Mining&nbsp;the&nbsp;Roles&nbsp;of&nbsp;Wheat (Triticum&nbsp;Aestivum)&nbsp;SnRK&nbsp;Gene&nbsp;Family in&nbsp;Biotic&nbsp;and&nbsp;Abiotic&nbsp;Responses

doi:10.21203/rs.3.rs-104118/v1

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Mining the Roles of Wheat (Triticum Aestivum) SnRK Gene Family in Biotic and Abiotic Responses

https://doi.org/10.21203/rs.3.rs-104118/v1

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Background

Sucrose non-fermenting-1-related protein kinase (SnRK) is a class of Ser/Thr protein kinases and plays vital functions in the plant stress responses. However, little is known about the SnRK in Triticum aestivum (TaSnRK).

Results

In this study, 149 TaSnRKs were identified from wheat and divided into three subfamilies, which may be due to the polyploidization induced gene duplication and high rate of homologous retention. A combination of public microarray datasets and quantitative real-time quantitative PCR (qRT-PCR) have further revealed the distinct expression patterns of TaSnRKs under specific abiotic/biotic stress responses. TaSnRK2.4-B, a member of SnRK2 subfamily, was located in the nucleus, cytoplasm, and cell membrane and showed ubiquitous expression in wheat life cycle, suggesting the possible response to polyethylene glycol (PEG), NaCl, heat, and cold stress, as well as the high concentrations of abscisic acid (ABA) application. Besides, transient Agro-infiltration assays showed that TaSnRK2.4-B was also involved in the resistance to pathogen.

Conclusions

These results imply that TaSnRK2.4-B may act as a multifunctional regulatory factor involved in multiple stress response pathways. Overall, our study provides new insights into the roles of TaSnRKs in biotic and abiotic responses.

Integrative & Complementary Medicine

Triticum aestivum

SnRK

Genome-wide analysis

biotic and abiotic stress

TaSnRK2.4-B

Plants have evolved a set of defending mechanisms, such as protein phosphorylation and dephosphorylation, as a response to myriad stresses including hormonal, pathogenic, and environmental stresses [1–2]. Sucrose non-fermenting-1-related protein kinase (SnRK) is a class of Ser/Thr protein kinases, modulating target proteins through phosphorylation to regulate the interrelationship of multiple signaling pathways in plants and plays a vital role in the stress responses [3–4].

SnRKs are widely found in plant kingdom and are strongly conserved among species. Three subfamilies, SnRK1, SnRK2, and SnRK3 have been divided according to the structural characteristics of proteins [5–6]. All plant SnRK subfamilies share a common S_TKc domain (Serine/Threonine protein kinases, PF00069), linked with a C-terminal kinase associated domain 1 (KA1 domain, PF02149) in SnRK1 [7]. SnRK2 contains SnRK2-specific box (glutamine-303 to proline-318) [8], while NAF domain (Asn-Ala-Phe, PF03822) is presents in the SnRK3 subfamily [9]. SnRK1, a heterotrimeric complex composed by α, β, and γ subunits, is homologous to the yeast SNF1 gene, which was first discovered and well-known by its role in the global regulation of carbon metabolism including glucose repression, and lipid accumulation [10]. SnRK2 and SnRK3, also known as CIPK (calcineurin B-like calcium sensor-interacting protein kinases), are unique existence in the plant kingdom. There are compelling pieces of evidence indicating that the divergence of SnRK2 and SnRK3 subfamilies is evolved after the duplication of SnRK1, which has enabled plants to form networks connecting metabolic and genetic responses to stresses, hormone, and calcium signal [4].

In Arabidopsis thaliana, the SnRK family contains 38 members, including three genes in SnRK1 subfamily, 10 SnRK2 genes, and 25 SnRK3 genes [5]. SnRK1 is related to increasing carbon and/or nitrogen assimilation and sensing nutrient and energy [11–12]. Over-expressing of MhSnRK1 in Pingyitiancha (Malus hupehensis Rehd. var. pinyiensis Jiang) could regulate fruit development and increase the assimilation rate of both the carbon and nitrogen [11]. To date, increasing evidence have shown that SnRK2 and SnRK3 members contribute particular regulatory roles in hyperosmotic stress responses and ABA signaling. Among the ten SnRK2 genes identified in Arabidopsis, nine of them (except SnRK2.9) can be activated by hyperosmotic and salinity stresses, and five of the nine (SnRK2.2, SnRK2.3, SnRK2.6, SnRK2.7, and SnRK2.8) can be activated by ABA [6, 13]. AtSnRK2.6 is particularly activated by ABA and involved in the ABA-regulated gene expression [14–15]. Several SnRK3 members are well described. The best-studied member is SOS2 (salt overly sensitive 2), which is involved in responses to salt stress and ABA signaling and is required for Na⁺ and K⁺ homeostasis and abiotic stress tolerance [16–17].

Bread wheat (Triticum aestivum) is one of the world’s most important food crops, accounting for more than half of the total human food consumption [18]. The function of SnRK in wheat is also reflected in many fields. Transcription of TaSnRK1 increased wheat FHB (Fusarium Head Blight) resistance in coordination with the deoxynivalenol (DON) induced TaFROG (Triticum aestivum Fusarium Resistance Orphan Gene) expression [19]. Overexpression of TaSnRK2.8 enhanced cell membrane stability and enhanced tolerance to drought, salt, and cold stresses in Arabidopsis [20]. The SnRK3/CIPK protein kinase WPK4 positively regulates the transcription factor TabZIP2, which regulates the rearrangement of carbon and nutrient flows when wheat suffers drought [21]. Although several research on SnRKs in wheat had been reported [22–23], this protein family was never elucidated. In this study, the gene members belonging to the SnRK family in wheat (TaSnRK) and their potential roles under specific stress responses were characterized. A combination of both the bioinformatic and molecular research methodologies have allowed the member identification of the TaSnRK family whilst uncovering their expression patterns under specific responses under ABA, NaCl, PEG, powdery mildew, and Fusarium graminearum stresses. Our results has presented useful information for the understanding of TaSnRK family and provided clues for the future genetic improvement in wheat stress resistance.

