Genome-Wide Identification and Expression Analysis of Sucrose Nonfermenting 1-Related Protein Kinase (SnRK) Genes in Salvia miltiorrhiza in Response to Hormone

Abstract

The SnRK gene family is the chief component of plant stress resistance and metabolism through activating the phosphorylation of downstream proteins. S. miltiorrhiza is widely used for the treatment of cardiovascular diseases in Asian countries. However, information about the SnRK gene family of S. miltiorrhiza is not clear. The aim of this study is to comprehensively analyze the SnRK gene family of S. miltiorrhiza and its response to phytohormone. Here, 33 SmSnRK genes were identified and divided into three subfamilies (SmSnRK1, SmSnRK2 and SmSnRK3) according to phylogenetic analysis and domain. SmSnRK genes within same subgroup shared similar protein motif composition and were unevenly distributed on eight chromosomes of S. miltiorrhiza. Cis-acting element analysis showed that the promoter of SmSnRK genes was enriched with ABRE motifs. Expression pattern analysis revealed that SmSnRK genes were preferentially expressed in leaves and roots. Most SmSnRK genes were induced by ABA and MeJA treatment. Correlation analysis showed that SmSnRK3.15 and SmSnRK3.18 might positively regulate tanshinone biosynthesis; SmSnRK3.10 and SmSnRK3.12 might positively regulate salvianolic acid biosynthesis. RNAi-based silencing of SmSnRK2.6 down-regulated the biosynthesis of tanshinones and biosynthetic genes expression. An in vitro phosphorylation assay verified that SmSnRK2.2 interacted with and phosphorylated SmAREB1. These findings will provide a valuable basis for the functional characterization of SmSnRK genes and quality improvement of S. miltiorrhiza.

1. Introduction

Plants have developed a complicated and multilayer system to recognize and react to abiotic stresses, including drought, salinity and extreme temperature [1]. The post-translational modification of proteins, especially the phosphorylation and dephosphorylation process, is vital for plants to deal with different stresses [2]. The sucrose non-fermenting 1-related protein kinases (SnRK) are Ser/Thr protein kinases, which phosphorylate downstream target proteins for the transduction of signal pathways [3]. Based on sequence similarity and protein structure, SnRK could be subdivided into three subfamilies in plants: SnRK1, SnRK2 and SnRK3 [4]. Due to the Ser/Thr kinase domain located on the N-terminal of SnRKs, the similarity of the N-terminal was higher than the C-terminal. The function of three subfamilies was not independent and the interaction of these genes forms a large and sophisticated regulation network [5].

The plant SnRK1, heterotrimeric complexes consisting of three subunits (a catalytic α-subunit and regulatory β- and γ- subunits), is an ortholog of the sucrose nonfermenting 1 (SNF1) of yeast and AMP-activated protein kinase (AMPK) of mammals [6]. Plant SnRK1 contains a protein kinase domain, ubiquitin-associated domain (UBA) and kinase-associated 1 domain (KA1) [7]. The SnRK1 family is a relatively small subfamily, containing three members in Arabidopsis thaliana and four members in Oryza sativa. SnRK1 kinase is a central regulator of energy management and drives a large array of metabolic and transcriptional reprogramming in the response of metabolic signaling and stress. SnRK1 is rapidly activated by stress and energy deficiency, thereby phosphorylating metabolic enzymes or bZIP transcription factors (TFs), leading metabolism-related gene activation or depression. Moreover, the function of SnRK1 is not limited to stress responses, and many studies have shown that SnRK1 balances metabolism, growth and development [8,9,10].

The SnRK2 subfamily is a plant-specific gene family. Both A. thaliana and O. sativa have 10 members in this subfamily. Most of SnRK2s in A. thaliana and O. sativa are activated by ABA and osmotic stress [11,12]. SnRK2s contain a Pkinase domain at the N-terminal and a dissimilar regulatory domain at the C-terminal [13]. SnRK2s play an important role when plants are confronted with abiotic stresses and they are central kinases in ABA signaling [14]. ABA could release the SnRK2s from the suppression of PP2Cs. These activated SnRK2s phosphorylate target proteins [15,16,17], such as SLAC1 [18], KAT1 [19], AtRbohF [20] and TFs [14,15]. SnRK2.2, SnRK2.3 and SnRK2.6 are central and positive regulators of ABA signaling in A. thaliana. The triple mutant of snrk2.2, snrk2.3 and snrk2.6 is extremely insensitive to the ABA-mediated inhibition of seed germination [21]. Studies have also shown that SnRK2s could enhance tolerance to salinity and drought in plants [22,23].

