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. 2022 Sep 3;28(9):1695–1711. doi: 10.1007/s12298-022-01222-3

Genome-wide identification and characterization of the C2 domain family in Sorghum bicolor (L.) and expression profiles in response to saline–alkali stress

Jiangshuai Niu 1, Zhijiang Li 2, Jiarui Zhu 1, Rong Wu 1, Lingxin Kong 1, Tingli Niu 1, Xueying Li 1, Xinran Cheng 1, Jianying Li 3, Lingyan Dai 1,✉
PMCID: PMC9636366  PMID: 36387979

Abstract

The C2 domain family proteins in plants has been recently shown to be involved in the response to abiotic stress such as salt and drought stress. However, less information on C2 domain family members has been reported in Sorghum bicolor (L.), which is a tolerant cereal crop. To elaborate the mechanism of C2 domain family members in response to abiotic stress, bioinformatic methods were used to analyze this family. The results indicated that 69 C2 domain genes belonging to 5 different groups were first identified within the sorghum genome, and each group possessed various gene structures and conserved functional domains. Second, those C2 family genes were localized on 10 chromosomes 3 tandem repeat genes and 1 pair of repeat gene fragments were detected. The family members further presented a variety of stress responsive cis-elements. Third, in addition to being the major integral component of the membrane, sorghum C2 domain family proteins mainly played roles in response to abiotic and biotic stress with their organic transport and catalytic activity by specific location in the cell on the basis of gene ontology analysis. C2 family genes were differentially expressed in root, shoot or leaf, and shown different expression profiling after saline–alkali stress, which indicated that C2 family members played an important role in response to saline–alkali stress based on the transcription profiles of RNA-seq data and expression analysis by quantitative real-time polymerase chain reaction. Besides, most C2 family members were mainly located in cytoplasmi and nucleus. Weighted gene co-expression network analysis revealed three modules (turquoise, dark magenta and pink) that were associated with stress resistance, respectively. Therefore, the present research provides comprehensive information for further analysis of the molecular function of C2 domain family genes in sorghum.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-022-01222-3.

Keywords: Sorghum bicolor (L.), C2 domain family genes, Bioinformatics, WGCNA, Saline–alkali stress

Introduction

As the fifth most cultivated grain crop in the world, Sorghum bicolor (L.) grown in saline–alkali soil areas offers strong growth ability and high yield (Li et al. 2021). With the expansion of saline–alkali land, it is of great importance to understand the molecular mechanism of the response to abiotic stress in sorghum.

Plants can sense and respond to various environmental signals and then initiate appropriate responses, which will lead to changes in gene expression. The Ca2+ concentration in the cytoplasm of plant cells has been proven to increase briefly and rapidly under various environmental stresses, and then couple with downstream signal transduction pathways through Ca2+-binding proteins (Bartels and Sunkar 2005; Kopka et al. 1998; Hetherington and Brownlee 2004). Most of these Ca2+-binding proteins contain C2 domains involved in targeting proteins to cell membranes. C2 domain was initially identified in mammals as one of the two conserved domains (C1–C2) in Ca2+-dependent protein kinase C (PKC) (Nalefski and Falke 1996), while many other C2 domain families have no calcium binding activity (Zhang and Aravind 2010, 2012). As a large family, the C2 domain can bind to many different ligands and substrates, such as Ca2+, phospholipids, inositol polyphosphates and intracellular proteins. With the development of genetic, biochemical, molecular and cell biological methods in plants, great progress has been made in identifying calcium-sensitive proteins and understanding the functions of some calcium-regulatory proteins. Although plants have several unique Ca2+-sensing proteins, the downstream components of Ca2+ signals in plants are still poorly understood (Reddy 2001). Most studies have shown that C2 domain proteins in plants play important roles in stress responses and signal transduction during biotic and abiotic stresses, such as drought tolerance (Schapire et al. 2008; De Silva et al. 2011; Pak Dek et al. 2020), salt stress tolerance and membrane targeting (Kim et al. 2008). The C2 domain protein V3-PLC3 in the mung bean has been suggested to play an important role in membrane transport in response to abiotic stress (Kim et al. 2004; Wang 2002). Under abiotic stress, such as salt stress, CaSRC2, which encodes a C2 domain protein in pepper, was significantly upregulated (Kim et al. 2008). In rice, overexpression of OsSMCP1, a small protein encoding a single C2 domain, can improve salt tolerance in transgenic Arabidopsis thaliana (Yokotani et al. 2009). OsC2DP, encoding a new protein containing a C2 domain, was significantly downregulated as a salt-responsive gene in rice roots, while others found that OsC2DP was necessary for salt tolerance in rice (Cotsaftis et al. 2011). Those reports indicated that the roles of C2 domain proteins in response to abiotic stress were important. Therefore, it is necessary to study the function of C2 domain genes in plants.

Although C2 domain families have been analyzed in several plant species (Chen 2019; Sun et al. 2021), there is still less information on sorghum. With the announcement of sorghum genome, it might be a possible breakthrough to identify new genes regulating salt and alkali tolerance. Bioinformatics methods were used in the present research to identify and characterize the C2 domain family on to analyze their phylogenetic relationship, chromosomal distribution, motif composition and cis-elements. The expression patterns of some C2 domain family numbers were further analyzed under saline–alkali stress. This research might present a theoretical reference for the transcriptional regulation mechanism and functional analysis of C2 domain family members and then provide candidate genes for the molecular breeding of sorghum to tolerate salt alkali stress.