Identification and phylogeny analysis of TaSnRK genes

After genomic retrieval by BLASTp with 38 AtSnRK and 47 OsSnRK proteins (Table S1), 1240 putative TaSnRK proteins (734 HC members and 506 LC members) were hunted [5, 24–25]. However, after validation by NCBI CDD and SMART online tools, only 186 protein sequences (transcribed from 149 genes), including splice variants (181 HC members and 5 LC members), were confirmed. The SnRK family which contained 21 SnRK1 proteins (20 HC members and 1 LC member), 38 SnRK2 proteins (38 HC members), and 127 SnRK3 proteins (123 HC members and 4 LC members) (Fig. 1; Table S2). Because the naming method of SnRK genes in wheat was not consistent currently, with several genes having several synonymous names, we renamed all genes according to their subfamily (SnRK1, SnRK2, and SnRK3) and sub-genome location (A, B, or D) (Table S2). Gene’s name started with an abbreviation for the acronym of species name Triticum aestivum (Ta), followed by subfamily (SnRK1, SnRK2, and SnRK3). Genes belonging to triads share the same gene number, but use suffixes A, B, and D to distinguish subgenomes they located. Consecutive minuscules separated by a semicolon distinguished splice variants (e.g., TaSnRK1.3-B;a and TaSnRK1.3-B;b) [26]. Phylogenetic trees generated by SnRK protein sequences of Arabidopsis, rice, and T. aestivum showed that TaSnRKs belong to well-defined subfamilies (Fig. 1). A previous study found that 94 proteins from 30 genes of the TaSnRK3 subfamily were identified [23]. This article matched all these genes except TaCIPK6 because we did not retrieve its gene or CDS (Coding Domain Sequence) sequence. The remaining 29 genes belonging to the SnRK3 subfamily were firstly identified in this paper (Fig. 1; Table S2).

Chromosomal localization, gene duplication, synteny and Ka/Ks analysis

TaSnRK genes are located on 21 chromosomes, except gene TaSnRK3.44-U in an unanchored contig (Fig. 2a; Table S2). And their chromosome distributions are uneven. There are eight, seven, and eight genes on the 1A, 1B, and 1D chromosomes, respectively, and three, one, and two genes on the 7A, 7B, and 7D chromosomes, respectively. That may be related to the different sizes and structure of the chromosomes. Overall, TaSnRK genes tended to located in the more central segments of the chromosomes (R2a, R2b, and C; 73.3% of genes) were more than genes in the distal telomeric parts of the chromosomes (R1 and R3; 26.7% of genes) (Fig. 2a) [26].

Compared with the number of SnRK genes in Arabidopsis (47) and rice (38), the number in wheat was much higher (149) (Fig. 3a-c). This partially due to the hexaploid nature of wheat. However, even when corrected for ploidy-level, the number of SnRK genes in bread wheat was significantly higher than in Arabidopsis and rice (1.71- and 1.38-fold higher, respectively). Specifically, genes in SnRK1 and SnRK3 subfamilies are largely more than expectation (Fig. 3a-d). To better understand why SnRK genes are so abundant in the wheat, we analyzed homoeologous groups in detail (Table 1). At the genome-wide level, 35.8% of wheat genes were present triads [26]. By contrast, the triad proportion of TaSnRK genes is as high as 84.86%. If only “HC” SnRK genes (HC defines genes with high confidence in the wheat genome) were considered, this ratio was even higher (85.42%, Table 1). Moreover, the loss of one homolog was less pronounced in SnRK genes (2.68% vs. 13.2%, Table 1). And only 19 genes were singletons (12.75%; 11.81% for HC only) because they could not be identified as groups. Therefore, the high homologous retention rate could partly explain the high number of TaSnRK genes. That is also evidence that genome-wide doubling events (WGD) contribute to the expansion of gene families.

Table 1

Groups of homoeologous SnRK genes in wheat.
Homoeoligous group (A:B:D)	HC-only¹	all wheat genes²	wheat SnRK (excluding splice variants, HC and LC)			wheat SnRK (excluding splice variants, HC-only)
Homoeoligous group (A:B:D)	HC-only¹	all wheat genes²	number of groups	number of genes	% genes	number of groups	number of genes	% genes
1:1:1	41.3%	35.8%	42⁵	126	84.6%	41	123	85.4%
n:1:1/1:n:1/1:1:n³		5.7%	/	/	/	/	/	/
1:1:0/1:0:1/0:1:1		13.2%	2⁶	4	2.7%	2	4	2.8%
orphans/singletons		37.2%	19	19	12.8%	17	17	11.8%
other ratios⁴		8.0%	/	/	/	/	/	/
total		99.9%		149	100.0%		144	100.0%
^1,2according to IWGSC (2018); ³ n > 1; ⁴ either n:1:n or 0:1:n, n > 1; ⁵ Compare to HC-only, one triads (TaSnRK3.30-A, TaSnRK3.30-B, TaSnRK3.41-D) was added; ⁶ Compare to HC-only, one triads (TaSnRK3.30-A, TaSnRK3.30-B) was changed to another triads (TaSnRK3.31-A, TaSnRK3.43-D)

Furthermore, to investigate the gene duplication events in wheat, tandem and segmental duplication were also analyzed. Through screening sequence’s similarity and matching rate, 49 paralogous pairs belonging to segmental copies were found, and there was no tandem repetition (triads were not considered) (Fig. 2b; Table S3). Combined with the phylogenetic tree (Fig. 1), we found that the segment duplications occurred within each subfamily (SnRK1, 17 pairs; SnRK2, three pairs; SnRK3, 29 pairs). Additionally, synteny analysis between wheat, Arabidopsis, and rice were also compared to explore the evolutionary constraints acting on the SnRK gene family (Fig. 3e). The results revealed that 15 (wheat-Arabidopsis; SnRK1, 0 pairs; SnRK2, two pairs; SnRK3, 13 pairs) and 159 (wheat-rice; SnRK1, nine pairs; SnRK2, 35 pairs; SnRK3, 115 pairs) orthologous pairs were detected, respectively (Fig. 3e; Table S4). That reflects the conservative evolution nature of the SnRK gene family in the plant.

