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Article

Genome-Wide Identification and Expression Analysis of SnRK Gene Family under Abiotic Stress in Cucumber (Cucumis sativus L.)

by
Yanyan Luo
,
Yuan Niu
,
Rong Gao
,
Chunlei Wang
and
Weibiao Liao
*
College of Horticulture, Gansu Agricultural University, Lanzhou 730070, China
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(7), 1550; https://doi.org/10.3390/agronomy12071550
Submission received: 18 April 2022 / Revised: 17 June 2022 / Accepted: 26 June 2022 / Published: 28 June 2022
(This article belongs to the Special Issue Breeding, Genetics, and Genomic of the Genus Cucumis)
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Abstract

:
Sucrose non-fermenting 1-related protein kinases (SnRKs) are a kind of plant-specific serine/threonine (Ser/Thr) protein kinase, which play an important role in plant stress resistance. However, the scale analysis of SnRK in the cucumber genome is currently unclear. In the study, a total of 30 CsSnRK genes were identified from genomic data. They were distributed on six chromosomes, including 1 CsSnRK1, 10 CsSnRK2s and 19 CsSnRK3s. According to the analysis of gene structure and motif composition, CsSnRKs showed obvious differences among the three subfamilies. The ratio of synonymous (Ks) and nonsynonymous (Ka) nucleotide substitutions (Ka/Ks) of three paralogues indicates that the CsSnRK gene family undergoes a purifying selection. The analysis of cis-acting elements shows that the promoter region of each CsSnRK gene contained different classes of hormone and stress-related cis-acting elements. Furthermore, based on RNA-sequencing data from the Short Read Archive (SRA) database of NCBI, the expression patterns of CsSnRK genes in six tissues were investigated, indicating that the expression of multiple CsSnRK genes was prevalent in these tissues. Transcription levels of CsSnRK genes after drought, methyl jasmonate (MeJA) and abscisic acid (ABA) treatments were analyzed by quantitative RT-PCR, and the results show that most of the CsSnRK genes responded to these stresses. However, under different treatments, individual genes played a major role. For example, under ABA treatment, CsSnRK2.2 and CsSnRK2.3 played a major role in the response to ABA. These results provide clear evidence that CsSnRKs may be involved in cucumber growth, development and stress response, and provide valuable information for future functional studies of CsSnRKs.

1. Introduction

Plants are constantly exposed to various unfavorable conditions in the natural environment, including salt, drought and low temperature stresses [1]. Plants form a variety of molecular defense mechanisms to deal with abiotic stresses, which helps plant to maintain optimal growth and development conditions [2]. Protein kinases and phosphatases are the main components of intracellular signal transduction and play an important role in stress response [3]. In recent years, protein kinases, such as calcium-dependent protein kinases (CDPK) [4], mitogen-activated protein kinase (MAPK) [5] and sucrose non-fermenting 1 related protein kinases (SnRKs) [6,7,8], have been extensively researched. Among them, SnRKs play important roles in regulating metabolism. In plants, SnRKs proteins can be divided into three subfamilies according to their sequence and structure, including SnRK1, SnRK2 and SnRK3 [9]. SnRKs are highly conserved in the organism and not only participate in plant metabolism regulation, but also in stress response [10,11,12]. All members of the SnRKs gene family have similar lengths and sequences and similar kinase domains. The kinase domain is located near the N-terminus, the middle is the junction domain and the C-terminus is the regulatory domain [9]. SnRKs is a key kinase in plant sugar signal transduction, stress response, seedling growth and seed germination and other biological processes. SnRK genes induced by drought, salt and cold stress are also activated by abscisic acid (ABA).
SnRK1 exists not only in plants, but also in yeast and mammals. Its subfamily has a highly conserved catalytic domain, which is a homolog of SNF1 in yeast and AMPKs in mammals [10]. SnRK1 is first found in rye (Secale graine L.) with a total length of 502aa [13]. Study has shown that SnRK1 is related to sugar and ABA signaling pathways and plays a central role in metabolic regulation [14]. The decreased expression of SnRK1 increases sucrose accumulation and reduces maturity in pea seeds [15]. SnRK1 also regulates gene activity and causes the change of gene expression to nutrient starvation [16]. Plants also contain two other SnRK protease subfamilies, SnRK2 and SnRK3, which also contain catalytic domains, but these two subfamilies are far less similar to SNF1 and AMPK than SnRK1 9. The SnRK2 and SnRK3 subfamily are unique in plants and are more diverse than the SnRK1 subfamily. SnRK2 subfamily members have kinase domain, ATP binding domain, serine/threonine active site and four N-fourteen sites [17]. SnRK2 mainly participates in the signal transduction process of plant abiotic stress, and SnRK2 kinase plays a role in an ABA-dependent or independent manner in plants [18]. In Arabidopsis, high osmotic pressure and salt stress can induce the expression of 8 SnRK2 genes, and ABA can activate the expression of 5 SnRK2 genes [19]. The SnRK2 subfamily also plays a key role in the regulation of gene expression by activating the basic region leucine zipper (bZIP) transcription factor. The bZIP transcription factor is related to the epigenetic mechanism that controls gene activation or suppression [20]. SnRK3 kinases are named CIPKs (CBL-interacting protein kinases), which interact with the calcium sensor calcineurin b-like protein (CBLs) to mediate the calcium signaling pathway. In general, CIPKs are composed of the Pkinase domain conserved at the n-terminal, the NAF domain and the PPI domain of the C-terminal regulatory region [21]. The first SnRK3 gene identified is also from wheat and is originally named wpk4. The SnRK3 gene family is even larger than the SnRK2 family 9. SnRK3 protein can interact with CBL protein to form a calcium signal complex, which plays an important role in responding to various stresses such as salt, drought, cold and ABA [22]. For example, the overexpression of BnCBL1-BnCIPK6 can enhance high salt tolerance and low potassium tolerance in Brassica napus [23]. Thus, more and more evidence indicates the importance of SnRKs function in nutrient utilization and stress response, but the specific function may vary with different plant species. Therefore, it is necessary to study its role in different types of plants.
Cucumber (Cucumis sativus L.) is a widely cultivated crop species and occupies an extremely important economic position in China’s agricultural production [24]. It belongs to the Cucurbitaceae family. The function of the SnRKs family has been widely studied in various plants. However, genome-wide information on members of the CsSnRKs family has not been reported. In the light of the important role of SnRKs in regulating metabolism and stress signals, we identified SnRK members in cucumber. A total of 30 CsSnRK members were identified and then their physicochemical properties, evolutionary relationship, gene structure, chromosomal location and cis-acting element distributions of SnRK genes in cucumber were analyzed. Members of the SnRK2 subfamily are major players in plant responses to osmotic stress and abscisic acid (ABA)-dependent developmental processes [25]. The best known signaling in which SnRK3s are involved is the Salt Overly Sensitive (SOS) pathway, which regulates plant salt tolerance [26]. More importantly, through cis-acting elements analysis, we found that cucumber SnRKs mainly were enriched in drought stress response, so CsSnRK2s were chosen to analyze their expression pattern. We determined the tissue-specific expression patterns, and the expression patterns under drought stress and hormonal conditions. Therefore, our results provide important information on the CsSnRK genes family and lay the foundation for the study on the abiotic stress tolerance in cucumber.