Materials and methods

Identification of C2 domain family members in sorghum genome

The genome sequence, protein sequence and annotations of sorghum were available from the Ensembl database (http://plants.ensembl.org/index.html) (Finn et al. 2016). The Raw Stockholm format file with characteristic domain alignment information of C2 proteins was downloaded from the Pfam website by retrieval number PF00168 for further application of HMMER software to retrieve the sequence of sorghum C2 domain proteins (Johnson et al. 2010). First, the hmm build module in HMMER software was used to transform the Raw Stockholm format file to a Hidden Markov Model (HMM) format file. The HMM file was then used as a query for secondary retrieval in the FASTA format file with representative protein sequences of sorghum using the hmm search module, and the screening of the E-value threshold adopted the default value. The outputs of protein sequences of sorghum C2 domains were submitted to Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi), SMART (http://smart.embl-heidelberg.de) and Pfam database (Finn et al. 2016) for further screening to exclude the predicted protein sequences lacking C2 domains. C2 domain family member proteins in sorghum were finally obtained from all nonredundant and high confidence genes and were separately renamed according to their positions on the chromosomes. The length of sequences, molecular weights and isoelectric point prediction of the identified C2 proteins were obtained by scripts written by the Omicsgene Company.

Phylogenetic relationship, chromosomal distribution and gene replication event analysis of C2 domain genes

Multisequence alignment of C2 domain genes in sorghum was performed by ClustalW software. The paired distance and neighbor connection (NJ) algorithm were used to construct a phylogenetic tree through MEGA 7.0 (Kumar et al. 2016) for 1000 iterations, in which the Evolview online tool (https://evolgenius.info//evolview-v2/#login) was used to construct the phylogenetic tree. All C2 domain genes were mapped to sorghum chromosomes based on physical location information of the sorghum genome from the Ensembl database by Mapchart 2.32 software (Voorrips 2002). Gene duplication events were analyzed using the Multiple Collinearity Scan toolkit (MCScanX) (Wang et al. 2012) of TBtools (Chen et al. 2020) with the default parameters. Each repeat fragment with a C2 domain gene was selected to generate a synthetic map by CIRCOS (Krzywinski et al. 2009). Syntenic analysis maps that can exhibit the synteny relationship of the orthologous C2 domain genes obtained from sorghum and other selected species were constructed by Dual Systeny Plotter software (https://github.com/CJ-Chen/TBtools).

Gene structure, motif composition and cis-acting element analysis of C2 domain genes

The gene structure maps were generated using the GSDS online website (http://gsds.gao-lab.org/). The motif information was obtained from MEME Suit 5.3. 3 (Bailey et al. 2009). Those maps were then imported into TBtools 10 to generate the cluster composition. The upstream sequences with 1.5 kb of the C2 domain gene coding sequence were selected from the sorghum genome and then submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plancare/html/) to identify cis-acting elements related to abiotic stress.

GO annotation and RNA-seq data analysis

Blast2GO 6.0.1 software (https://david.ncifcrf.gov/) was used to execute gene ontology (GO) analysis of sorghum C2 domain family genes. The full-length amino acid sequences of sorghum protein containing the C2 domain were uploaded to the program, and seed plants were selected as reference databases for analysis of molecular function, cellular composition and biological process.

The RNA-seq data of sorghum C2 domain family genes under saline–alkali stress at 0 h, 4 h, 24 h and 72 h and the corresponding control treatments were all from our research group. The raw fragments per kilobase per million (FPKM) values were equalized by the average of the whole data. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Chen et al. 2021) in National Genomics Data Center (CNCB-NGDC Members and Partners 2022), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA007686) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa. The processed data were converted by log2, and then Omicstudio platform (http://www.omicstudio.cn/tool/4) was used to generate the expression heatmap of C2 domain genes.

RNA extraction and qRT-PCR analysis

Wild-type sorghum P898012 was selected as the raw material. When the radicles grew to 2–3 cm length, the seeds were transferred to a hydroponic device and cultured in Hoagland nutrient solution with a photoperiod of 16 h light/8 h dark at 25 ± 1 °C. The saline–alkali stress solution was prepared by using Hoagland nutrient solution as solvent, and NaHCO3 and Na2CO3 in a molar ratio of 5:1 were dissolved together to a concentration of 40 mM. When the seedlings grew to three leaves and one heart, the treated group was cultured with saline–alkali stress solution, and the control was still cultured in Hoagland nutrient solution. To verify the expression profiling of C2 domain family genes under saline–alkali stress, the plants were obtained and frozen in liquid nitrogen at 0 h, 6 h, 12 h, 24 h, 48 h and 72 h with three biological replicates, respectively. While the plants obtained at 4 h after saline–alkali stress were used to study the tissue-specific expression patterns of C2 domain genes. RNA was extracted using TRIzol reagent.