Meanwhile, we calculated Ka (non-synonymous substitution rate), Ks (synonymous substitution rate), and the Ka/Ks ratio of each pair (triad pairs are taken into account) to estimate evolutionary rates and determine the relative divergence times [27–29]. Except that the Ka/Ks ratios of two gene pairs (TaSnRK3.27-B/OsSnRK3.26, TaSnRK3.27-D/OsSnRK3.26) were > 1, the remaining pairs were all less than 0.87, implying that the SnRK genes were under different natural selection during plant adaptation to terrestrial environments (Fig. 3f; Table S3-4). Also, these genes' duplication events were estimated to have occurred around ~ 10.05 MYA (million years ago; Ks values average at 0.13). In addition, the Ka/Ks ratios of the orthologous gene-pairs between wheat and other two model plants were also calculated (Fig. 3g; Table S3-4). The average Ka/Ks value was maximum between wheat and Arabidopsis (0.54), followed by rice (0.13), suggesting the genes pairs between wheat and those two species appeared to have undergone extensive, intense purifying selection. The divergence time was about 41.50 and 10 MYA for Arabidopsis and rice, respectively. On the whole, the differentiation of gene pairs is concentrated in the α WGD period, which flowering plants experienced together (Fig. 3g) [27].

Motif composition, exon-intron structure, protein feature and structure analysis

The conserved domain and gene structure analysis provide information regarding gene duplication and their functional conservation during evolution [30]. We analyzed TaSnRKs according to the following criteria: (1) one gene of the triads was retained, (2) only the first variant was kept, the 65 representative TaSnRKs were chosen (Fig. 4). First, we found six of 20 conserved motifs related to the functional domains in plant SnRK proteins (Fig. 4b; Fig. S1-2, Table S5). Motif 2 and motif 4 contained Ser/Thr active site and ATP-binding region, respectively. Motif 17, motif 15, and motif 8 contained specific conserved domain of SnRK1 (KA1), SnRK2 (glutamine-303 to proline-318), and SnRK3 (NAF), respectively. Besides, ABA-specific box hid in motif 18 (Fig. 4b; Fig. S1-2, Table S5). Overall, the same subfamily members shared similar motif compositions, supporting the clustering of the phylogenetic tree. Second, we found that all genes of the SnRK1 and SnRK2 subfamily had introns. However, there were 40% of SnRK3 genes without the intron. Furthermore, there were 28.6%, 5.3%, and 18.9% genes from SnRK1, SnRK2, and SnRK3 had UTR (untranslated region), respectively (Fig. 4c). We also noted that the exon number of TaSnRKs varied from 1 to 13. In short, the difference of gene and protein structure within three subfamilies illustrates the possibility related to the existence of gene subfunctionalization or neofunctionalization in the TaSnRK family.

To further understand the features of TaSnRKs, protein properties of 186 TaSnRK proteins were analyzed (Table S2). The length of TaSnRKs ranged from 103 (TaSnRK1.10-B) to 885 (TaSnRK3.32-B;c) amino acids (aa). The average molecular weight (MW) of SnRK1, SnRK2, and SnRK3 subgroup proteins were 53, 40, and 51 kDa, respectively. Meanwhile, the theoretical isoelectric point (pI) were ranged from 4.38 (TaSnRK2.8-D;a) to 9.51 (TaSnRK3.12-B and TaSnRK3.21-B). Compared to the other two subfamilies, the members of SnRK2 have lower MW values, and pIs are all less than 7 (Fig. 5a). All the TaSnRKs belonged to hydrophilic proteins due to their grand average of hydropathicity (GRAVY) values are negative. The secondary and tertiary structure analyses revealed the prominence of helices and loops in TaSnRK proteins (> 73%) (Fig. 5b; Table S2). In addition, SnRK1 proteins matched models 6b1u.2 and 6c9h.1, while SnRK2s matched three different models (3zuu.1, 3ujg.1, and 5wax.1). Members of the SnRK3 were matched to five models, two of which were shared with SnRK1s, and the remaining three were 5iso.1, 4czt.2, and 6c9d.1, respectively. Generally, protein properties were similar between the SnRK1s and SnRK3s, and SnRK2s were significantly different from them, which supports the functional differentiation of this gene family.

Promoter analysis

After analyzing the 1.5 kb upstream region of TaSnRK genes, we found that most of them have been broadly categorized into growth and development relate cis-acting elements. CAAT-box, a common element in promoter and enhancer regions that plays an essential role in transcription, is predominant among these genes (98.5%) [31]. Other cis-acting elements, such as A-box, AT-rich sequence, CAT-box, CCAAT-box, GCN4_motif, and O2-site, were also found in various genes and known for participating in multiple growth regulation processes (Fig. 6a; Fig. S3, Table S5) [31–33]. Furthermore, many identified elements are related to hormone signaling pathways, namely auxin (AuxRR-core and TGA-element), abscisic acid (ABRE), gibberellin (TATC-box, GARE-motif, and P-box), salicylic acid (TCA-element and SARE), and methyl jasmonate (CGTCA-motif and TGACG-motif) [31, 34]. In addition, a few elements were predicted to be involved in abiotic stresses, such as wounding (WUN-motif), cold (LTR), and light (Box 4, GATA-motif, G-Box, I-box, Sp1, and MRE) [31–34]. We also found that the number of cis-acting elements distributed in the TaSnRK genes varied from 2 (TaSnRK3.42-B and TaSnRK3.44-U) to 63 (TaSnRK3.11-A) (Fig. 6b). The different numbers and types of cis-elements presenting indicated the diversified regulatory networks that the TaSnRK genes may involve.