2. Materials and Methods

2.1. Identification and Phylogenetic Analysis of the CsSnRK Gene Family in Cucumber

The genome and protein sequence information file of Arabidopsis and cucumber were obtained from the National Center for Biotechnology Information (NCBI). Based on the third-generation high-throughput sequencing genome of cucumber, the protein sequences of three subgroup members in the Arabidopsis SnRK family were used as probes. Local BLASTP (E-value-20) searches were performed based on Hidden Markov Model (HMM) profiles of SnRK gene domains from the Pfam database (http://pfam.janelia.org/; accessed on 4 January 2022). Redundant and incomplete sequences were removed, and the remaining sequences containing the protein kinase domain (PF00069) were considered as candidate SnRK members. The candidate genes were obtained and used to construct a phylogenetic tree with SnRK of Arabidopsis and rice. The candidate genes clustered with Arabidopsis and rice SnRK were identified as putative CsSnRK. To further ensure the existence of SnRK conserved structures, we used the online software SMART (http://smart.embl-heidelberg.de/; accessed on 4 January 2022), HMMER (https://www.ebi.ac.uk/Tools/hmmer/, accessed on 4 January 2022) and Pfam (http://pfam.xfam.org/; accessed on 4 January 2022) on the structure of candidate members (Pkinase/PF00069 and Pkinase_Tyr/PF07714) for reconfirmation. The members that contain Pkinas (PF00069), UBA (PF00627) and KA1 (PF02149) domains were grouped into subgroup I, and Pkinase (the members of the PF00069) domain were classified into subgroup II, and the members containing both the Pkinase (PF00069) and NAF (PF03822) domains were classified into the third subgroup. Finally, we obtained the members of the cucumber SnRK genes family, and used ExPASy (https://web.expasy.org/protparam/, accessed on 11 January 2022) to analyze the physical and chemical properties of these genes. The subcellular localization of CsSnRK protein was predicted using the online software PlantmPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/; accessed on 11 January 2022).
The ClustalX software was used for multiple sequence alignment of 30 CsSnRK proteins [27]. Based on the alignments, the phylogenetic tree of CsSnRK between cucumber, Arabidopsis, rice and barley was constructed using MEGA 11 software [28] with the neighbor-joining method with 1000 replicates of bootstrap values [29]. The EvolView tool (http://www.evolgenius.info, accessed on 12 January 2022) was used to draw the phylogenetic tree.

2.2. Gene Structure and Protein Motif Analysis

The online Gene Structure Display Server (GSDS; http://gsds.cbi.pku.edu.cn; accessed on 11 January 2022) was used to assess the distribution of introns and exons and intron phase patterns [30]. The online software Multiple Expectation Maximization for Motif Elicitation (MEME; http://meme-suite.org/tools/meme; accessed on 11 January 2022) was used to analyze the conserved motifs. The optimized parameters were as follows: number of repetitions, any; maximum number of motifs, 20; and optimum width of each motif, between 6 and 100 residues.

2.3. Chromosomal Location and Gene Duplication Analyses

The chromosomal location of the CsSnRK genes information was extracted by BLAST (Basic Local Alignment Search Tool; https://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 8 January 2022), and the distribution of the geneson chromosomes was drawn using the MG2C v.2 program (http://mg2c.iask.in/mg2c_v2.0/; accessed on 8 January 2022). Gene duplication, including segmental and tandem duplication, was analyzed using the MCScanX program [31]. Tandem duplication events were defined as ≥2 adjacent genes on the same chromosome. A segmental duplication event was defined as duplicate pairs located on different chromosomes [32]. The ratio of synonymous (Ks) and nonsynonymous (Ka) nucleotide substitutions between paralogues gene pairs was calculated by using the KaKs Calculator 2.0.

2.4. Cis-Acting Elements Analysis CsSnRK Genes

In order to analyze the cis-acting elements of CsSnRK family, the up-stream 2000 bp DNA sequences of CsSnRK genes were submitted to PlantCARE Database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/; accessed on 13 January 2022). Excel was used to count the number of the cis-acting elements and made the stacked chart.