Primer Premier 5 software was used to design gene-specific primers. The cDNA was reverse transcribed with a First Strand cDNA Synthesis Kit, ReverTra Ace-α (TOYOBO, Shanghai, China). All operating procedures were carried out according to the kit instructions. Before qRT-PCR analysis, cDNA was diluted to 500 ng/μL with nuclease-free water. qRT-PCR was performed on a Bio-Rad CFX96 Real Time PCR System using SYBR Green real-time PCR Master Mix (TOYOBO, Shanghai, China). Each PCR was carried out in a 20-μL reaction volume comprising 10 μL KAPA SYBR, 0.5-μL 10-μm primer solution, 1 μL diluted cDNA, and 8 μL dd H2O. The PCR program was set as follows: 95 °C for 2 min, 95 °C for 15 s, 57 °C for 15 s, 72 °C for 45 s, and 40 cycles. The melting curve was analyzed from 65 to 95 °C, increasing at 0.5 °C every 5 s. The actin-3 (XM_002463521) gene was used as an internal reference gene, and the relative expression level of the gene was calculated by the 2−ΔΔCt method. The results are shown as the mean ± standard deviation (SD).

Subcellular localization analysis

Subcellular location prediction of all identified C2 proteins was carried out by the CELLO website (http://cello.life.nctu.edu.tw/). In all predicted results, the subcellular component with the highest reliable-index score was selected. Meanwhile, the subcellular location of SbC2-29 protein was analyzed. An expression vector with green fluorescent protein (GFP) marker was constructed (Lampropoulos et al. 2013). The coding sequence length (CDS) of SbC2-29 gene was specifically amplified by PCR with GG-SbC2-29-F (aacaggtctcaggctCCATGGCGACGACGCGGAC) and GG-SbC2-29-R (aacaggtctcactgaGAAATCAAAGCCGCCATCGAA), then the final expression vector was constructed by Green gate method, and transformed in Escherichia coli to extract the fusion plasmid of pGG-SbC2-29-GFP. The recombinant plasmid of pGG-SbC2-29-GFP was transformed into Agrobacterium tumefaciens. Finally, Agrobacterium cells were suspended in 3 mL of dip solution (containing 10 mM MgCl2, 10 mM MES, 150 μM acetosyringone, pH = 5.6), and kept at room temperature for 3 h. After a small opening was gently made on the back of the tobacco leaf by a needle, the bacterial solution was injected into the wound of the leaf, and the water-collapsed area of the tobacco leaf was marked. Then the injected plants were cultured at about 21 °C for 2 days, the lower epidermis of the marked area in tobacco leaves was torn off, and the position of the fluorescence was observed using a fluorescence microscope (Leica, Germany).

Physiological measurements of sorghum under saline–alkaline stress

Three seedlings with the same growth status were picked and weighed from the control group and the treatment group, respectively. According to the ratio of tissue mass (g) and extract volume (mL) to 1:10, three seedlings were homogenized in ice bath and centrifuged 12,000 rpm for 10 min at 4 °C. Finally, the supernatant containing the crude enzyme extract of tissue was obtained and measured according to Bradford method (Bradford 1976). The enzymatic activity of CAT, POD and SOD in different samples were detected using kits (Nanjing Jiancheng Bioengineering Institute, China), respectively. The malondialdehyde (MDA) content was detected according to the trichloroacetic acid method.

Weighted gene co-expression network analysis (WGCNA)

The previous studies have shown that C2 domain family genes are involved in the production of reactive oxygen species. In present research, according to the FPKM value of differentially expressed genes from the RNA-seq data of sorghum under saline–alkali stress as the expression data, and the physiological indicators of sorghum resistance (POD, SOD, CAT and MDA) as the phenotypic data, WGCNA analysis was conducted to construct co-expression networks of genes. The cutreeStatic function was used to remove the offending sample. The soft thresholding power β was chosen based on the lowest power for which the scale-free topology fit index reached a high value. Module-trait associations were estimated using the correlation between the module eigengene and saline–alkali/control treatments. Cytoscape (version 3.9.1) was used to export the network edge and node information of genes in each module, and to visualize the network. The plug-in cytoHubba was used to identify the top 20 hub genes with maximal clique centrality (MCC) computing method.

Results

Screening, identification and phylogenetic analysis of C2 domain gene family members in sorghum

After screening through the CDD, SMART and Pfam databases, 69 potential C2 domain family members were finally identified in the sorghum genome, which come from Sorghum_bicolor_NCBIv3 in Ensembl. Statistical results showed that the protein sequence lengths of 69 C2 domain genes were in the range of 133–2144 amino acid residues. The predicted isoelectric point (pI) of C2 domain family proteins ranged from 3.86 to 11.4, and the molecular weight (MW) ranged from 14.5 to 229.6 kDa (Tab. S1). To analyze the evolutionary relationships among the C2 domain family members, a rootless NJ phylogenetic tree was constructed. According to their genetic relationship, 69 C2 domain family members were classified into 5 distinct groups (I–V) or 8 subgroups (A–H). Among them, each Groups I, II and V included two subgroups. Group V containing 22 proteins was the largest one, while the smallest group, Group IV, contained only 3 proteins (Fig. 1).

Fig. 1.

Fig. 1

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Phylogenetic relationship of C2 domain genes in sorghum. The phylogenetic tree was generated by comparing the amino acid sequences of the C2 domain proteins in MEGA 7. Using the NJ adjacency method and setting the bootstrap value to 1,000, all genes were classified into five groups (I, II, III, IV, and V)

Chromosome distribution and collinearity analysis of C2 domain family genes

According to the position of their corresponding genes on chromosomes, the member genes of the sorghum C2 domain family were distributed on all 10 chromosomes, while the distribution on chromosomes was uneven (Fig. 2), and most C2 domain genes were located at the proximal or distal ends in sorghum chromosomes. The largest members, fifteen C2 domain genes, were localized on chromosome 2, and only one gene was on chromosome 8.