Expression patterns of tissue developmental stages and stress conditions

To determine TaSnRK genes’ expression patterns, we analyzed 209 RNA-seq samples under nonstress conditions of ‘Azhurnaya’ (a hexaploid wheat variety) (Fig. 7a; Fig. S4, Table S7-8) [26]. About 80.65% (genes) TaSnRKs were expressed at least one developmental stage with a broad expression range from 1 to 285 TPM (Transcripts Per Kilobase of exon model per Million mapped reads). The remaining 18.81% (35 genes) showed a deficient expression with a TPM < 1 and were considered not expressed (SnRK1s, six genes; SnRK2s, three genes; SnRK2s, 26 genes). TaSnRK genes were expressed in multiple tissues, including leaves, roots, sheaths, spikelets, anthers, and grains (Fig. 7a; Table S8), which was consistent with previous reports in Arabidopsis [4–5, 35], tomato [11], potato [36], pea [37], and maize [38–39]. To verify the expression patterns in wheat, we made hierarchical clustering according to expression similarity, and then group the data into 15 different expression modules (Fig. 7b; Fig. S4). The result showed that only four expression patterns in SnRK1, while the SnRK3 contained the most types of expression patterns (up to 12). Moreover, the expression modules I and V were the most widely distributed, with multiple gene anastomoses in all three subfamilies (Fig. 7b-c). In general, the genes of TaSnRK1s and TaSnRK2s were both ubiquitously expressed during the plant life cycle (Fig. 7c). In addition to being generally expressed (39/127, 30.7%), the TaSnRK3 genes were also explicitly highly expressed in reproductive organ spikelets (modules XIII (66/127, 60.0%) and XIV (2/127, 1.6%)). Interestingly, not or low-expressed genes (module VIII) were also concentrated in the SnRK3 subfamily (20/127, 15.7%). That may be related to a large number of genes and functional redundancy in this subfamily. The multi-tissue and multi-stage expression suggest the TaSnRK gene family's critical role in wheat growth and development.

To verify the response pattern of TaSnRKs to abiotic and biotic stresses in wheat, nine genes belonging to different subfamilies with universally expression patterns were randomly selected. Their expression patterns under various stresses were further illustrated by heatmap and analyzed by qRT-PCR.

At low temperature, TaSnRK2.4-B’ expression was decreased (Fig. 8a), which was similar to the expression pattern of ZmSnRK2.10 in maize [40]. In the SnRK3 subfamily, two genes were responsive to cold stress. One is TaSnRK3.35-A, whose expression was notably increased, may form a complex with CBL1 to regulate cold stress like homologous gene AtSnRK3.10 [41]. Another is TaSnRK3.16-D, which was drastically down-regulated than control, implying that TaSnRK3 genes participate in cold response in various ways. Also, TaSnRK1.1-A, TaSnRK2.4-B, and TaSnRK2.7-A responded to phosphorus deficiency only in the root. Under drought treatment (Fig. 8a), the expression of TaSnRK2.4-B, TaSnRK2.7-A, and TaSnRK3.35-A were increased significantly. In heat stress, except for TaSnRK3.37-D, the remaining eight genes all reached a peak at six hours. When subjected dual stress of drought and heat, TaSnRK2.7-A and TaSnRK3.37-D showed a gradual increase, and the remaining seven genes were down-regulated first and then up-regulated. In the qRT-PCR result (Fig. 8b), these nine genes also respond to drought stress. Under osmotic stress, the expression levels of TaSnRK1.2-A, TaSnRK2.4-B, TaSnRK2.10-A, TaSnRK3.16-D, TaSnRK3.35-A, and TaSnRK3.37-D changed significantly. For ABA treatment, each gene notably responded to the ABA signal but reached its maximum expression value at different times.

For biotic treatment, the response patterns of the nine genes were also varied (Fig. 8a). In the infection of Zymoseptoria triticipowdery in leaf, all TaSnRK genes showed down-regulated expression after 14 days post inoculation (dpi), showing the rife response pattern. Moreover, three different modes of expression were observed when threatened by stripe rust. First, the expression of TaSnRK1.2-A, TaSnRK3.35-A, and TaSnRK3.37-D was increased over time. Second, TaSnRK1.1-A, TaSnRK2.4-B, TaSnRK2.7-A, TaSnRK2.10-A, and TaSnRK3.16-A were decreased first and then increased gradually. Finally, the expression of TaSnRK1.9-A was lower and lower. When inoculated with powdery mildew, TaSnRK1.2-A, TaSnRK2.7-A, TaSnRK2.10-A, and TaSnRK3.16-A had significant positive responses to stress. In contrast, the expression of TaSnRK2.4-B decreased with the infestation time. Interestingly, in our qRT-PCR results (Fig. 8c), the genes that maintain continuous response to powdery fungus are TaSnRK1.1-A, TaSnRK3.16-A, and TaSnRK3.37-A. Similarly, through the RNA-seq data, we only found two genes that significantly responded to Fusarium graminearum, namely TaSnRK1.2-A and TaSnRK2.7-A. However, in our study, all nine genes were responded to Fusarium graminearum infection. The inconsistencies in response patterns may be related to the difference between cultivars and stress treatment time. In conclusion, wheat SnRK genes are involved in varieties of abiotic and biotic stress regulation.

Gene Ontology (GO), Kyoto Encyclopedia of Gene and Genome (KEGG) and Protein-protein interaction(PPI)network Analysis

To understand the functions of the biological processes, all TaSnRKs were searched for GO and KEGG databases. In total, 86.5% (129/149) TaSnRKs were assigned to one or more GO terms in the biological process (129 genes), molecular function (129 genes), and cellular component (102 genes) categories (Fig. 9a-b; Fig. S5, Table S10). We also mapped the TaSnRKs in the KEGG pathway database (Fig. 9b). The result revealed that most genes were enriched in signal transduction of environmental information processing pathways, supporting the previous studies [1–2, 4–5]. By grouping comparison, we found that the members from the TaSnRK1 and the TaSnRK3 subfamilies may perform similar functions on plant physiological regulation (Fig. S6). The reason is they shared eight identical pathways, except the pathways of “Folding, sorting and degradation” and “Membrane transport”, which were only annotated with the SnRK3 subfamily. By contrast, the TaSnRK2 members were only enriched on the “signal transduction pathway” of environmental information processing. That signals functional differentiation among the TaSnRK subfamilies and supports the SnRK genes regulation network drawn by Cramer et al. [42].