2.5. Expression Analysis of CsSnRK2 Genes in Different Plant Tissues

The expression data of cucumber CsSnRK2 genes in six different tissues was obtained from the Short Read Archive (SRA) database of NCBI (accession number SRP071224). The differential expression analysis of CsSnRK2 subfamily was performed according to the method of Wei et al. [33]. Gene expression levels were calculated with Log (RPKM + 1). The heat map of the expression profile CsSnRK2s was created by TBtools software.

2.6. Expression Analysis of CsSnRK Genes under Different Abiotic Stresses

2.6.1. Plant Materials and Treatments

Cucumber seeds (“China Long” inbred line 9930) were provided by Shenzhen Institute of Genomics, Chinese Academy of Agricultural Sciences. We soaked the seeds in water at 55 °C for 15 min, then placed them on damp filter paper and germinated overnight in a 25 °C incubator. After the cotyledons were fully expanded, they were cultivated in a hydroponic box. The environment of the growth room was controlled to have a photoperiod of 14/10 h (light/dark), an air temperature of 28/18 °C (day/night), and a light intensity of 200 µmol∙m−2∙s−1. The nutrient solution was changed every three days. As for ABA, methyl jasmonate (MeJA) and drought stress treatments, the selected seedlings were transferred to nutrient solution containing 15% (w/v) PEG6000 [34], 100 µM MeJA or 100 µM ABA [35]. Plant of the control group was grown in the nutrient solution without adding other reagents. All the seedlings were grown in the same conditions of a growth chamber. Then leaves and roots were collected at 0, 3, 12, 24 and 48 h and immediately frozen in liquid nitrogen. Finally, samples were stored at −80 °C for qRT-PCR experiment [36]. There were three biological replicates per treatment.
Meanwhile, the roots, stems, young leaves, old leaves, female flowers and male flowers of untreated seedlings were collected for CsSnRK2 genes expression analysis. There were three biological replications collected under the identical experimental condition. Then the collections were frozen with liquid nitrogen and stored at −80 °C.

2.6.2. RNA Extraction and Quantitative RT-PCR

Total RNA was isolated from cucumber leaves with TRIzol reagent. Then, according to the manufacturer’s instructions, total RNA was reverse-transcribed to cDNA using cDNA Synthesis Kit (Tiangen, Beijing, China). The primers were designed with Primer 5 and their specificities were confirmed by a BLAST search. The cucumber ACTIN gene [37] was used to normalize relative expression levels, as shown in Table S1. RT-PCR was performed using the SuperReal PreMix Plus kit (Tiangen, Beijing, China) and Roche LightCycler instrument. There were three biological replicates per gene. The 2−∆∆Ct method [38] was used to analyze the relative expression level of each CsSnRK gene.

3. Results

3.1. Identification of CsSnRK Gene Family in Cucumber

Using a Hidden Markov model, according to the sequence alignment of Arabidopsis and rice, a total of 30 CsSnRK members were finally identified from the cucumber genome database, including 1 CsSnRK1, 10 CsSnRK2s and 19 CsSnRK3s. According to the conserved domains and nomenclature in Arabidopsis, they were named CsSnRK1.1, CsSnRK2.1 to CsSnRK2.10 and CsSnRK3.1 to CsSnRK3.19. The physical parameters of these genes were summarized in Table 1. 30 CsSnRK members encoded 340 to 673 amino acids, with a molecular weight (MW) of 38.38 to 77.01 kDa. The isoelectric point (pI) of CsSnRKs was from 4.44 to 9.64. The predicted subcellular localization showed that CsSnRK1.1 and CsSnRK2s were localized to the cytoplasm and nucleus, whereas most CsSnRK3s were localized to cytoplasm or chloroplast. Among them, only CsSnRK3.15 was at the nucleus.

3.2. Phylogeny Analysis of CsSnRK Gene Family

We constructed a phylogenetic tree using the amino acid sequences of Arabidopsis (39), rice (50), barley (50) and cucumber (30) in order to analyze their evolutionary relationships (Figure 1). Based on the phylogenetic tree, all SnRK proteins were divided into three groups: SnRK1, SnRK2, SnRK3. In details, the SnRK1 subfamily had the fewest members and all family members contained Pkinase (PF00069 of Pfam), UBA (PF00627) and KA1 (PF02149) domains, while SnRK2 proteins contained pkinase domain. The SnRK3 subfamily had the largest number of members, and all family members contain Pkinase and NAF (PF03822) domains. The assignation of each cucumber SnRK to the groups confirmed the Pfams analysis. The study found that most of the CsSnRK proteins had high homology with Arabidopsis. Furthermore, some SnRK members within species clustered together, implying expansion after speciation.

3.3. Gene Structure and Conserved Motifs Analysis of CsSnRK Gene Family

We analyzed 10 conserved motifs of 30 CsSnRK proteins using an online MEME program. In this study, except for CsSnRK3.2, which lacked motif 3, all other genes contained 3 motifs (1, 2, 3) (Figure 2B). In detail, the motif of the CsSnRK1 subfamily was 1, 2, 3, 5, 6 and 9; CsSnRK2 subfamily had motif 1, 2, 3, 4 and 7; the motif of the CsSnRK3 subfamily was 1, 2, 3, 5, 6, 8, 9 and 10, except that the motif of CsSnRK3.2 was 1, 2, 5, 8, 9 and 10. Taken together, CsSnRK proteins had a conserved motif composition and similar gene structure within the same subfamily, which strongly supported the reliability of phylogenetic analysis to classify this subfamily.
To further understand the gene structure, we combined phylogenetic tree and the Gene Structure Display server (GSDS) analysis to determine the intron/exon structure of the CsSnRK genes (Figure 2A,C). CsSnRK1.1 had 10 exons, while CsSnRK2 subfamily had 9–18 exons. However, the number of exons in the CsSnRK3 subfamily varies. The 10 CsSnRK3 genes contained 1 exon, and the 9 CsSnRK3 genes contained 12 to 20 exons. Therefore, CsSnRK3 genes can be divided into two subgroups, exon-rich and exon-poor subgroups, based on the number of exons. CsSnRK genes exhibited similar exon-intron structures in each subfamily, confirming their close evolutionary relationship and subfamily classification.