Fig. 2.

Fig. 2

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Chromosome mapping of C2 domain family genes in sorghum. The scale on the left represents the chromosome length, and the number corresponding to each gene represents the position of the gene on the chromosome. The percentage in brackets on each chromosome represents the distribution frequency of C2 domain family genes on this chromosome. Genes labeled with different colors indicate that they are classified into different group. Red represents Group I, purple represents Group II, blue represents Group III, orange represents Group IV and black represents Group V

To identify the repeated events in the sorghum C2 domain family, collinearity analysis of C2 domain family genes were performed (Fig. 3A). Based on the definition criteria, 6 C2 domain genes (SbC2-55/SbC2-41, SbC2-03/SbC2-15 and SbC2-35/SbC2-48) were clustered into three tandem duplication event regions on sorghum linkage Groups I, II, and V. Twelve pairs of independent repeat genes were located on various chromosomes, among which SbC2-05 had collinearity with SbC2-55, SbC2-22 and SbC2-41, which indicates that some C2 domain family genes might be produced by gene replication and that segmental replication events were the main driving force for the evolution of C2 domain family genes. To better understand the evolutionary constraints acting on the C2 domain gene family, the Ka/Ks values of C2 domain gene pairs were calculated. The Ka/Ks values of both the SbC2-03 and SbC2-15 genes were less than 1 (Ka/Ks = 0.0998 < 1), and those values of most of the orthologous C2 domain gene pairs were more than 1, indicating that the C2 domain gene family in sorghum might have experienced strong positive selection pressure during evolution.

Fig. 3.

Fig. 3

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Collinearity analysis of C2 domain family in sorghum. A Only chromosomes containing C2 domain genes are represented in the circle. Chromosomes are painted in different colors. The approximate location of the C2 domain gene is shown by a short black line on the circle. B Collinearity of the C2 domain family in different species. The red curve linking C2 domain genes represents repeated events in the sorghum C2 domain family

To further infer the evolutionary mechanism of the sorghum C2 domain family, a comparative collinearity map related to two representative species was constructed, including a dicotyledonous plant (Arabidopsis thaliana) and a monocotyledonous plant (maize) (Fig. 3B). A total of 58 C2 domain genes in sorghum were collinear with those in maize, while 12 genes were collinear with those in Arabidopsis thaliana. According to this result, there are multiple C2 domain genes with high homology within the same species or among different species. We speculate that these genes might have played an important role during the evolution process. In addition, some collinear gene pairs were identified between sorghum and Arabidopsis thaliana or maize, which did not exist in different species at the same time, indicating that these orthologous pairs were formed after the differentiation of dicotyledonous plants and monocotyledonous plants. Other identification of some collinear gene pairs among Sorghum bicolor and Arabidopsis thaliana or maize were collinear, indicating that those pairs might have already existed before ancestral differentiation.

Gene structure and motif analysis of C2 domain family genes

Gene structure plays a vital role during the evolution of multiple gene families. A NJ phylogenetic tree was first constructed with MEGA7 (Fig. 4A). The number of introns ranged from zero to more than a dozen after analysis of the gene structure of C2 domain family genes (Fig. 4B). Among them, 19 genes (27.5%) were intron-free, and the other genes had two or more introns, which may be due to the closely related replication relationship, and tandem duplication genes contain the same number of introns, such as SbC2-55/SbC2-41, SbC2-03/SbC2-15 and SbC2-35/SbC2-48. However, intron–exon structural changes may result in duplicate gene differentiation and amino acid insertion and deletion. Afterwards the MEME program was used to identify the conserved motifs of C2 domain family proteins, the predicted patterns were annotated at the same time, and 10 highly conserved motifs were identified (Fig. 4C). Each C2 domain family protein contained different numbers of conserved motifs ranging from 1 to 8. Generally, the closely related C2 domain family proteins on adjacent branches of the phylogenetic tree have the same or similar motif structure, which further supports the phylogenetic classification of the C2 domain family. Combining evolutionary relationship and motif analysis, numerous members belonging to the same subclass can be seen to repeatedly appear to have the same sequence structure, which indicates that members of the same subclass may have the same or similar biological functions or regulate similar biological processes. The diversity of conserved sequences outside the C2 domain and twelve different permutations and combinations of motifs were found in the sorghum C2 domain family, indicating that the C2 domain family undergoes extensive domain shuffling after genome replication.

Fig. 4.

Fig. 4

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Phylogenetic relationship, conserved motifs and gene structure analysis of C2 domain family members in sorghum. A Phylogenetic trees of 69 C2 domain proteins. B Exon–intron organization of C2 domain genes. Yellow boxes represent exons, and black lines of the same length represent introns. The upstream and downstream regions of C2 domain genes are indicated by green boxes. The sizes of exons can be estimated by the scale at the bottom. C Arrangements of conserved motifs in the C2 domain proteins. Ten predicted motifs are represented by different colored boxes, and motif sizes are indicated by the scale at the bottom

To further study the potential regulatory mechanisms of the C2 domain family during the abiotic stress response, the promoter region of the C2 domain family gene was obtained and then submitted to PlantCARE to detect cis-elements. The results indicated that C2 domain family members contained a variety of response elements related to abiotic stress, including ABA response elements ABRE4 and ABRE, GA response element W-BOX, MeJA response element CGTCA-motif, drought response element MYB, and low temperature response elements LTR and AP2 (Fig. S1). These results might further indicate that those genes were involved in responses to different abiotic stresses.

