vector, then inserted into the pCam-bia1305-GFP vector, which contains the green fluorescent protein (GFP) reporter gene driven by the CaMV 35S promoter, using homologous cloning technology as per the manufacturers' instructions (Vazyme, Nanjing, China) (Table S3).The 1305-GFP fusion constructed plasmid was transformed into N. benthamiana epidermal cells by Agrobacterium tumefaciens (strain GV3101) bacteria and incubated in darkness at 22 °C for 48 h.Then, the fluorescence signals were observed under confocal microscopy (LSM780, Zeiss, Oberkochen, Germany) according to the methods described by Wang and Zhang [76,77].

Genetic transformation
SnRK2.9-V was cloned into the plant expression vector pWM110 (driven by the CAMV 35S promoter) to generate vector pWM110:SnRK2.9-V.Then, the vector pWM110:SnRK2.9-Vwas transformed by A. tumefaciens to young embryos of wheat cultivar Fielder [65].Regenerated plants were transplanted in the greenhouse and used for further characterization [63], and they were analyzed via PCR using conjugated gene-specific primers (Table S3) to identify positive transgenic plants.

Statistical analysis
All statistical analyses were performed using SPSS version 13.0 (SPSS, Chicago, IL, USA).Data are presented as means and standard error (SE), and analyzed by a student's t-test to check for quantitative differences between two different treatments [77].P < 0.05 was set as the significance cut-off.

Fig. 5
Fig. 5 Protein sequence analysis of SnRK2 in H. villosa.ATP binding site, alpha C helix, serine/threonine protein kinase active-site, activation loop and SnRK2 Domain were shown in yellow box, magenta box, red box, black box and blue box respectively.Alpha helix was shown in green helix.Beta strand was shown in blue arrows.The predicted phosphorylation sites were indicated by red asterisks.Species abbreviations: At: A. thaliana; Os: O. sativa; -V: H. villosa

Fig.
Fig.6 The predicted 3D structure of SnRK2-V proteins.ATP binding sites, Alpha C helixs, Serine/threonine protein kinase active-sites, Activation loops and SnRK2 boxs (domain I) were shown in yellow, magenta, red, black and blue, respectively

Fig. 9
Fig.9 Transcription profiling of SnRK2-V genes after treatments with abiotic and biotic stresses.The transcription profiling of SnRK2-V genes in response to drought (a) and salt stress (b).The transcription profiling of SnRK2-V genes in response to Bgt (c) and Pst (d).The scale bar was showing transcription level of the genes.Abbreviations: Bgt: Blumeria graminis f. sp.tritici; Pst: Puccinia sriiformis f. sp.tritici

Fig. 12
Fig.12 Physiological indices of the transgenic wheat plants overexpressing SnRK2.9-Vunder drought and salt stress.Two-week-old wheat plants grown on 1/2 MS medium were transferred to hydroponics for seven days with 1/2 MS medium supplemented with the 150 mM NaCl solution and15% PEG6000 solution.Leaves (a) and roots (b) of control and OESnRK2.9-V-T 2 plants under drought and salt treatments were sampled for the assessment of H 2 O 2 and MDA.Significant differences are indicated as **P < 0.01, *P < 0.05.Data are mean ± SE (n ≥ 3)