3.4. Chromosomal Location and Gene Duplication Analyses

In order to clarify the distribution of CsSnRK genes on the chromosomes, we used the cucumber genome annotation information and TBtools software to visualize the distribution of chromosomes (Figure 3). Among 30 CsSnRK genes, the 29 CsSnRK genes were distributed on 6 out of 7 chromosomes and the number of genes on each chromosome was irrelevant of the size of the chromosome. CsSnRK2.9 gene, which could not be conclusively mapped to any chromosome, was mapped to scaffold94. Chromosome 6 (Chr 6) contained the largest number of cucumber genes (8 CsSnRK genes) whereas Chr7 contained only one. No CsSnRK genes were detected on Chr5. The CsSnRK2 genes were mainly distributed on Chr4, while the CsSnRK3 genes were mainly distributed on Chr 6. CsSnRK1.1 was distributed on Chr 6.
To identify duplications of the CsSnRK genes, we analyzed the segmental duplications and tandem duplications of the cucumber genome during evolution. As shown in Figure 4, there were three segmental duplication events (CsSnRK3.1/CsSnRK3.12, CsSnRK3.3/CsSnRK3.17 and CsSnRK3.6/CsSnRK3.9) between different chromosomes. Simultaneously, the Ka/Ks ratio between these duplicated gene pairs were calculated. The Ka/Ks values of the three gene paralogs were less than1 (Table 2), which indicates that they had undergone purifying selection during evolution. We also calculated the KaKs values of the segmental duplicated CsSnRK genes in other species of Cucurbitaceae (Table S2). The Ka/Ks values were less than 1, which indicates that the similar segmental duplication happened in other closely related cucumber species.

3.5. Cis-Acting Regulatory Elements Analysis in the Promoter Region of CsSnRK Genes

To investigate the potential functions and regulatory mechanisms of CsSnRK genes, we analyzed cis-acting regulatory elements in the 2000 bp promoter regions using the Plant CARE database. The results show that cis-acting regulatory elements were mainly divided into two categories: hormone-responsive and abiotic stress-responsive (Figure 5 and Table S2). Among them, hormone response elements included ABRE, TGA-element, AuxRR-core, P-box, ERE, TCA-element and CGTCA-motif. Almost all CsSnRK genes contained the ABA-responsive element (ABRE) and the ethylene-responsive element (ERE), indicating that it may be sensitive to hormones. Abiotic stress response elements included MBS, MYC and ARE. MYC was a cis-expression element that regulated drought stress and was contained in all genes, indicating that CsSnRKs played an important regulatory role in drought stress.

3.6. Expression Profiles Analysis of CsSnRK2 Genes

3.6.1. Expression Patterns of CsSnRK2 Gene Family in Different Tissues

To understand the roles of CsSnRK2 subfamily members in osmotic stress and hormone response, we analyzed the expression of CsSnRK2 genes in different tissues (leaf, root, stem, and flower) (Figure 6). The expression level was based on the RNA sequencing data from SRA database of NCBI (accession number SRP071224) (Figure 6A). CsSnRK2.2, CsSnRK2.3, CsSnRK2.4, CsSnRK2.8, CsSnRK2.9 and CsSnRK2.10 showed high expression in all tissues, whereas the expression of CsSnRK2.5 and CsSnRK2.6 were very low in roots, stems and leaves. CsSnRK2.7 was specifically expressed in roots at very low levels. More significantly, CsSnRK2.1 was not expressed in roots and stems. CsSnRK2.3 and CsSnRK2.4 were highly expressed in female and male flowers. Meanwhile, CsSnRK2.5, CsSnRK2.6 and CsSnRK2.7 were not expressed in male flower tissues. These results suggest that CsSnRK2 genes displayed diverse expression patterns and CsSnRK2 genes might play special roles in different growth stages and different plant organs. To validate the RNA-seq data, qRT-PCR (quantitative real-time polymerase chain reaction) was performed. Total RNA from different tissues collected at developmental stage (Figure 6B), was used to measure the transcription levels of CsSnRK2 in different tissues. The qRT-PCR data were consistent with the RNA-seq data. The close correlation of these results indicated that the RNA-seq results were accurate in representing the gene expression levels.
To validate the RNA-seq data, qRT-PCR was performed. Total RNA from different tissues collected at developmental stage (Figure 6B), was used to measure the transcription levels of CsSnRK2 in different tissues. The qRT-PCR data was consistent with the RNA-seq data. The close correlation of these results indicated that the RNA-seq results were accurate in representing the gene expression levels.