GO annotation of C2 domain proteins

The highly divergent sequences outside the conserved C2 domain indicate that C2 domain family proteins are involved in multiple biological processes. To understand the function, a GO annotation of sorghum C2 domain proteins was performed with seed plants as the reference species. The results are shown in Fig. 5. Molecular functional analysis showed that most of the C2 domain proteins were active at the molecular level in the form of binding activity (28, 60.87%) and enzyme catalytic activity (23.50%). At the same time, most of the C2 domain proteins (49, 90.74%) were located on the membrane, but some were located in intracellular anatomical structure (23, 42.59%), nucleus (6, 11.11%) and other anatomical structures. In addition, some C2 domain proteins existed in multiple cell components. For example, SbC2-02 was located on three cell components: plasma membrane, integral component of membrane and vesicles, which may reflect its multifunction in various biological processes. The analysis of biological processes also showed that C2 domain proteins were involved in a variety of biological processes. Among these processes, the responses to cellular processes (23, 45.1%), stress biological processes (27, 52.94%) and biological regulation processes (24, 47.06%) involve the largest number of C2 domain proteins. The C2 domain protein could also respond to stress. Additionally, some members were involved in the regulation of catalytic activity, signal transduction, primary metabolic processes, localization and development. Therefore, based on the analysis of three aspects of GO analysis, C2 domain proteins may play a role in the development of various tissues and organs, signal transduction and cell communication in response to abiotic and biotic stress with their organic transport and catalytic activity by specific location in the cell.

Fig. 5.

Fig. 5

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Gene ontology (GO) annotation of C2 domain proteins. The annotation was performed on three categories: A molecular function, B biological processes and C cellular component

Expression profiling of C2 domain family genes under saline–alkali stress

To determine the dynamic expression of C2 domain family members in sorghum under saline–alkali stress, their expression levels from differential transcriptome data during various saline–alkali treatment periods were analyzed, and differential expression patterns were constructed (Fig. S2). The RNA-seq data were from sorghum seedlings treated with saline and alkali at the three-leaf and one-heart stages. A total of 22 differentially expressed genes (DEG) in response to saline–alkali stress were screened from 69 C2 gene families in sorghum. Among these families, several C2 domain family genes indicated obvious responses to stress; for example, the transcription levels of SbC2-25, SbC2-46 and SbC2-18 gradually increased with prolonged stress time, SbC2-02 gradually decreased, and SbC2-40 fluctuated in waves.

Verification of C2 domain family genes by qRT-PCR

To investigate the response of C2 domain family genes to saline–alkali stress at the transcriptional level, twelve genes (SbC2-17, SbC2-18, SbC2-19, etc.) were selected for further expression profiling after saline–alkali stress by qRT-PCR (Fig. 6). In general, expression of the genes changed in different growth periods. Compared with the control, some C2 domain genes were significantly induced/inhibited by saline–alkali treatment and showed different expression patterns under the same treatment. For example, some C2 domain genes, such as SbC2-19 and SbC2-63, were significantly induced by saline–alkali treatment, while SbC2-60 were significantly inhibited by saline–alkali stress. In contrast, the transcription level of SbC2-43 and SbC2-67 showed wave-like changes. In addition, the response speed of different genes with the same trend to salt stress was also different. For example, compared with SbC2-19, SbC2-63 responded faster and lasted longer with salt stress. These results indicated that the expression of C2 domain family genes did have different responses to alkali and salt stress. Among all the genes, some were not expressed in all the tested samples, which may be pseudogenes or special spatiotemporal expression patterns that we have not seen.

Fig. 6.

Fig. 6

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Expression profiles of 12 selected C2 domain genes in response to saline-alkali treatment in different processing periods. Quantitative RT-PCR was used to investigate the expression levels of C2 domain genes, and the results are represented by means ± standard deviations. The vertical coordinate is multiple changes, and the horizontal ordinate is the processing time. Asterisks indicate that the corresponding gene was significantly up- or down-regulated compared with the untreated control (*P < 0.05, **P < 0.01, Student’s t-test)

Tissue-specific expression patterns of C2 domain genes

To comprehend the tissue-specific expression patterns of sorghum C2 domain genes at 4 h after saline–alkali stress, the expression pattern of above selected 12 genes in different tissues were obtained by qRT-PCR (Fig. 7). Most sorghum C2 domain genes showed diverse transcript levels in various tissues. In particular, SbC2-67 was not expressed in the leaves, and SbC2-19 was only expressed in root after stress. SbC2-58 was most expressed in root, SbC2-19, SbC2-40 and SbC2-43 in leaves, and SbC2-18, SbC2-50, SbC2-63 were expressed mainly in root and leaves. It’s easy to draw a conclusion that C2 domain genes play important roles in plant growth.

Fig. 7.