Furthermore, we used the model plant Arabidopsis protein database as a reference to constructed the interaction network of the three TaSnRK subfamilies, respectively. At first, 26 protein pairs were predicted with confidence to interact (score > 0.47) between 21 TaSnRK1s and 12 other proteins in the SnRK1 subfamily (Fig. 9c; Table S11). Among these protein pairs, AT4G16360, AKINBETA1, SEX4, and SNF4 code subunit β, and SNF4 code subunit γ, together with subunits α to form TaSnRK1 heterotrimers (Table S11) [7, 10]. And, TaSnRK1s maybe regulate sugar signals and control plant energy transfer through interacting with B3-domain transcription factor FUSCA3 (FUS3), which have been verified by experiments in Arabidopsis [35, 43]. Simultaneously, TaSnRK1s also bind to a myristoylated 2C-type protein phosphatase ABI1, which contributes to balance carbon and nitrogen and ABA-free signaling pathways [44]. Another PP2C protein that binds TaSnRK1s is PP2C74, which involved the early development in the plant as one of the substrates of AtNMT1 in Arabidopsis [45]. Secondly, 52 protein pairs associated with TaSnRK2s were identified. Of these, four pairs occurred within the TaSnRK2 subfamily, and 48 pairs were associated with 38 TaSnRK2s and other five functional proteins (Fig. 9c; Table S11). TaSnRK2s and their interaction proteins are all concentrated in the PYR/PRL-PP2C-SNRK-ABF signaling pathway [8, 13–15, 22, 24–25, 35, 43–44]. When ABA content is low, the activities of SnRK2s were inhibited by clade A PP2C phosphatases (ABI1, ABI2, and HAB1) [46–48]. After the concentration increases, ABA is bound to receptor PYR/PRL and PP2C phosphatases to form regulating complexes and release the inhibition of SnRK2s and downstream stress signaling [46–48]. Meanwhile, uninhibited SnRK2s [49–51] activate downstream bZIP-like transcription factor (ABF), induces ABA response gene expression, and regulates plant growth and development [52]. Ultimately, the signal transduction process is inhibited by SnRK-Calcium-binging Sensor (OZS1) and scaffold protein ABA Terminator (Fig. 9c, 10a) [53–54]. Finally, the SnRK3 subfamily has been shown to participate in complex networks of interactions. We identified 135 protein pairs involving 24 SnRK3 proteins and ten other proteins. Among them, 18.52% (25/135) of interactions occurred within the subfamily, while the remaining protein pairs (81.48%, 110/135) were concentrated in the calcium-dependent CBL-SnRK3 signaling pathway (Fig. 9c; Table S11) [1, 3, 9, 16–17, 23, 25, 41–42, 55–58]. We speculate that the complex of TaSnRK3s and CBL can regulate Arabidopsis K⁺ transporter (AKT1), which balances the absorption and release of K⁺ in root cells under low K⁺ concentration [55]. Meanwhile, TaSnRK3s may participate in the SOS (SOS1, SOS2, SOS3, SIP3, and SIP4) pathway to regulate the transport of Na⁺/H⁺ reverse transporters (NHX1) and NO₃^- (NRT1.1) to enhance tolerance to abiotic stress [16–17, 23, 56–60].

Path analysis, subcellular localization and transient Agro-Infiltration assays of TaSnRK2.4-B

In previous studies, TaSnRK2.7 (renamed as TaSnRK2.4-D in this study) responded to polyethylene glycol and NaCl stress, but not to ABA application [59]. However, TaSnRK2.4-B, the triplet of TaSnRK2.4-D, showed significant change under ABA stress (100 uM) (at 2 h, 12 h, and 24 h), which was at odds with previous research (Fig. 9b) [59]. To investigate whether TaSnRK2.4-B indeed responds to ABA signaling, we quantified the expression levels of several key genes involved in the ABA pathways concerning to SnRK2 subfamily (Fig. 10a; Fig. S7) [8, 22, 24, 44, 60]. On the one hand, TaPYR1 (homologous to AtPYR1) was weakly expressed after ABA treatment, and TaPYR4 (homologous to AtPYR4) and TaPP2C (homologous to AtPP2CA and AtABI2) was continuously decreased with stress time. Moreover, TaSnRK2.4-B and TaABF (homologous to AtABF) both increased and reached a peak at 12 h and then decreased continuously. On the other hand, in the SCS-SnRK2 signaling pathway [60], TaSCS (homologous to AtSCS-A and AtSCS-B) was highly expressed at all time points, especially at 12 h, whose expression level was 731 times compared to control. Furthermore, its expression trend is utterly consistent with TaSnRK2.4-B. That suggests that ABA signaling may cause an outbreak of ROS (Reactive Oxygen Species), which induce SCS overexpression in a calcium- or non-calcium- dependent manner [60]. After that, TaSCS may inhibit the expression of TaSnRK2.4-B by directly interacting with it, as similarly reported in Arabidopsis [60].

To validate the subcellular localization of TaSnRK2.4-B, we successfully constructed the part27-35S:TaSnRK2.4-B-GFP fusion expression vector into Agrobacterium GV3101 and transferred it to tobacco leaves. The results showed that TaSnRK2.4-B was located in the cell membrane, cytoplasm, and nucleus (Fig. 10b), which was consistent with the localization results of its homologous gene TaSnRKs [22, 60].

In this study, we have demonstrated that TaSnRK2.4-B responds to many biotic stresses, and we wanted to examine whether this response exists in Phytophthora infestans (P. infestans) challenge. Therefore, the mature protein-coding regions of TaSnRK2.4-B were cloned and transiently expressed in N. benthamiana using Agrobacterium-mediated expression followed by a P. infestans infection. After six days post inoculation (dpi), significantly smaller lesions were observed in areas expressing TaSnRK2.4-B compared to that of free GFP (Fig. 10c-d; P < 0.05), suggesting that the expression of TaSnRK2.4-B inhibited P. infestans invasion to the host. This inhibition is likely to enhance plants’ ability to resist stress by responding to signal transduction pathways of hormones such as salicylic acid or gibberellin [61–62]. However, the specific mechanism of action still needs further experimental investigation.

SnRK family was highly conserved in eukaryotes, working as a sensor in the cellular energy metabolism [10–12, 35, 43, 62–63]. PKABA1, the primary member of the wheat SnRK family, was discovered and cloned from ABA-induced wheat embryo cDNA [19]. After that, five PKABA1-like protein kinase genes (TaPK3, W55a, W55b, W55c, and TaSRK2C1), activated by multiple stresses related to ABA signals, were excavated from wheat [21, 64–66]. However, compared with Arabidopsis and rice, the research on SnRKs in wheat is limited. So far, there has been no genome-wide analysis of the SnRK family in wheat. We have thereby developed a comprehensively evolutionary analysis and functional classification of the three SnRK subfamilies in wheat.