3.6.2. Expression Profiles of CsSnRK2 Gene Family under Different Abiotic Stresses

SnRK2s was confirmed to participate widely in response to various environmental elicitors. To investigate the potential function of CsSnRK2s at seedling stage in response to exogenous factors, the expression patterns of 10 CsSnRK2 genes in roots and leaves under PEG, ABA and MeJA treatments were examined using qRT-PCR assays (Figure 7 and Figure 8). In leaves (Figure 7), except for CsSnRK2.7, all CsSnRK2 genes could be induced by various abiotic stresses. All genes were up-regulated after ABA treatment and reached the highest value after 24 h of treatment. Among them, the expression level of CsSnRK2.2 and CsSnRK2.3 had the most significant change. After PEG treatment, the expression level of CsSnRK2.6 was down-regulated. However, other 8 CsSnRK2 genes reached their highest values and were significantly up-regulated after 12 and 48 h of treatment. Under MeJA treatment, the transcription level of 9 CsSnRK2 genes almost had no change after 3 and 12 h treatment, but it was significantly up-regulated and reached the highest expression level at 24 or 48 h. Among them, the expression levels of CsSnRK2.3 and CsSnRK2.8 were significantly increased by more than 16 times. Taken together, the three abiotic stress treatments induced changes in the abundance of most CsSnRK2 genes transcripts, especially for the expression of CsSnRK2.3 and CsSnRK2.8, and the expression patterns of CsSnRK2 genes suggest that CsSnRK2 genes may play a role in abiotic stress responses in cucumber.
CsSnRK2.1 and CsSnRK2.7 were not expressed in roots (Figure 8) under different treatments, which was consistent with the results of tissue expression. It also further illustrates that the RNA-seq results are accurate in representing gene expression levels. The expression of CsSnRK2.2, CsSnRK2.3 and CsSnRK2.9 genes was up-regulated by ABA treatment and reached the highest value after 24 h of treatment. The expression of other genes was down-regulated by ABA stress. In PEG treatment, except for CsSnRK2.5, the expression level of all genes decreased after 3 h of treatment and reached the highest value after 12 h. The expression pattern of each gene was different in MeJA treatment. Among them, CsSnRK2.9 changed most significantly after 12 h of treatment. In addition, we also observed the phenotype of cucumber seedlings after 48 h of different treatments (Figure 9). Drought stress caused the seedlings to wilt and dwarf, while ABA and MeJA treatments inhibited the growth of the seedlings compared to the control. The expression patterns of CsSnRK2 genes in roots and leaves were different, indicating that CsSnRK2s play different roles in different tissues at seedling stage under abiotic stress.