The SnRK3 subfamily is also plant-specific, and is also known as CIPK (CBL-interacting protein kinase). Calcineurin B-like proteins (CBLs) are calcium sensors, interacting with SnRK3 to transfer cellular calcium signals. A. thaliana and O. sativa have 26 and 33 members in this subfamily [24,25]. SnRK3 contains a Pkinase domain at the N-terminal and an NAF domain at the C-terminal. When plants are confronted with stresses, CBLs detect and bind to Ca²⁺ ions, interacting with the NAF domain of SnRK3s to activate it [26,27]. The CBL-CIPK module plays an important role in the salt stress of plants. The first discovered CBL-CIPK module was the SOS (salt overly sensitive) module. In A. thaliana, the cell membrane located SOS3 (AtCBL4) recognized and bound to a calcium signal that was produced by salt stress. Then, SOS3 formed a complex with SOS2 (AtCIPK24), which activated the AtCIPK24 kinase activity to phosphorylate SOS1 (Na⁺/H⁺ antiporter) to excrete Na⁺ from plant cells and protect plant cells from the damage caused by a high concentration of Na⁺ [28,29,30]. Moreover, CBL-CIPK modules are also involved in ABA signaling. The CBL1 and CIPK15 modules negatively regulated seed germination, stomatal movement and ABA-mediated gene expression [31]. CIPK1 and CIPK23 also played a negative role in ABA-regulated seed germination and stomatal movement, respectively [32,33]. CIPK11 phosphorylated and activated ABI5 (ABA-Insensitive 5) to regulate seed germination [34].

The traditional Chinese medicinal plant S. miltiorrhiza (also known as Danshen) is widely used alone or in combination with other medicines in clinics for the management of cardiovascular diseases in China and even in some other Asian countries [35]. Many drugs containing S. miltiorrhiza material have been developed and marketed in China for preventing and treating cardiovascular diseases, such as Fufang Danshen tablets and Compound Danshen Dripping Pills [36]. With the genome of S. miltiorrhiza sequenced [37,38,39], many gene families have been identified and investigated in S. miltiorrhiza, such as WRKY [40], bHLH [41], TIFY [42], laccases [43], B-box [44] and MADS-box [45]. The information about SnRK genes in S. miltiorrhiza is not clear despite SnRK genes being key components of energy management and stress resistance in plants. Moreover, the function of SnRK genes in S. miltiorrhiza is also obscure. To better understand the information and function of SnRK genes in S. miltiorrhiza, we systematically analyzed SmSnRK genes with bioinformatics.

In this study, we identified thirty-three SnRK gene family members in S. miltiorrhiza, including three members in SnRK1, seven members in SnRK2 and twenty-three members in SnRK3. Tissue expression pattern, gene structure, phylogenetic relationships, hormone response and chromosome location were systematically analyzed. Based on correlation and function analysis, we found that SmSnRK2.6 positively regulated tanshinones biosynthesis and gene expression. These results will provide important insights into the molecular mechanisms of SnRK for stress tolerance and molecular breeding in S. miltiorrhiza.

2. Results

2.1. Identification, Phylogenetic Analysis, Motif and Chromosomal Location of SnRK Genes in S. miltiorrhiza

A total of 33 SmSnRK genes were identified in the genome of S. miltiorrhiza with BLAST search and HMMscan, and the longest protein sequence was selected for further study. All of the 33 SmSnRK genes possessed a Ser/Thr kinase domain (Pkinase, PF00069) and can be divided into three subfamilies (SmSnRK1, SmSnRK2 and SmSnRK3) based on different domains contained in these genes. SmSnRK1.1, SmSnRK1.2 and SmSnRK1.3 were placed into the SnRK1 subfamily, due to these genes containing the Pkinase domain (PF00069), UBA domain (PF00627) and KA1 domain (PF02149) domain. SmSnRK2.1–SmSnRK2.7 were identified as part of the SnRK2 subfamily, as these seven members showed high similarity with the SnRK2 family in A. thaliana and O. sativa. The 23 members (SmSnRK3.1–SmSnRK3.23) grouped into SnRK3 subfamily had an NAF domain (PF03822). The residues of the amino acids of the SmSnRK genes ranged from 341 (SmSnRK2.1) to 513 (SmSnRK1.1) and the molecular weight of the SmSnRK genes ranged from 38.78 to 58.52 kDa. The isoelectric point of the SmSnRK gene family spanned from 4.838 to 9.633. Based on subcellular localization prediction results, most of the SmSnRK proteins were located in the cytoplasm and nucleus, followed by the chloroplast (Table S1).