Fig. 7

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Expression of 12 selected C2 domain genes in response to saline-alkali treatment in various tissues. Quantitative RT-PCR was used to investigate the expression levels of C2 domain genes, and the results are represented by means ± standard deviations. Different lowercase letters indicate significant differences (P < 0.05)

Subcellular localization

In order to further understand the function of C2 domain family proteins, subcellular localization prediction analysis was performed. The results showed that most members of this family were mainly located in cytoplasmic and nuclear compartments, few were in cytoskeletal and extracellular (Fig. 8A). SbC2-29 was selected to verify the subcellular localization due to their response to saline–alkali stress at the transcriptional level. The results showed that the NLS-3 × mcherry control was detected red fluorescent signal in the nucleus, while SbC2-29-GFP could also be detected green fluorescent signal in the nucleus, which indicated that SbC2-29-GFP and NLS-3 × mcherry could be co-localized in the nucleus. In addition, the blue fluorescence signal of AtHA1-BFP localized in the cell membrane could be superimposed with the green fluorescence signal of SbC2-29-GFP. The results indicated that SbC2-29 is mainly localized in the cell membrane and nucleus (Fig. 8B).

Fig. 8.

Fig. 8

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Subcellular localization of C2 domain proteins. A The prediction of subcellular localization of 69 C2 domain proteins. B Subcellular localization of SbC2-29 protein in N. benthamiana. The fluorescence signal of SbC2-29-GFP could be detected in the same subcellular localization of NLS-3 × mcherry and AtHA1-BFP respectively, indicating that SbC2-29 is mainly localized in the cell membrane and nucleus

Detection of physiological indicators of sorghum under saline–alkaline stress

When plants respond to abiotic stress, various antioxidant enzymes can play a crucial role in scavenging the production of reactive oxygen species. The activities of CAT, POD and SOD in the whole sorghum plant were detected (Fig. S3). The results showed the activity changes of CAT and POD were all same between control and treated group, that is, within 0–4 h, the enzyme activity increased, and the enzyme activity of the treatment group was significantly higher than that of the control group. And at 72 h, there was no significant difference between the treatment group and the control group. For SOD at 4 h, the enzyme activity of the treatment group was significantly lower than that of the control group. Salinity–alkali stress would increase the degree of damage to sorghum membrane system, and treatment at 4 h may be a turning point. There was no significant difference in the contents of MDA between the treatment group and the control group before 4 h, but at 24 h and 72 h, the content of MDA in the treatment group was significantly higher than that in the control group, respectively.

WGCNA identifies candidate modules associated with seedling saline–alkali resistance traits

All 31,237 differentially expressed genes from RNA-seq were retained for WGCNA unsigned co-expression network analysis. The soft threshold power of 12 (β = 12) was selected according to the preconditions of approximate scale-free topology. The analysis identified thirty distinct co-expression modules (labeled with diverse colors) shown in the dendrogram, of which turquoise module was positively associated with MDA and CAT, with correlation coefficient (r) of 0.81 and 0.58 respectively. While darkmagenta modules positively associated with the SOD (correlation coefficient is 0.58) and pink modules positively associated with the POD (correlation coefficient is 0.8) (Fig. S4). These results indicates possible correlations between genes and some physiological indicators contributed to the improvement of salt alkali resistance.

Network visualizing

The top 10 hub genes in each module were identified via the cytoHubba plug-in (Fig. 9). Genes in three modules had different functions, such as RING-H2 finger protein ATL80 (Sb10g020990) in turquoise module, putative E3 ubiquitin-protein ligase (Sb06g026880) in darkmagenta module, serine carboxypeptidase II-3 (Sb01g033780) and aspartic proteinase nepenthesin-1 (Sb10g006870) in pink module were participated in the progress for post translational modification protein turnover, chaperones, amino acid transport and metabolism. And rho GTPase-activating protein 3 (Sb03g047080) in darkmagenta module and oligopeptide transporter 7 (Sb10g0015300) in pink module were participated in the progress of signal transduction mechanisms. While sucrose transport protein SUT5 (Sb07g028120) and glucose-6-phosphate 1-dehydrogenase (Sb06g020640) in pink module were about carbohydrate transport and metabolism.

Fig. 9.

Fig. 9

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The top 10 genes in each module are calculated by MCC algorithm of cytohubba. A Top 10 genes in turquoise module. B Top 10 genes in darkmagenta module. C Top 10 genes in pink module. The network interface will display the connections of these hub nodes in the network. The darker the node color, the higher the score

Discussion

The current study identified 69 C2-domain genes and analyzed their structure, chromosomal location, phylogeny, gene duplication, stress-related cis-elements and expression patterns in different tissues and abiotic stresses. This study provides comprehensive information on the C2-domain gene family and will aid in understanding the functional divergence of C2-domain genes in sorghum. Previous research identified 180 (Chen 2019) and 90 C2 domain proteins (Sun et al. 2021) in Soybean and Arabidopsis, respectively, and 69 C2 domain genes in Sorghum bicolor. The difference in the number of C2-domain genes in different species may be related to their genome size and evolutionary relationship (Boulesteix et al. 2006). In the evolution of sorghum, tandem duplication, segment duplication and positive selection pressure may jointly promote the expansion of the C2 domain family.