Based on the BLAST and HMM algorithm, 149 non-redundant SnRK genes were identified at the whole genome level of wheat (Fig. 1; Table S2). The number of TaSnRKs was 3.92 and 3.17 times higher than in Arabidopsis and rice, which could generally be explained by its hexaploidy and fragment replication (Fig. 3a-d). The Ka/Ks ratio test explains that the wheat SnRK family has experienced positive selection, but displayed different evolutionary branch selection pressures (Fig. 3f; Table S2). Meanwhile, we calculated that 77.9% (116/149) SnRK members had collinear relationships with rice, and these pairs were all distributed in the three subfamilies (Fig. 3e; Table S4). More interestingly, the same TaSnRK gene co-linear with multiple rice genes and vice versa. However, only 10.1% (15/149) of the SnRK members had collinearity pairs with Arabidopsis (Fig. 3e; Table S4). In general, the rice SnRK genes showed a more intimate genetic relationship with wheat than Arabidopsis, consistent with the evolution tendency. Three branches of the SnRKs existent in Arabidopsis and rice (Fig. 3f), reflecting that the evolution of SnRK2 and SnRK3 from SnRK1 has already been completed before the arose of monocot and eudicot plants.

The protein property and structure of the wheat SnRK family were analyzed concerning the interrelationship with the protein function. It was found in our study that the N-terminus of the SnRK protein was more conservative in each subfamily in the full-length sequence alignment (Fig. 4; Fig. S2). By contrast, TaSnRKs shared a lower similarity in C-terminal sequences among the three subfamilies. The C-terminal of SnRK sequences generally includes KA1, SnRK2-specific box, and NAF domain [7–9], which is common in Arabidopsis [6, 10, 24], Oryza sativa [24], Zea mays [40], and Brachypodium distachyon [25]. However in wheat, motif 17 with the KA1 domain is distributed only in the SnRK1 subfamily, and motif 8 containing the NAF domain is distributed in the SnRK3 subfamily (Fig. 4; Fig. S2, Table S5). Motif 15 containing the SnRK2-specific box is conserved in the TaSnRK2 family and distributed in each member sequence. This result indicated that the wheat SnRK protein sequences were conserved in each subfamily. The tertiary structure prediction result further revealed the same subfamily members share similar protein models, suggesting that members of the same subfamily may also share similar functions (Fig. 5b). Nevertheless, the differences between the TaSnRK subfamilies cannot be ignored, which may relate to one or more domains at the C-terminal end.

At present, most of the SnRK proteins reported are found to be located in the cell membrane, cytoplasm, and nucleus as the regulation of SnRK kinase activity at different levels of transcription, post-transcription, protein translation, and post-translational modification [3, 10, 19, 46, 48, 52, 55, 58, 64]. For example, wheat SnRK2 member PKABA1 was not regulated by ABA in the protein level but was regulated by ABA in the mRNA leve1 [19]. This multilevel regulatory ability has determined that the SnRK family plays a vital role in the plant regulatory network as an essential protein kinase [4, 6–7, 43]. For a better understanding of the plant SnRK families, we drew a schematic diagram of the regulatory network of plant SnRK family (Fig. 10e) [1, 4, 6–7, 10–11, 24–25, 42, 46–47, 54, 60]. Not only the SnRKs has multiple roles in sugar signal transduction [10, 12, 43], ABA signal regulation [14–15, 37, 39, 43–44, 46], osmotic stress response [1, 6, 13, 16, 52], but also the stomatal closure [6, 13], abiotic stress [6, 67], growth and development regulation [11, 35–36] are involved.

In this research, we have identified multiple cis-acting elements that responded to wheat growth and development regulation, as well as the hormone and stress response in the promoter regions of the three TaSnRK subfamily members (Fig. 6). Simultaneously, TaSnRKs were annotated to the three types of GO items and enriched to multiple KEGG pathways (Fig. 9a-b). The nine genes focused on were ubiquitously expressed during the wheat life cycle, except for TaSnRK3.16-D, whose expression level was low in general, but relatively higher in the reproductive organs. Overall, the TaSnRKs may play a role in various aspects of vegetative growth and reproductive development, and is not just limited to regulating root development as previously reported [22, 59].

For biotic stress, all nine genes responded to the infection of Zymoseptoria triticipowdery and stripe rust, according to public microarray datasets. In the powdery mildew and Fusarium graminearum treatment, there were discrepancies showed between the public data and our qRT-PCR results, which may be possibly due to the difference in experimental materials. But generally, some members of the three TaSnRK subfamilies responded to the invasion of powdery mildew and Fusarium graminearum, which was consistent with the former reports to a certain extent [21, 62]. Still, further studies are needed for the verification.

Under the abiotic stress, TaSnRK2.4-B, TaSnRK3.16-D, and TaSnRK3.35-A both demonstrated the responses to cold stress. Except for TaSnRK3.37-D, the remaining eight genes were induced to express after high temperature treatment for 1 hour (Fig. 8a). Besides, these nine genes all responded to PEG, NaCl, ABA (100 uM), and phosphorus deficiency variously. In previous studies, the SnRK2 family was divided into three groups mainly based on the gene responding pattern to ABA signals (Fig. S8) [22, 24, 48, 59, 67]. SnRK2s in Group I cannot activated by ABA treatment and Group II SnRK2s responded weakly to ABA, whereas SnRK2s in Group III were strongly induced by the ABA signal. In 2011, Zhang et al. [59] identified a Group I gene, TaSnRK2.7 (renamed as TaSnRK2.4-D), that did not respond to ABA (5 uM). However, in our study, it was found the TaSnRK2.4-B, a triad gene of TaSnRK2.7, responded to ABA (100 uM) signal significantly (Fig. 8b). By adjusting the ABA application concentration in the experiment, we concluded that the Group I genes could be more sensitive to high concentration of ABA. Also, from the signaling pathway, TaSCS were significantly induced after the ABA application. We speculated that it might interact with and inhibit the expression of TaSnRK2.4-B through Ca²⁺ dependent or independent means (Fig. 10e) [60]. Furthermore, TaSnRK2.4-B was found to be located in both the cell nucleus, cytoplasm, and membrane, and its overexpression in tobacco has enhanced tolerance to P. infestans invasion (Fig. 10c-d). Consequently, it could be reasoned that TaSnRK2.4-B has the very potential to improve the crop tolerance to stresses, underlying a practical breeding utility in the wheat production.