To further study the phylogenetic relationships of SmSnRK genes, we constructed a phylogenetic tree using the Neighbor-Joining (NJ) method with the full-length amino acid sequences of the SnRKs genes from S. miltiorrhiza, A. thaliana and O. sativa. As shown in Figure 1, all SnRKs genes were clustered into three groups, which was basically in accordance with previous studies. SmSnRK genes were more related to the SnRKs genes of A. thaliana than O. sativa. In detail, SmSnRK2.2, SmSnRK2.3 and SmSnRK2.6 had a close relationship with AtSnRK2.2, AtSnRK2.3 and SmSnRK2.6, respectively, but not OsSAPK8. SmSnRK1.1 and SmSnRK1.2 were clustered with AtSnRK1.1.

Twelve conserved motifs were identified in SmSnRK proteins and all SmSnRK proteins had the conserved Pkinase domain, including motif 1, 2, 3, 4, 5 and 7. Moreover, SmSnRK proteins within the same subfamily retained a similar composition pattern. Motif 2, 6 and 9 were the NAF domain, specifically found in the SmSnRK3 subfamily members. Motif 14 (UBA domain) and 15 (KA1 domain) were specifically contained in SmSnRK1 subfamily members (Figure S1a). The conserved motif compositions supported the subfamily classifications.

Gene structural analysis showed that the members in the subgroup shared a similar distribution of exon numbers. All of the SmSnRK1 members have ten exons. All SmSnRK2 members have nine exons, except for SmSnRK2.2 and SmSnRK2.3 which both clustered in a subgroup and contained eight exons (Figure S1b).

A chromosomal distribution map of SmSnRK genes was established and it was found that SmSnRK genes were distributed randomly on eight chromosomes. In addition, only the SmSnRK3.15 gene was located on chromosome LG2, whereas most of the SmSnRK genes (eight genes) were located on chromosome LG8 (Figure S2).

2.2. Analysis of Cis-Elements in the Promoter of SmSnRK Genes

To understand the potential biological regulatory mechanisms of the SmSnRK genes, the promoter sequence (2 kb region upstream of ATG) was analyzed with PlantCARE. The cis-elements in the promoter of SmSnRK genes can be classified into light response, stress response (low temperature stress and drought stress) and hormone response (ABA, JA, Auxin, GA and SA). Except for SmSnRK3.20, the promoter of all SmSnRK genes contained an ABRE cis-element, indicating that most SmSnRK genes may be involved in ABA signaling. Moreover, about 67%, 51.5%, 36% and 30% of the promoters of SmSnRK genes contained MeJA, GA, auxin and SA responsive motifs, respectively. The drought response cis-elements were enriched in the SmSnRK2 and SmSnRK3 subfamily, but not in SmSnRK1 (Figure 2). The promoter analysis showed that most of the SmSnRK genes may participate in diverse hormone responses and drought stress. These results suggested that different subfamilies may be regulated by distinct pathways.