The C2 domain protein family was clearly studied in present research. According to recent studies, C2 domain protein family genes were involved in regulating plant responses to environmental stress and pathogen infection (De Silva et al. 2011). However, information about the function of the C2 domain family in sorghum is still limited. To understand the unknown gene functions, it is necessary to predict their possible molecular functions and involvement in biological processes on the basis of gene sequences. With the development of bioinformatics technology, researchers have built a variety of methods to predict the function of unknown genes. All genes of the sorghum C2 domain family were identified by various bioinformatics analysis methods in present research, their sequences were characterized from multiple angles, and the expression profiles in response to saline–alkali stress were systematically analyzed. This information might provide useful clues for the functional characterization of C2 domain family genes, especially their role in stress tolerance.

In present study, 69 C2 domain family members were identified in the sorghum genome. Compared with the results of 180 and 90 C2 domain genes identified in Soybean and Arabidopsis thaliana, the difference in the number of C2 domain genes in different species may be related to their genome size and evolutionary relationship (Boulesteix et al. 2006). According to the phylogenetic tree, C2 domain family genes can be divided into 5 groups and 8 subgroups. Most of the C2 domain proteins were mainly located on the cell membrane, few were located in the intracellular anatomical structure, organelle, cell periphery, cell junction and protein-containing complex according to cell composition analysis, which indicated that they were versatile in various biological processes.

The sorghum C2 domain members, in comparison with other C2 domain proteins from the same species, were more closely related to members from different species belonging to the same family, which meant that the evolutionary relationship between different species belonging to the same family was closer. Gene organization plays a vital role in the evolution of multiple gene families (Mao et al. 2019). Through the joint analysis of evolutionary relationships and motifs, motif analysis was basically consistent with phylogenetic analysis. Members of the C2 domain family in each subgroup usually shared subclass-specific motifs. In addition, different types of C2 domain family members contained different numbers of exons. Combined with the results of gene family duplication doubling analysis, we can infer that tandem repeats, segment repeats and positive selection pressure may promote the expansion of the sorghum C2 domain family together, resulting in the production of different numbers of exons and structural differences in genes with common evolution (Cannon et al. 2004). In some studies, genes with few or no introns are considered to have enhanced expression levels in plants. To respond to various stresses in a timely manner, genes must be activated rapidly, which would be assisted by a compact gene structure with fewer introns (Zhao et al. 2018). In our study, most C2 domain genes were highly induced under saline–alkali stress, and the response time of SbC2-63 with 8 introns to saline–alkali stress was later than the response time of SbC2-19 with 2 introns (Fig. 6 and Fig. 4).

In our study, GO analysis revealed multiple functions of C2 domain proteins. Our results showed that C2 domain family members contain a variety of response elements related to abiotic stress (Fig. S1), and qRT-PCR results further verified that many C2 domain family members respond to saline–alkali stress, but the expression patterns of different members under saline–alkali stress are quite different (Fig. 6). Salt and high osmotic pressure induce an increase in intracellular Ca2+, which is related to the expression of pressure-responsive genes (Zhao et al. 2010). Although plant C2 domain genes are still unknown, many reports have indicated that they are involved in Ca2+ signaling and gene expression through plant nuclear calcium signaling and calcium-regulated transcription (Xie et al. 2011; Galon et al. 2010; Charpentier and Oldroyd 2013). Among these genes, synaptotagmin-5 (e.g., SbC2-49) may be involved in Ca2+-dependent exocytosis of secretory vesicles through Ca2+ and phospholipid binding to the C2 domain or may serve as Ca2+ sensors in the process of vesicular trafficking and exocytosis (Haberman et al. 2003). Calcium-dependent lipid binding (e.g., SbC2-12) may be involved in membrane trafficking and act as a repressor of abiotic stress (e.g., drought and salt) responses by binding specifically to the promoter of THAS1 to regulate its transcription (De Silva et al. 2011).

When plants are subjected to various stresses, abiotic stresses induce a rapid accumulation of reactive oxygen species (ROS), which will lead to an imbalance in the production and removal of active oxygen in the plant body (Huang et al. 2019; Pizzino et al. 2017). The imbalance between systems may cause excessive accumulation of ROS, which in turn damages proteins, membrane lipids and other cellular components and causes oxidative damage to plants (Poljsak et al. 2013). In rice, ROD1 can interact with catalase CatB to promote CatB activity and transfer it from the peroxisome to the plasma membrane to degrade ROS produced during the immune process, and this function depends on the calcium binding ability of ROD1 (Gao et al. 2021). C2-DOMAIN ABA-RELATED-like proteins (e.g., SbC2_18) have been reported to be able to mediate the transient calcium-dependent interaction of PYR/PYL/RCAR abscisic acid (ABA) receptors with the plasma membrane and thus regulate ABA sensitivity (Diaz et al. 2016; Rodriguez et al. 2014). In addition, the protein can stimulate the GTPase/ATPase activities of YchF1 and regulate its subcellular localization. In addition, the protein can also promote tolerance toward salinity stress by limiting the accumulation of ROS (Cheung et al. 2013).

Previous studies have shown that C2 domain proteins can interact with Ca2+ because there are five conserved aspartic acid (Asp) residues in the protein sequence (Rizo and Südhof 1998). Ca2+ was also reported to play a vital role in coping with abiotic stress as the second messenger (Zheng et al. 2014). Although most C2 domains function in a Ca2+-dependent manner, some of the C2 domains may function without binding to Ca2+ (Xie et al. 2011). For example, CaSRC2-1 (SbC2-13 in sorghum), encoding a new protein containing a C2 domain, can also be closely related to the abiotic stress response, even though the C2 domain plays an important role in the localization of the plasma membrane (Kim et al. 2008). The C2 domain of CaSRC2-1 did not contain five Asp residues involved in Ca2+-dependent binding of other C2 domain-containing proteins, while the expression of CaSRC2-1 depended on Ca2+ (Kim et al. 2003, 2008). These results suggest that different members of the C2 domain in sorghum may be involved in different regulatory mechanisms and signaling pathways and may have evolved to have different functions.