4. Discussion

Phosphorylation of plant protein kinases plays a very important role in the mechanism of response to stress. However, SnRKs as a type of Ser/Thr protein kinase represent an interface between stress and ABA signal transduction [39]. It has been shown that 39, 48, 50 and 44 SnRK genes were identified in Oryza sativa, Brachypodium distachyon [3], Arabidopsis thaliana [40] and Hordeum vulgare [41], respectively. In this study, we were the first to comprehensively identified SnRK members of cucumber genome, which will provide crucial information to research their functions in the future. Bioinformatic analysis of SnRK family has isolated a total of 30 CsSnRKs including 1 CsSnRK1, 10 CsSnRK2s and 19 CsSnRK3s in cucumber. SnRK2 and SnRK3 subfamilies were larger and relatively more diverse than SnRK1. These results support the previous view that the SnRK2 and SnRK3 subfamilies evolved from the SnRK1 subfamily and that their numbers have increased during evolution [8].
To explore the evolutionary relationship between CsSnRK members and other species, we analyzed the phylogenetic tree containing SnRK members of cucumber, Arabidopsis, rice and barley. CsSnRKs were clearly divided into three subgroups and clustered with the corresponding subgroups in rice, Arabidopsis and barley, suggesting that the structure and function of CsSnRK are highly conserved during plant evolution. CsSnRKs from the same subgroup had similar conserved domains, motif composition and gene structure, with differences among different subgroups. Different numbers of exons/introns played important roles in the evolution and function of different gene families. The CsSnRK1.1 had 10 exons, which was the same as that reported for EgrSnRK1s and BdSnRK1s [3,7]. CsSnRK2 subfamily had 9–18 exons. Previous study showed that SnRK3 subfamily can be divided into two clades according to the number of exons [42]. The CsSnRK3 subfamily was also subdivided into the exon-rich and exon-poor type. Study has also shown that the SnRK3 subfamily originated from green algae, and the intron-poor group first appeared in seed plants [6]. These results were consistent with phylogenetic tree studies showing that SnRKs exon-intron numbers were highly conserved during the evolution of each subfamily. Different subfamilies of SnRK genes contained different conserved domains, but all SnRK genes in cucumber contained an N-terminal protein kinase domain. Each domain had its own unique function, for example, CsSnRK3 subfamily genes contained NAF domains and have been reported to interact with CBLs in a calcium-dependent manner. Furthermore, the NAF domain as a target of the CBL calcium sensor protein was defined a group of heterologous kinases involved in a variety of signal transduction processes [21]. This study also found that different CsSnRKs subfamily genes shared different types of conserved domains. This may indicate that there was functional diversity in the CsSnRK gene family according to their domains.
In our current research, the identified CsSnRK genes were unevenly distributed across the six chromosomes of the cucumber genome. In addition, based on the distribution of CsSnRKs in chromosomes, we identified gene duplication events. Gene duplication plays an irreplaceable role in the evolution of plant genomes and genetic systems [43]. Duplicated genes can generate new genes and corresponding new functions in the form of segmental duplications, tandem duplications and translocation events. Segmental and tandem duplications can lead to the expansion of gene families [44]. Our research results demonstrate that in the cucumber genome, three duplication events were observed between chromosomes, which evolved from segment duplication. These segmental duplications might have contributed greatly to the expansion and evolution of the CsSnRK gene family.
When plants are stimulated by external factors, some transcription factors are activated, and the activated factors combine with the cis-acting elements of the downstream target gene promoters to change the expression pattern of genes. The study of gene cis-acting elements is particularly important for mining the potential function of gene. To date, many cis-acting elements have been well characterized and divided into distinct groups [45]. Many cis-acting elements responsive to hormone responses were found in the promoter sequence of the CsSnRK genes, such as ABRE, ERE, CGTCA-motif and TGA-element. These results indicate that the CsSnRK genes might respond to a variety of hormone signals and interact with other metabolic pathways. A large body of evidence indicates that the SnRK family was widely involved in the response to various biotic and abiotic stresses, including drought, salt and temperature stresses. The cis-acting element in response to drought stress, such as MBS and MYC, were also found in CsSnRK genes. The widespread presence of these cis-acting elements suggests a critical role for CsSnRK members in responding to abiotic stress and hormonal stimuli. For instance, under ABA treatment, CsSnRKs was not expressed, which was consistent with the distribution of ABA-responsive elements. However, some conflicting results also emerged in our study. The promoter region of CsSnRK2.6 did not contain an AREB binding site, but it responded to ABA treatment at a low level. Therefore, gene activity is often associated with differences in cis-acting elements in the promoter region [46].
Numerous studies demonstrated that SnRK2s were involved in multiple abiotic stress responses. In Arabidopsis, SnRK2 genes (SRK2C, SRK2D, SRK2E, SRK2F and SRK2I) were activated by ABA [47,48]. In cotton, GhSnRK2.3, GhSnRK2.7, GhSnRK2.8, GhSnRK2.9 and GhSnRK2.10 were notably up-regulated under salt and PEG treatment [49]. In pepper, CaSnRK2.1, CaSnRK2.3 and CaSnRK2.8 were significantly induced by PEG and NaCl treatment [50]. In this study, in order to verify the response of CsSnRK genes to abiotic stress, we used qRT-PCR to detect the expression of 10 CsSnRK2 genes in leaves and roots. All CsSnRK2s were found to respond to PEG, ABA and MeJA stress except for CsSnRK2.7 of leaves and CsSnRK2.1 and CsSnRK2.7 of roots. In cucumber seedling leaves, CsSnRK2.3 was significantly up-regulated under ABA and MeJA treatments. CsSnRK2.2, CsSnRK2.5 and CsSnRK2.9 were significantly up-regulated under PEG treatment. In roots, CsSnRK2.2, CsSnRK2.3 and CsSnRK2.9 were significantly up-regulated by ABA and MeJA treatments. CsSnRK2.5 was significantly up-regulated by PEG treatment. The above results indicate that CsSnRK2.3 might regulate ABA and MeJA synthesis in leaves, while CsSnRK2.2, CsSnRK2.5 and CsSnRK2.9 might play a major role in regulating drought stress in leaves. Meanwhile, CsSnRK2.5 might play a major role in the regulation of drought in roots, CsSnRK2.2, CsSnRK2.3 and CsSnRK2.9 regulate ABA and MeJA synthesis in roots. In Arabidopsis, AtSnRK2.2 also responded to drought [51]. CsSnRKs showed different expression patterns for different treatments, suggesting that CsSnRKs may be specific during evolution. Therefore, further studies on the interacting proteins of the CsSnRK2 family and phenotypic changes in transgenic plants will help to clarify the precise biological role of each member of the CsSnRK2 family in vivo, and, thus, determine the involvement of the ABA pathway on CsSnRK2s.
In plants, SnRK genes have been widely transcribed in many tissues, but different SnRK members have different expression levels at different growth and development stages [3]. The extensive expression of SnRK genes indicates that SnRKs could be necessary for plants to grow and develop different tissues. For example, there were eight SnRK2 genes that were widely transcribed in a variety of tissues in Brachypodium distachyon [3]. In apples, MdCIPK4, MdCIPK9, MdCIPK15 and MdCIPK32 were highly expressed in flowers, and MdCIPK29 was highly expressed in fruits, indicating that they had different biological functions in their respective tissues [52]. In this study, transcriptome data from different tissues of CsSnRK2 genes were used to study the expression patterns. Six genes were highly expressed in flower tissues, while CsSnRK2.5, CsSnRK2.6 and CsSnRK2.7 were very poorly expressed. All genes were expressed in roots except CsSnRK2.1. The above results indicate that CsSnRK genes had tissue-specific and developmental stage-specific expression in different tissues.

5. Conclusions

In this study, the identified 30 cucumber SnRK gene families were comprehensively studied and divided into 3 subfamilies with high similarity in gene structure and motif composition. The evolutionary relationship of CsSnRK genes in cucumber and other species was established, showing that these subfamilies were evolutionarily conserved in both structure and function. Moreover, the cis-acting elements, tissue-specific analysis of CsSnRK family were also determined. Last but not least, we provided evidence that cucumber SnRKs were involved in alleviating drought stress and hormonal responses. The present genomic and bioinformatics analyses of CsSnRK genes provided a solid foundation for further investigation of CsSnRK gene functions. In addition, these results will also help to expand our knowledge to identify candidate genes that improve plant architecture under stressful conditions, and open up possibilities for breeding and genetic improvement of other agricultural crops.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12071550/s1. Table S1: Primer sequence of CsSnRK2 for real-time RT-PCR. Table S2: cis-acting regulatory elements in CsSnRK promoters of cucumber.

Author Contributions

Formal analysis, Y.L. and W.L.; Funding acquisition, C.W. and W.L.; Software, Y.N.; Visualization, R.G. and W.L.; Writing—original draft, Y.L.; Writing—review and editing, Y.L. and W.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key Research and Development Program (2018YFD1000800); the National Natural Science Foundation of China (Nos. 31860568, 31560563 and 31160398); the Research Fund of Higher Education of Gansu, China (No. 2018C-14); the Post-Doctoral Foundation of China (Nos. 20100470887 and 2012T50828) and the Natural Science Foundation of Gansu Province, China (Nos. 1606RJZA073 and 1606RJZA077).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All relevant data are contained within the article.