2.3. Expression Patterns of SmSnRK Genes

To gain insight into the relative expression level of SmSnRK genes in different tissues, the flower, leaf, stem and root were sampled for qRT-PCR. SmSnRK genes exhibited differential expression in different tissues, even in the same subgroup. SmSnRK1.1 and SmSnRK1.3 showed a similar expression pattern with the main expression in leaves, followed by stem, flower and root. Most of SmSnRK2 subfamily members showed high expression in leaves, including SmSnRK2.2, SmSnRK2.3, SmSnRK2.4 and SmSnRK2.6. SmSnRK2.1 and SmSnRK2.5 were mainly expressed in flowers. The expression pattern of SmSnRK3s could be grouped into three categories. SmSnRK3.1, SmSnRK3.3, SmSnRK3.11, SmSnRK3.12 and SmSnRK3.16 showed high expression in flowers. SmSnRK3.2, SmSnRK3.8, SmSnRK3.14, SmSnRK3.15, SmSnRK3.18 and SmSnRK3.22 were mainly expressed in roots. SmSnRK3.13, SmSnRK3.20 and SmSnRK3.23 showed relatively equivalent expression levels in the tested tissues. The diverse expression levels of SmSnRKs in different tissues suggested that SmSnRKs exhibit functional diversity in plant development and stress response (Figure 3).

It was reported that MeJA (methyl jasmonate) [46] and ABA (abscisic acid) [47] were important hormones in promoting tanshinone and salvianolic acid accumulation. Moreover, the promoter of SmSnRK genes was enriched with ABA-responsive and MeJA-responsive cis-elements [48]. To determine whether MeJA and ABA affect the expression of SmSnRK genes, we treated Salvia miltiorrhiza with ABA and MeJA. Quantitative real-time PCR (qRT-PCR) showed that most of the SmSnRK genes (45%) were up-regulated by ABA treatment and several members (24%) were down-regulated by ABA treatment. The expression of SmSnRK3.1, SmSnRK3.12 and SmSnRK3.17 was highly induced by ABA (Figure 4). One-third of SmSnRK gene expression was induced by MeJA, of which SmSnRK3.18 and SmSnRK3.19 were extremely induced. A limited number of SmSnRK members were down-regulated by MeJA treatment (Figure 5). The expression of SmSnRK3.2, SmSnRK3.10, SmSnRK3.18, SmSnRK3.19 and SmSnRK3.21 was responsive to both ABA and MeJA. These results suggested that the expression of SmSnRKs in different tissues and the response to hormones were varied, even in the same subgroups.

2.4. Gene Ontology Analysis of SmSnRKs

Gene ontology analysis revealed that the molecular function of all of the SmSnRK genes was closely related to ATP binding and protein serine/threonine kinase activity, followed by protein binding, catalytic activity and ion binding. The biological processes of most SmSnRK genes were associated with cell progress and intracellular signal transduction, indicating that SmSnRK genes played an important role in the signaling pathway. Moreover, several SmSnRK members were involved in ABA signaling, calcium-mediated signaling and calcium/potassium homeostasis (Figure 6).

2.5. Correlation Analysis of SmSnRK Genes with Tanshinones and Salvianolic Acids Biosynthesis Genes

To further study whether SmSnRK genes had potential regulatory roles in the expression of tanshinone biosynthesis genes, we employed a co-expression analysis of SmSnRK genes and tanshinone biosynthesis genes, based on the tissue expression patterns and MeJA transcriptome (Figure S3). SmSnRK3.15 and SmSnRK3.18 were relatively highly co-expressed with key tanshinone biosynthesis genes. SmSnRK3.8, SmSnRK3.14 and SmSnRK3.22 showed moderate co-expression with key tanshinone biosynthesis genes, suggesting that SmSnRK genes could be participating in tanshinone accumulation (Figure 7a). Moreover, we also used the ABA transcriptome-based (Figure S4) correlation analysis to explore the relationship between SmSnRK genes and salvianolic acids biosynthesis genes. It was observed that SmSnRK3.10 and SmSnRK3.12 had strong positive correlations with all of the salvianolic acids biosynthesis genes, except SmPAL1 and SmHPPR, suggesting that these two SmSnRK genes might regulate the salvianolic acid biosynthesis genes expression (Figure 7b).