Totally, we completed genome-wide identification and classification of the wheat SnRK family. Replication events analysis showed that both WGD and fragment duplication contributed to the expansion of the TaSnRK family. Ka/Ks value suggested that most TaSnRKs were undergoing positive selection and the pressure intensity varied among the subfamilies. The GO annotation, KEGG, PPI network, transcriptomic profiling, and qRT-PCR analyses indicated the TaSnRK family played the role of multifunctional regulators responding to diversified biotic and abiotic stresses. Subcellular localization showed the presence of TaSnRK2.4-B in the cell membrane, cytoplasm, and nucleus. Expression patterns revealed that TaSnRK2.4-B responds to PEG, NaCl, and high concentration ABA. Moreover, transient agro-infiltration assays showed that TaSnRK2.4-B was involved in the resistance to the pathogen. As a potential gene in crop tolerance, its function needs further study. Overall, our study brings new insight for transcriptional regulatory mechanisms of TaSnRKs and transgenic breeding in crop plants.

Identification and classification of TaSnRK protein sequences

The computer-based method was used to identify SnRK gene family members from the wheat reference genome IWGSC RefSeq v1.1 assembly (https://wheat-urgi.versailles.inra.fr/Seq-Repository/Assemblies) [30]. The known 38 AtSnRK proteins and 47 OsSnRK proteins sequences were collected and used as query sequences for BLASTp searching against the IWGSCv1.1 [5,24-25]. The first uncurated candidate hits were further screened using SMART (http://smart.embl-heidelberg.de/) and NCBI CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to predict domains which belong to SnRK gene family. All SnRK protein sequences contained the S_TKc domain (Serine/Threonine protein kinases, catalytic domain). Besides, SnRK1 subfamily contained KA1 (C-terminal kinase associated domain 1) [7], SnRK2 subfamily contained SnRK2-specific box (glutamine-303 to proline-318) [8], and SnRK3 subfamily contained NAF (Asn-Ala-Phe) [9]. According to these specific domains, TaSnRKs were identified and classified.

Phylogenetic analyses of the SnRK gene family

To further explore the evolutionary relationship of plant SnRKs, multiple sequence alignments and phylogenetic analysis of the SnRK proteins were performed with Clustalw [68]. And neighbor-Joining (NJ) was used to perform phylogenetic analyses of the SnRK proteins with 1000 replicated-bootstraps in MEGA 7.0 [69-70]. The midpoint rooted base tree was drawn and cleaned up via Interactive Tree of Life (ITOL, version 3.2.317, http://itol.embl.de).

Chromosomal location, gene duplication, and Ka/Ks analysis of TaSnRKs

The start and end location information of TaSnRKs extracting from the reference IWGSCv1.1 GFF3 file was used to draw the physical map via MapInspect software [31-32]. Tandem duplication events were identified using the following evaluation criteria: (1) length of the aligned sequence > 80% of the length of each sequence; (2) identity > 80%; (3) threshold ≤ 10⁻¹⁰; (4) only one duplication event could be admitted when genes were linked closely; (5) the intergenic distance was less than 25 kb. If genes satisfied criteria (1), (2), and (3) and located on different chromosomes, they were judged as segmental duplications [30-31]. The evolutionary rates (Ka, Ks, and Ka/Ksratio) were estimated by TBtools (https://github.com/CJ-Chen/TBtools) [71]. On the basis of the synonymous substitutions per year (λ) of 2.6 × 10^-9 for wheat, the divergent time of the duplicated SnRK genes was estimated (T = Ks/2λ × 10^-6MYA) [72].

Protein sequences analysis

The characteristic features of TaSnRK proteins were predicted using ExPASy Server10 (SIB Bioinformatics Resource Portal, https://prosite.expasy.org/PS50011). Signal peptide lengths were predicted with SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/), and Plant-mPLoc executed the prediction of subcellular localization (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/). The SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) was used to predicted the secondary and three-dimensionally structure by the intensive mode. Structure validation was performed using Ramachandran plots (http://www.mordred.bioc.cam.ac.uk/rapper/rampage.php/). The MEME v4.9.1 (http://meme-suite.org/index.html) and SMART motif search tool (http://smart.embl-heidelberg.de/) were used to identify conserved motifs of TaSnRK proteins. These motif patterns were drawn by TBtools software [71]. The GSDS2.0 (http://gsds.cbi.pku.edu.cn/index.php) was used to draw gene exon-intron structures.

Cis-elements analysis and expression Pattern Analyse

We are using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) identify cis-acting elements in the upstream sequences (1-1.5 kb) of the 186 TaSnRKs. Then, the predicted results were organized and displayed by the R package “pheatmap” [30]. RNA-seq data of wheat SnRK genes were downloaded from Wheat Expression Browser (http://www.wheat-expression.com/ and http://bar.utoronto.ca/efp_wheat/). The MORPHEUS (https://software.broadinstitute.org/morpheus/) was used to generate heatmaps of TaSnRKs fold change based on the log2 (TPM+1) values. Genes were clustered according to their expression using K-means (K-means = 15) [73].

GO annotation, KEGG annotation, and PPI network prediction

GO annotations of TaSnRK proteins was performed by Blast2GO [74]. KEGG pathways analysis of TaSnRKs was performed using the KEGG web server (http://www.kegg.jp/). The PPI network was constructed using the STRING database (https://string-db.org/), and the genes with confidence score ≥ 0.4 were reserved.

Plant materials, treatments, and qRT-PCR analysis

Seeds of Yangmai 20, a hexaploid common wheat cultivar, were surface-sterilized with 1% hydrogen peroxide, rinsed thoroughly with distilled water, and germinated in an incubator at 20 ℃ for two days then placed in 1/2 strength Hoagland nutrient solution [75]. When wheat grew to one heart and one leaf, the plants were treated with 150 mM sodium chloride (NaCl), 20% polyethylene glycol 6000 (PEG 6000), and 100 uM ABA. The plants grow at 26 ℃ and 16 h/8 h (day/night) environment during application. Roots were sampled at two, 12, 24, 48, and 72 h after treatments.