Acknowledgments

We would like to thank Wei Li (Shenzhen Institute of Genomics, Chinese Academy of Agricultural Sciences, China) for providing us with cucumber seeds.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic relationship of the SnRK homologs in different species. The four differently-colored shapes represent SnRK proteins from four species. The orange rectangle, green pentacle, blue triangle and purple round represent Arabidopsis, rice, barley and cucumber SnRK proteins, respectively. Different color sectors represent SnRKs from SnRK1 subfamilies (blue), SnRK2 (pink) and SnRK3 (green).
Figure 1. Phylogenetic relationship of the SnRK homologs in different species. The four differently-colored shapes represent SnRK proteins from four species. The orange rectangle, green pentacle, blue triangle and purple round represent Arabidopsis, rice, barley and cucumber SnRK proteins, respectively. Different color sectors represent SnRKs from SnRK1 subfamilies (blue), SnRK2 (pink) and SnRK3 (green).
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Figure 2. Phylogenetic relationships, architecture of conserved protein motifs and gene structure of CsSnRK genes. (A) The phylogenetic tree of 30 CsSnRK genes was built with MEGA7 choosing the NJ method. Pink, green and blue background color represents SnRK1, SnRK2 and SnRK3 subgroups, respectively. (B) The motif compositions of 30 CsSnRK proteins. The motifs were identified using the MEME program. Boxes of different colors represent motif 1 to 10, respectively. The length of amino acid sequences can be estimated by the scale at the bottom. (C) Gene structures of 30 CsSnRK genes. Green boxes represent exons (CDS), yellow boxes represent the upstream/downstream region (UTR), and black lines represent introns. The length of nucleotide sequences of exons/introns/UTRs can be inferred by the scale at the bottom.
Figure 2. Phylogenetic relationships, architecture of conserved protein motifs and gene structure of CsSnRK genes. (A) The phylogenetic tree of 30 CsSnRK genes was built with MEGA7 choosing the NJ method. Pink, green and blue background color represents SnRK1, SnRK2 and SnRK3 subgroups, respectively. (B) The motif compositions of 30 CsSnRK proteins. The motifs were identified using the MEME program. Boxes of different colors represent motif 1 to 10, respectively. The length of amino acid sequences can be estimated by the scale at the bottom. (C) Gene structures of 30 CsSnRK genes. Green boxes represent exons (CDS), yellow boxes represent the upstream/downstream region (UTR), and black lines represent introns. The length of nucleotide sequences of exons/introns/UTRs can be inferred by the scale at the bottom.
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Figure 3. Chromosomal locations of CsSnRK genes. The length of chromosomes can be estimated using the scale on the left.
Figure 3. Chromosomal locations of CsSnRK genes. The length of chromosomes can be estimated using the scale on the left.
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Figure 4. The duplication analysis of CsSnRK family in cucumber. Gray lines indicated all synteny blocks in the cucumber genome, and the orange lines indicated duplicated CsSnRK gene pairs. The chromosome number is indicated on each chromosome. Scale bar marked on the chromosome indicates chromosome lengths (Mb). Chr, Chromosome.
Figure 4. The duplication analysis of CsSnRK family in cucumber. Gray lines indicated all synteny blocks in the cucumber genome, and the orange lines indicated duplicated CsSnRK gene pairs. The chromosome number is indicated on each chromosome. Scale bar marked on the chromosome indicates chromosome lengths (Mb). Chr, Chromosome.
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Figure 5. Predicted cis-acting regulatory elements in CsSnRK promoters. Different colors represented different cis-elements.
Figure 5. Predicted cis-acting regulatory elements in CsSnRK promoters. Different colors represented different cis-elements.
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Figure 6. Expression patterns of 10 CsSnRK2 genes in six tissues of cucumber. (A) The expression level based on the RNA sequencing data from SRA database of NCBI (accession number SRP071224). (B) The relative expression levels of CsSnRK2 genes in different tissues from qRT-PCR analysis. Color bars represent expression values: red and blue indicate high and low expression levels, respectively.
Figure 6. Expression patterns of 10 CsSnRK2 genes in six tissues of cucumber. (A) The expression level based on the RNA sequencing data from SRA database of NCBI (accession number SRP071224). (B) The relative expression levels of CsSnRK2 genes in different tissues from qRT-PCR analysis. Color bars represent expression values: red and blue indicate high and low expression levels, respectively.
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Figure 7. The expression pattern of CsSnRK genes in leaves treated with (A) abscisic acid (ABA), (B) PEG and (C) methyl jasmonate (MeJA). The sampling time points were 0, 3, 12, 24 and 48 h. Error bars represent the standard error (SE) of three replicates. The relative expression of each gene in different treatment is expressed as mean ± SE (n = 3). Bars with different lowercase letters were significantly different by Duncan’s multiple range tests (p < 0.05).