2.6. SmSnRK2.6 Positively Regulated Tanshinones Biosynthesis and Genes Expression

Previous studies have demonstrated that SmSnRK2.6 regulates the accumulation of phenolic acids and is involved in the ABA signaling and stress response in S. miltiorrhiza [49]. Moreover, based on our correlation analysis, we found that SmSnRK2.6 was related to tanshinones biosynthesis, indicating that SmSnRK2.6 may participate in tanshinones biosynthesis. To investigate whether SmSnRK2.6 participated in regulating tanshinone biosynthesis, we silenced SmSnRK2.6 expression in transgenic hairy root lines with the RNAi method. Ri-2.6-5, Ri-2.6-7 and Ri-2.6-8 with substantial a down-regulation of SmSnRK2.6 expression were selected for further study (Figure S5). The phenotype and extract color of hairy roots hinted that the content of tanshinones in Ri-SmSnRK2.6 strains was lower than that in the control strain (Figure 8a,b). Metabolic content analysis showed that the content of dihydrotanshinone (DHT), cryptotanshinone (CT), tanshinone I (TI) and tanshinone IIA (TIIA) in the control lines was about 2.54–4.33, 2.28–6.29, 1.83–2.26 and 1.83–2.26 times higher than in the Ri-SmSnRK2.6 lines, respectively (Figure 8c). Moreover, the expression level of most tanshinone biosynthetic genes was decreased in the Ri-SmSnRK2.6 lines, among which SmGGPPS1 and SmCPS1 were significantly down-regulated, with the lowest decrease of 0.26- and 0.43-fold that of the control lines, respectively (Figure 8d). These results confirmed that SmSnRK2.6 positively regulated tanshinone biosynthesis.

2.7. SmSnRK2.2 Interacts with and Phosphorylates SmAREB1

A previous study has shown that SmSnRK2.3 and SmSnRK2.6 interacted with SmAREB1 [49]. To test whether SmSnRK2.2 interacts with SmAREB1, yeast two hybrid (Y2H) assays and bimolecular fluorescence complementation (BiFc) assays were applied. Y2H assays showed that SmSnRK2.2 interacts with SmAREB1 in yeast (Figure 9a). BiFc assays showed that SmSnRK2.2 can interact with SmAREB1 in plant cells (Figure 9b). Furthermore, we also tested whether SmSnRK2.2 could phosphorylate SmAREB1 by using a Phos-tag reagent. As shown in Figure 9c, the mobility shift was quite slow compared with the control, indicating that SmSnRK2.2 could phosphorylate SmAREB1 in vitro.

3. Discussion

An increasing number of studies have revealed that the SnRK family is a crucial component in plant signaling transduction and response to abiotic stress [50,51]. Moreover, the SnRK family members of many plants have been clarified, including Liriodendron chinense [52], Brassica napus [53], Casuarina equisetifolia [54], Brachypodium distachyon [55], cotton [56], Triticum aestivum [57] and Phaseolus vulgaris [58]. However, only a few SnRK genes of S. miltiorrhiza were cloned and studied in previous studies [59]. In this study, we systematically identified a total of thirty-three SmSnRK members in the genome of S. miltiorrhiza, including three SmSnRK1 genes, seven SmSnRK2 genes and twenty-three SmSnRK3 genes. The component ratio of SmSnRK subfamily genes in all SmSnRK genes were similar to other species, in which SmSnRK1 subfamily genes made up a small percentage of SmSnRK genes and SmSnRK3 subfamily genes accounted for a large proportion of SmSnRK genes.

It was reported that SmSnRK genes belong to the serine/threonine kinase family. Based on protein motifs analysis, we found that all SmSnRK proteins had a serine/threonine kinase domain at the N-terminal, which further verified the accuracy of the SmSnRK family identification. Moreover, the SmSnRK1 and SmSnRK3 subfamilies contained unique domains at the C-terminal within their subfamily, as did SmSnRK2. This might due to the C-terminal of SmSnRK2 genes being divergent and it was not annotated in this study. Previous studies have shown that the exon–intron structure of the SnRK2 subfamily in higher plants is conserved and most of the SnRK2 genes have nine exons [60]. In S. miltiorrhiza, all SmSnRK2 subfamily members had nine exons, in addition to SmSnRK2.2 and SmSnRK2.3. Phylogenetic tree and exon–intron structure revealed that SmSnRK2.2 and SmSnRK2.3 were closely related and both had eight exons, indicating that the evolution and function of SnRK2 might be relatively conserved. The SmSnRK3 subfamily could be divided into an intron-rich group and an intron-poor group, indicating that the structural diversification of SmSnRK3 may have been caused by gain or loss intron events, similarly to what has been suggested by results reported previously [61]. Previous studies found that the expression level of genes with fewer introns was higher than that of those with more introns, and the response to stress was more timely [62]. However, this was not observed for SmSnRK genes in this study.