Although Ca2+-binding proteins can be identified rapidly by bioinformatics, progress in elucidating the functions of these proteins is still limited (Reddy 2001). In addition, the functions of known C2 domain-containing proteins in plants have not been characterized, and it is likely that many C2 domain Ca2+-sensing proteins are involved in the transduction of plant stress signals as positive or negative regulators of the stress signal cascade. The study of these proteins may lead to new biotechnology strategies to improve the stress resistance of crops.

A large number of studies have shown that the expression of C2 domain genes are unlike in diverse plants, for example, CaSRC2-1 (homologous gene of SbC2-29) was mainly expressed in roots (Kim et al. 2008). We found the expression of SbC2-29 was the highest one in roots and leaf, and most sorghum C2 domain genes showed diverse transcript levels in various tissues (Fig. 7).

When plants are subjected to various stresses, a large amount of active oxygen is produced. A primary mechanism of harm to plants is the imbalance of the production and removal of active oxygen in the plant, which in turn damages proteins, membrane lipids and other cellular components, as well as causes oxidative damage to plants (Adamipour et al. 2020). AtSRC2, as homologous gene of SbC2-29, is a novel activator of Ca2+-dependent AtRbohF-mediated ROS production with a role in cold response (Kawarazaki et al. 2013). Our study shows that SbC2-29 is located in the nucleus and cell membrane, that means the response of SbC2-29 to abiotic stresses may be involved in the multiple signaling pathways. In order to mine genes related to ROS scavenging, we used WGCNA to perform association analysis of RNA-seq data with resistance indicators. WGCNA is a progressive analysis method in which variable genes are divided into co-expression modules through an unsigned network. In present study, we identified three modules were associated with resistance metrics and constructed a network of correlations. Among them, sulfate transporter 3.1 (Sb01g046410) in turquoise module as H+/sulfate cotransporter that may play a role in the regulation of sulfate assimilation (Cao et al. 2013). Protein IQ-DOMAIN 14 (Sb02g035710) in darkmagenta may be involved in cooperative interactions with calmodulins or calmodulin-like proteins andrecruits calmodulin proteins to microtubules, thus being a potential scaffold in cellular signaling and trafficking (Bürstenbinder et al. 2017). It is worth noting that glucose-6-phosphate 1-dehydrogenase (Sb06g020640) in pink module can provide reducing power (NADPH) and pentose phosphates for fatty acid and nucleic acid synthesis which are involved in membrane synthesis and cell division (Wakao and Benning 2005). And peroxidase 9 (Sb03g010230) in pink module can play a role in removal of H2O2, oxidation of toxic reductants, biosynthesis and degradation of lignin, suberization, auxin catabolism, response to environmental stresses such as wounding, pathogen attack and oxidative stress. These functions might be dependent on each isozyme/isoform in each plant tissue (Ostergaard et al. 1998). In this study, the function of C2 domain proteins of sorghum need further elaborate.

Conclusions

Genome-wide analysis of the C2 domain family in sorghum was carried out, and 69 C2 domain family genes were identified. Similar exon–intron structures and motif arrangements of C2 domain proteins in subcones further supported the classification of the phylogenetic tree prediction. C2 domain family genes were distributed on 10 sorghum chromosomes. Three pairs of C2 domain family genes were identified as duplicates, indicating that these duplications played an important role in the expansion of the C2 domain family. GO analysis showed that C2 domain family proteins had multiple functions. RNA-seq and qRT-PCR results showed that several C2 domain family genes responded to saline–alkali stress at the transcriptional level. The expression levels of some C2 domain family genes showed significant changes during saline–alkali stress. At the same time, the expression of some C2 domain family genes did not change under saline–alkali stress, and the response time of some genes to saline–alkali stress was different. C2 family domain genes were differentially expressed in root, shoot or leaf. Most members of this family were mainly located in cytoplasmic and nuclear. WGCNA revealed three modules (turquoise, darkmagenta and pink) were associated with stress resistance respectively, in which many DEG from RNA-seq were involved. These results revealed the potential role of C2 domain family genes in response to saline–alkali stress and provided comprehensive information for further analysis of the functions of the C2 domain family. However, the specific function of the C2 domain gene needs further study. In summary, the purpose of this study was to explore the important role of C2 domain genes in sorghum under saline–alkali stress.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 14435 KB) (14.1MB, docx)

Funding

This work was supported by the National Key R&D Program of China (2018YFD1000702/2018YFD1000700), the Postdoctoral Start-up Science Foundation of Heilongjiang (LBH-Q19164), Heilongjiang Bayi Agricultural University Support Program for San Heng San Zong (ZRCLG201906), the Natural Science Foundation of Heilongjiang Province (C2016046), and Innovation Research Project for Master Student supported by Heilongjiang Bayi Agricultural University (YJSCX2021-Y105, YJSCX2021-Y107).

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher's Note

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Change history

10/9/2022

A Correction to this paper has been published: 10.1007/s12298-022-01233-0

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