Figure 7. The expression pattern of CsSnRK genes in leaves treated with (A) abscisic acid (ABA), (B) PEG and (C) methyl jasmonate (MeJA). The sampling time points were 0, 3, 12, 24 and 48 h. Error bars represent the standard error (SE) of three replicates. The relative expression of each gene in different treatment is expressed as mean ± SE (n = 3). Bars with different lowercase letters were significantly different by Duncan’s multiple range tests (p < 0.05).
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Figure 8. The expression pattern of CsSnRK2 genes in roots treated with (A) abscisic acid (ABA), (B) PEG and (C) methyl jasmonate (MeJA). The sampling time points were 0, 3, 12, 24 and 48 h. Error bars represent the standard error (SE) of three replicates. The relative expression of each gene in different treatment is expressed as mean ± SE (n = 3). Bars with different lowercase letters were significantly different by Duncan’s multiple range tests (p < 0.05).
Figure 8. The expression pattern of CsSnRK2 genes in roots treated with (A) abscisic acid (ABA), (B) PEG and (C) methyl jasmonate (MeJA). The sampling time points were 0, 3, 12, 24 and 48 h. Error bars represent the standard error (SE) of three replicates. The relative expression of each gene in different treatment is expressed as mean ± SE (n = 3). Bars with different lowercase letters were significantly different by Duncan’s multiple range tests (p < 0.05).
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Figure 9. Plant morphology of cucumber seedlings under different treatments. Cucumber seedlings at the two-leaf stage were treated with 15% (w/v) PEG6000, 100 µM methyl jasmonate (MeJA), 100 µM ABA, and photographed 48 h after treatments.
Figure 9. Plant morphology of cucumber seedlings under different treatments. Cucumber seedlings at the two-leaf stage were treated with 15% (w/v) PEG6000, 100 µM methyl jasmonate (MeJA), 100 µM ABA, and photographed 48 h after treatments.
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Table
Table 1. The characteristics of the CsSnRK gene family in cucumber.
Table 1. The characteristics of the CsSnRK gene family in cucumber.
Gene NameGene IDAmino Acid
(aa)		ExonsMW (kDa)pISubcellular
Localization
CsSnRK1.1CsaV3_6G006250.15151058.818.59cytoplasm
CsSnRK2.1CsaV3_2G014200.1361941.244.44nucleus
CsSnRK2.2CsaV3_2G016930.13631041.074.45nucleus
CsSnRK2.3CsaV3_3G002220.1340938.385.95cytoplasm
CsSnRK2.4CsaV3_4G030050.1355940.906.38nucleus
CsSnRK2.5CsaV3_4G030080.16731877.017.02cytoplasm
CsSnRK2.6CsaV3_4G030100.13721242.448.78nucleus
CsSnRK2.7CsaV3_4G030120.14861355.735.53nucleus
CsSnRK2.8CsaV3_4G030260.13551040.945.83nucleus
CsSnRK2.9CsaV3_6G045260.1344938.955.07cytoplasm
CsSnRK2.10CsaV3_7G008840.1365941.224.52cytoplasm
CsSnRK3.1CsaV3_1G028820.1430148.519.44cytoplasm
CsSnRK3.2CsaV3_1G030280.13561540.565.74chloroplast
CsSnRK3.3CsaV3_1G033260.1463252.409.63chloroplast
CsSnRK3.4CsaV3_1G036820.14641652.059.55cytoplasm
CsSnRK3.5CsaV3_2G003670.1433248.699.52cytoplasm
CsSnRK3.6CsaV3_2G013250.1430147.889.45chloroplast
CsSnRK3.7CsaV3_2G030480.14461450.857.79cytoplasm
CsSnRK3.8CsaV3_3G007670.14411450.088.42cytoplasm
CsSnRK3.9CsaV3_3G012720.1433148.609.64chloroplast
CsSnRK3.10CsaV3_3G015590.14421249.586.05cytoplasm
CsSnRK3.11CsaV3_4G017300.16392072.877.7cytoplasm
CsSnRK3.12CsaV3_4G036630.1443150.298.61cytoplasm
CsSnRK3.13CsaV3_6G000230.14361249.386.45cytoplasm
CsSnRK3.14CsaV3_6G000240.14311249.038.34chloroplast
CsSnRK3.15CsaV3_6G019060.1408446.148.41nucleus
CsSnRK3.16CsaV3_6G019070.1424148.148.86cytoplasm
CsSnRK3.17CsaV3_6G050960.1467153.018.06cytoplasm
CsSnRK3.18CsaV3_6G051970.14541551.179.22chloroplast
CsSnRK3.19CsaV3_UNG100750.1432148.946.63cytoplasm
Note: pI, isoelectric point; MW, molecular weight.
Table
Table 2. The KaKs values of the paired duplicated CsSnRK genes.
Table 2. The KaKs values of the paired duplicated CsSnRK genes.
Duplicated GenesKaKsKa/Ks
CsSnRK3.1/CsSnRK3.120.3134254.275160.073313
CsSnRK3.3/CsSnRK3.170.3227124.268530.075603
CsSnRK3.6/CsSnRK3.90.238431.751270.136146
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Luo, Y.; Niu, Y.; Gao, R.; Wang, C.; Liao, W. Genome-Wide Identification and Expression Analysis of SnRK Gene Family under Abiotic Stress in Cucumber (Cucumis sativus L.). Agronomy 2022, 12, 1550. https://doi.org/10.3390/agronomy12071550

AMA Style

Luo Y, Niu Y, Gao R, Wang C, Liao W. Genome-Wide Identification and Expression Analysis of SnRK Gene Family under Abiotic Stress in Cucumber (Cucumis sativus L.). Agronomy. 2022; 12(7):1550. https://doi.org/10.3390/agronomy12071550

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Luo, Yanyan, Yuan Niu, Rong Gao, Chunlei Wang, and Weibiao Liao. 2022. "Genome-Wide Identification and Expression Analysis of SnRK Gene Family under Abiotic Stress in Cucumber (Cucumis sativus L.)" Agronomy 12, no. 7: 1550. https://doi.org/10.3390/agronomy12071550

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