Cis-regulatory elements, which were bound to transcriptional factors, played important roles in regulating gene expression. The ABRE, ABA-responsive element, has an ACGT core sequence and is recognized by bZIP transcriptional factors, which are phosphorylated by ABA-responsive SnRK2 kinases [63]. The promoter of SmSnRKs mainly contained light response elements and each promoter of SmSnRK had an average of 1.81 ABRE and 1.06 MeJA response motifs, suggesting that the expression of SmSnRKs was correlated with hormone signaling. The chromosomal location of SmSnRK genes showed that these genes were randomly distributed. The chromosomal location of tanshinone or salvianolic acid biosynthesis genes was also mapped to find out whether the SmSnRK genes clustered with these genes. SmSnRK3.15 was clustered with SmCPS1 and SmKSL1. The SnRK2.2 and SnRK2.3 in S. miltiorrhiza were clustered on chromosome LG8, and these protein sequences shared an identity of 91%, suggesting that these genes were included in tandem duplication occurrences.

Tanshinones and salvianolic acids were the main bioactive compounds of S. miltiorrhiza. Previous studies have shown that MeJA [46] and ABA [47] up-regulated the biosynthesis of these two compounds. Moreover, several genes involved in JA and ABA signaling pathways have been proven to promote the accumulation of tanshinones and salvianolic acids. For example, SmJAZ3 and SmJAZ9, the key repressors of JA signaling, negatively regulated the accumulation of tanshinones and salvianolic acids [64]. SmMYC2, the core component in JA signaling, up-regulated salvianolic acids accumulation [65]. SmSnRK2.6 (homology of OST), a key component in ABA signaling, interacted with SmAREB1 to promote the biosynthesis of salvianolic acids [49]. In Artemisia annua, AaAPK1 was an SnRK2 family member and responsive to ABA treatment, which acted as a positive regulator of artemisinin by phosphorylating AabZIP1 [66]. The expression of genes could give insight into the gene function. According to the transcriptome data for the SnRK2 subfamily members, the transcripts of SmSnRK2.1, SmSnRK2.2 and SmSnRK2.6 were up-regulated by ABA treatment. However, the transcripts of other SmSnRK2 members were slightly down-regulated by ABA treatment. In A. thaliana, SnRK2 could be divided into ABA-dependent and ABA-independent groups [67]. Moreover, Jia et al. reported that the overexpression of SmSnRK2.6 enhanced the accumulation of salvianolic acids, whereas the overexpression of SmSnRK2.3 did not [49]. Based on these findings, we suggested that SmSnRK2 genes could also be divided into ABA-dependent and ABA-independent groups, and that SmSnRK2 genes possessed functional diversity. The transcripts of SmSnRK3.1, SmSnRK3.17 and SmSnRK3.18 significantly increased and reached their highest at 0.5–1 h after ABA treatment, indicating that SmSnRK3 genes might also be involved in ABA signaling. MeJA treatment decreased the transcripts of SmSnRK2.7, while other SmSnRK1 and SmSnRK2 genes remained almost unchanged. The transcripts of SmSnRK3.18 significantly increased and reached their highest at 1 h after MeJA treatment, which was about 38 times higher than the control. The results showed that several SmSnRK3 members might participate in ABA or MeJA signaling.

To further study the relationship between SmSnRK genes and tanshinone and salvianolic acid biosynthesis, a correlation analysis was conducted using the tissue expression analysis and hormone treatment of the SmSnRK gene with tanshinone and salvianolic acid biosynthesis genes. SmSnRK3.15 and SmSnRK3.18 were significantly related to the expression of the tanshinone biosynthesis gene; these genes were predominantly expressed in the roots of S. miltiorrhiza, indicating that SmSnRK3.15 and SmSnRK3.18 might regulate the expression of tanshinone biosynthesis. Based on correlation analysis, SmSnRK3.10 and SmSnRK3.12 were found to have a co-expression with salvianolic acid biosynthesis genes. The analysis revealed that most genes were involved in signal transduction biological processes, and we also found that SmSnRK2.2 interacted with and phosphorylated the SmAREB1 transcription factor, suggesting that several SmSnRK genes were important components in the signaling pathway for regulating tanshinone and salvianolic acid biosynthesis. More importantly, we identified that SmSnRK2.6 is a positive regulator in tanshinones biosynthesis, which was validated by our correlation analysis and shed lights on the role of the SmSnRK gene in tanshinones. This study provides comprehensive information about the SmSnRK family genes in S. miltiorrhiza and evidence for SnRK genes regulating plant metabolism.