The Yangmai 20 plants were cultivated in soil in a growth chamber at 20 ℃ under a 16 h light/8 h dark photoperiod. When wheat seedlings’coleoptiles reached 0.5 cm, they were point inoculated with either water (control) or Fusarium graminearum (strain PH-1). Plant growth, inoculation and infection conditions were described in detail previously [76]. The Blumeria graminis f. sp. Tritici (Bgt strain E09) was maintained on susceptible wheat ‘MingXian 169’ [77]. The 7-day-old seedlings were inoculated with Bgt conidia from ‘MingXian 169’ seedlings infected 10 days previously. The inoculated leaves of Yangmai 20 were harvested at one, three and five dpi, frozen immediately in liquid nitrogen and stored at −80 °C. The test was carried out with three biological replications.

Total RNA was isolated from 100 mg tissues by using TRizol reagent (Invitrogen, U.S.A) and digested with DNaseI (TaKaRa, U.S.A) to eliminate DNA. RNA was reverse-transcribed to cDNA by using RevertAid Reverse Transcriptase (Vazyme, China). Quantitative expression was performed with a qRT-PCR system (BIO-RAD, U.S.A.). Gene-specific primers were designed using Primer 5.0. Primers that met the two conditions, (1) containing single and correct size segments in ordinary PCR products, (2) showing single melt peak of qRT-PCR, were chosen to be used in the further experiment. The qRT-PCR reaction system and protocol were carried out as manufacturer's instructions of 2× SYBR Green qPCR Master Mix (Us everbright, China). Three technical replicates were used for each cDNA. The relative gene expression level was calculated with the 2^−△△Ct method.

Subcellular localization and transient Agro-Infiltration analysis of TaSnRK2.4-B

The full-length open reading frame (ORF) of TaSnRK2.4-B were fused upstream of GFP in the PART27 expression vector driven by the CaMV35S promoter. The recombinant vector were transformed into Agrobacterium tumefaciens strain GV3101 and then infected Nicotiana benthamiana leaves. After 48 hours, the injected leaves were placed on the glass slides and imaged by laser scanning confocal microscopy (OLYMPUS FV3000, Tokyo, Japan). The exciting light wavelength was 488 nm.

Agro-infiltration experiments were implemented on leaves of 6- to 8-week-old N. benthamiana. After 24 h, infiltrated N. benthamiana leaves were excised and transferred into sealed moist plastic trays and inoculated with 10 µl P. infestans strains (88069) zoospore suspension (100 zoospores/µl) at the infiltration range [78]. Photographing under ultraviolet light and recording lesion diameters of the inoculated leaves at six dpi. Assay consisted of three biological replications, and each biological replication contained ten technical duplications.

SnRK: Sucrose non-fermenting-1-related protein kinase; Ta: Triticum aestivum; qRT-PCR: quantitative real-time quantitative PCR; PEG: polyethylene glycol; ABA: abscisic acid; S_TKc domain: Serine/Threonine protein kinases; KA1 domain: C-terminal kinase associated domain 1; CIPK: calcineurin B-like calcium sensor-interacting protein kinases; FHB: Fusarium Head Blight; DON: deoxynivalenol; TaFROG: Triticum aestivum Fusarium Resistance Orphan Gene; CDS: Coding Domain Sequence; WGD: genome-wide doubling events; Ka: non-synonymous substitution rate; Ks: synonymous substitution rate; aa: amino acids; MW: molecular weight; pI: theoretical isoelectric point; GRAVY: grand average of hydropathicity; TPM: Transcripts Per Kilobase of exon model per Million mapped reads; dpi: days post inoculation; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; PPI: Protein-protein interaction; ROS: Reactive Oxygen Species; P. infestans: Phytophthora infestans; ORF: full-length open reading frame.

Acknowledgements

We thank the Instrument sharing platform of Yangtzeu University and lab members for their assistance in this study.

Authors’ contributions

Conceptualization, Z.W. Fang and D.F. Ma; methodology, J.H. Song and Y.Q. He; validation, D.F. Ma and J.L. Yin; formal analysis and investigation, W.D. Huang and Z.H. Hou; resources, Z.W. Fang and D.F. Ma; data curation, J.H. Song; writing original draft preparation, Y.Q. He and J.H. Song; writing—review and editing, J.L. Yin and Y.K. Liu; visualization, J.H. Song; supervision, Z.W. Fang; funding acquisition, Z.W. Fang and D.F. Ma.

Funding

This research was funded by “National Key R&D Program of China (2017YFD0100800)”, “The Major Program of Technological Innovation of Hubei Province (2018ABA085)”, and “Open Program of Hubei Key Laboratory of Food Crop Germplasm and Genetic Improvement ”(2018lzjj09).

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹ College of Agriculture, Yangtze University, Jingzhou 434025, Hubei, China. ²Institute of Food Crops, Hubei Academy of Agricultural Sciences, Wuhan 430064, Hubei, China.

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Mining the Roles of Wheat (Triticum Aestivum) SnRK Gene Family in Biotic and Abiotic Responses

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Abstract

Figures

Background

Results

Identification and phylogeny analysis of TaSnRK genes

Chromosomal localization, gene duplication, synteny and Ka/Ks analysis

Motif composition, exon-intron structure, protein feature and structure analysis

Promoter analysis

Expression patterns of tissue developmental stages and stress conditions

Gene Ontology (GO), Kyoto Encyclopedia of Gene and Genome (KEGG) and Protein-protein interaction(PPI)network Analysis

Path analysis, subcellular localization and transient Agro-Infiltration assays of TaSnRK2.4-B

Discussion

Conclusions

Methods

Identification and classification of TaSnRK protein sequences

Phylogenetic analyses of the SnRK gene family

Chromosomal location, gene duplication, and Ka/Ks analysis of TaSnRKs

Protein sequences analysis

Cis-elements analysis and expression Pattern Analyse

GO annotation, KEGG annotation, and PPI network prediction

Plant materials, treatments, and qRT-PCR analysis

Subcellular localization and transient Agro-Infiltration analysis of TaSnRK2.4-B

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Acknowledgements

Authors’ contributions

Funding

Availability of data and materials

Ethics approval and consent to participate

Consent for publication

Competing interests

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References

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