4. Materials and Methods

4.1. Identification and Characterization of SmSnRK Genes in the S. miltiorrhiza Genome

To identify the SmSnRK family members in S. miltiorrhiza, the amino acid sequences of SnRK proteins from A. thaliana and O. sativa were downloaded from the TAIR database (http://www.arabidopsis.org, accessed on 15 May 2023) and Rice Annotation Project (RAP) (https://rapdb.dna.affrc.go.jp/, accessed on 15 May 2023). The protein and genome sequences of S. miltiorrhiza were downloaded from the Genome Warehouse (https://ngdc.cncb.ac.cn/, accessed on 15 May 2023) (PRJCA003150). In order to search all of the candidate SmSnRK genes, the Hidden Markov Model (HMM) profile of the Pkinase domain (PF00069) was obtained from the Pfam database (http://pfam.xfam.org/, accessed on 15 May 2023) [68] to find genes with a Pkinase domain by HMMER 3.0. Then, SnRK protein sequences from A. thaliana and O. sativa were set as the query and BLASTP was used against genome sequences of S. miltiorrhiza. Possible SnRK candidate genes were selected manually using the same genes which could be found in both HMM and BLASTP results. These non-redundant sequences were further verified by checking for the presence of the SnRK-related domains within the Pfam database, NCBI conserved domain database (https://www.ncbi.nlm.nih.gov/Structure/cdd/, accessed on 18 May 2023) [69] and SMART (http://smart.embl-heidelberg.de/, accessed on 18 May 2023) [70]. The properties of SnRK proteins, including the isoelectric point (pI), amino acids length and molecular weights (MW) were calculated with the ExPASy-Compute pI/Mw tool (https://www.expasy.org/resources/compute-pi-mw, accessed on 18 May 2023). The subcellular localization of the SmSnRK proteins was predicted with WoLP PSORT (https://wolfpsort.hgc.jp/, accessed on 18 May 2023).

4.2. Phylogenetic Analysis of SnRK Genes

A multiple sequence alignment of all of the SnRK members from S. miltiorrhiza, A. thaliana and O. sativa was conducted with ClustalW [71], using the default settings. A phylogenetic tree was built using the Neighbor-Joining (NJ) method [72] and bootstrap was set to 1000 with MEGA7.0 [73]. The Interactive Tree of Life (iTOL) website (https://itol.embl.de, accessed on 23 May 2023) was used to create the phylogenetic tree in this study.

4.3. Protein Motif and Gene Structural Analysis of SmSnRKs

The conserved motif analysis of the SmSnRK proteins was searched on the MEME website (https://meme-suite.org/meme/, accessed on 4 June 2023) [74] with the motif number of 15 and range from 6–50 amino acids residues. The gene structural analysis of the SmSnRKs was conducted with the Gene Structure Display Server (http://gsds.gao-lab.org/, accessed on 10 June 2023) [75].

4.4. Promoter Analysis and Chromosomal Location

The promoter sequences (2000 bp upstream from the initiation codon) of SmSnRK genes were extracted from the S. miltiorrhiza genome. Then, the promoter sequences were analyzed for cis-elements by the PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 6 June 2023) website [76] and graphed by TBtools [77].

4.5. Expression Analysis of SmSnRK Genes

S. miltiorrhiza was cultivated in greenhouse at Zhejiang Chinese Medical University, Hangzhou. For tissue expression pattern analysis, different tissues including leaves, roots, stems and flowers of one-year-old S. miltiorrhiza plants were collected and frozen in liquid nitrogen immediately. After the samples were ground in liquid nitrogen, the RNApure Plant Kit (Tiangen, Beijing, China) was used for the RNA extraction of samples. For qRT-PCR analysis, cDNA was synthesized by the reverse transcription method (Vazyme). qRT-PCR was conducted on a StepOne Plus Real-Time PCR system (Applied Biosystems, Waltham, MA, USA) and ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) was used. The PCR program was followed with the protocol. The relative expression was calculated by the 2^−ΔΔCt algorithm. SmActin was used as an internal reference gene [78]. Primers used in this study are listed in Supplementary Materials Table S2. Triple biological repeats (each replicate containing five plants) were performed for each sample, and each reaction was measured in triplicate.

To study the expression pattern of SmSnRK genes responses to hormone, the transcriptome data of S. miltiorrhiza treated with ABA [79] or MeJA [80] were obtained from previous studies. The FPKM of SmSnRKs was used to analyze the expression of SmSnRK genes and the FPKM value was standardized by log₂ for heatmap presentation. The heatmap was based on the transcriptome data of hormone treatment and drawn by the TBtools [77].

4.6. Gene Ontology and Correlation Analysis

The Gene Ontology information, including the biological processes and molecular function of SmSnRKs, was analyzed on the PANTHER database (https://ngdc.cncb.ac.cn/databasecommons/, accessed on 23 September 2023) [81]. The correlation analysis of SmSnRKs and tanshinones biosynthesis genes was based on the Pearson correlation coefficient value with R > 0.8 and the p-value < 0.05. The relevance between SmSnRKs and tanshinone biosynthesis genes was drawn by Cytoscape_v3.7.2 software [82].

4.7. Yeast Two-Hybrid (Y2H) Assays

The Y2H assays were performed as described previously [83]. The coding region of SmSnRK2.2 and SmAREB1 were ligated into pGBKT7 and pGADT7 constructing the BD-SmSnRK2.2 and AD-SmAREB1, respectively. BD-SmSnRK2.2 and AD-SmAREB1 were co-transformed into yeast competent cells and the interaction was tested on selective medium (SD/-Trp-Leu and SD/-Leu-Trp-His-Ade). The combination of pGBKT7/AD-SmAREB1, pGADT7/BD-SmSnRK2.2 and pGBKT7/pGADT7 served as the negative control. Primers used in this study are listed in Supplementary Materials Table S2.

4.8. Bimolecular Fluorescence Complementation (BiFc) Analysis

The BiFc assays were performed as described previously [83]. The CDS sequence of SmSnRK2.2 was amplified and ligated into a pXY104 vector to generate the pXY104-SmSnRK2.2-cYFP construct. The CDS sequences of SmAREB1 were amplified and ligated into the vector pXY106 to generate the pXY106-nYFP-SmAREB1 construct, respectively. Then, these vectors were individually transformed into Agrobacterium tumefaciens GV3101, and co-infiltrated into tobacco leaves. After 48 h of infiltration, the fluorescence was captured by a confocal laser-scanning microscope (Zeiss LSM-710, Carl Zeiss, Shanghai, China). The combination of pXY104-SmSnRK2.2-cYFP/pXY106 and pXY106-nYFP-SmAREB1/pXY104 served as the negative control. Primers used in this study are listed in Supplementary Materials Table S2.

4.9. Phosphorylation Assays

For phosphorylation assays, the SmSnRK2.2 and SmAREB1 proteins were obtained by prokaryotic protein expression. First, the coding sequences of SmSnRK2.2 and SmAREB1 were inserted into the pGEX-4T-1 and pET32a vectors, respectively. Then, the vectors were transformed into E. coli BL21 (DE3) for protein production. The purified proteins of SmSnRK2.2 and SmAREB1 were co-incubated in kinase buffer (20 mM Tris-HCl pH 7.5, 20 mM MgCl₂, 1 mM DTT and 0.5 mM ATP) for 1 h, separated by SDS-PAGE with phos-tag (Wako) of 25 μM and 50 μM MnCl₂ and subjected to immunoblot analysis with an anti-GST antibody (Transgen, Beijing, China). Primers used in this study are listed in Supplementary Materials Table S2.

4.10. Trangenic Method

In order to generate the RNAi-SmSnRK2.6 construct, a 250 bp specific coding sequence of SmSnRK2.6 was inserted into the pFGC5941 vector between NcoI and AscI restriction sites. After that, the specific fragment was then reversibly inserted into the BamHI and XbaI restriction sites of pFGC5941, which already contained the specific coding sequence. Then, the construct was transformed into A. tumefaciens strain C58C1 and transgenic hairy roots production was based on our previous study [83]. Primers used in this study are listed in Supplementary Materials Table S2.