Genome-wide identification and gene expression analysis of Clade A protein phosphatase 2C family genes in Brassica juncea var. tumida

Abscisic acid (ABA) plays crucial roles in plant response to environmental stresses and development. The clade A phosphatases (PP2Cs) play a crucial role in ABA signaling. However, little is known about the details regarding PP2Cs family genes in Brassica juncea var. tumida. Here, 20 clade A PP2Cs family genes were identified in tuber mustard genome, including BjuABI1s, BjuABI2s, BjuAHG1s, BjuAHG3s, BjuHAB1, BjuHAB2s, BjuHAI1s, BjuHAI2s and BjuHAI3. The promoters of BjuPP2Cs family genes contained various of cis-acting elements, such as ABRE, GT1GMSCAM4, ARFAT and MYB1AT. We also analyzed the expression pattern of clade A BjuPP2Cs under abiotic stresses (low temperature, NaCl and ABA) treatment, pathogen Plasmodiophora brassicae treatment and different stages of stem swollen. The results suggested that clade A BjuPP2Cs regulated tuber mustard response to P. brassicae to mediate the formation of clubroot and might play roles in stem swelling and response to abiotic stresses. This study provides valuable information for further functional investigations of clade A PP2Cs family genes in B. juncea var. tumida.


Introduction
The reversible phosphorylation of proteins by protein phosphatases is a crucial process which modulates plant growth and development [1,2] . Protein phosphatases (PPs), removed the phosphate group of phosphorylated proteins, play important roles in regulation of protein function. PPs can be divided into two major subfamilies: protein tyrosine phosphatases and protein serine/threonine phosphatases. Based on the distinct amino acid sequences and crystal structures, the protein serine/threonine phosphatases can be classified into two groups: the phosphor-protein phosphatase (PPP) and the phosphoprotein metallophosphatases (PPM). The PP1, PP2A and PP2B subgroups belong to PPP family, whereas the PP2C subgroup belongs to the PPM family (Mg 2+ or Mn 2+ dependent) [3] .
ABA plays a crucial role in plant response to environmental stresses such as abiotic stresses (salinity and low temperature stresses) and biotic stresses (such as pathogen) [4][5][6] . Until now, the core components of the ABA signaling pathway include pyrabactin resistance (PYR)/PYR1-like (PYL) regulatory components of ABA receptor (RCAR) protein family (ABA receptors), the co-receptors clade A protein type 2C phosphatases (PP2Cs), and sucrose nonfermenting-1-related protein kinase class 2 (SnRK2s). In the absence of ABA, PP2Cs interact with SnRK2s to removing the phosphate group and inhibit the kinase activity of SnRK2s, which resulted in turning off ABA signaling pathway. In the presence of ABA, ABA binds the PYR/PYL/RCAR receptors and then interacts with PP2Cs, which inhibit the phosphatase activity of PP2Cs and eliminate the inhibitory effect of the phosphatases on SnRK2s to turn on the ABA signaling [7][8][9] . In Arabidopsis, nine clade A PP2Cs (AtABI1、AtABI2、AtAHG1、AtHG3、AtHAB1、AtHAB2、AtHAI1、AtHAI2 and AtHAI3) have been identified as the negative regulators in ABA signaling pathway.
The dominant mutations abi1-1 and abi2-1 showed the insensitive phenotype to ABA in germination and greening stages indicating that ABI1 and ABI2 were the negative regulators in ABA signaling [10] . The ahg1-1 and ahg3-1 mutant showed ABA hypersensitivity phenotype in germination and post germination growth stages, but not in adult plants. And the expression level of AHG1 was strongest in seeds indicating that it played an important role in response to ABA in seed [11,12] . HAB1 underwent alternative splicing and produced two splice variants, named HAB1.1 and HAB1.2. HAB1.1 and HAB1.2 played the opposing roles in ABA-mediated seed germination and post-germination stages [13] . In B.rapa the PP2C genes were structurally conserved based on the amino acid sequence alignment, phylogenetic analysis and conserved domains; and the gene expression levels were induced by heat, cold, ABA and drought treatment [14] . Twenty group A PP2C homologous genes of B. oleracea were identified; the genetic analysis corroborated the presence of two to three gene copies in B. oleracea in comparison to clade A PP2C genes in Arabidopsis thaliana; the gene expression patterns of PP2Cs in B. oleracea were significant differences [15] .
The swollen stem of tuber mustard is the raw material of Fuling mustard and is an important vegetable. However, little is known about the regulation mechanism of stem swelling [17] . And during the growth and development stages, tuber mustard frequently suffers from abiotic and biotic stresses, such as salt stress, cold stress and Plasmodiophora brassicae, which leads to the suppression of plant growth and development and the formation of clubroot. Clubroot is one of the most serious biotic stresses that plants need to cope with and usually results in the suppression of tuber mustard growth and stem swollen, leading to limitation in yield [18] . Therefore, illustrating the mechanisms underlying the stem swollen and the resistance to abiotic and biotic stresses will be helpful for improving the production of tuber mustard.
Clade A PP2Cs of ABA signaling pathway play important roles in response to abiotic and biotic stresses and regulation plant development. However, the gene structures, protein motifs, gene duplication events and functions of clade A PP2Cs in B. juncea var. tumida remains mainly unknown.
In this study, twenty clade A PP2C family genes were identified in B. juncea var. tumida genome. The phylogenic relationship, gene structures, and protein motifs were compared between clade A BjuPP2Cs and AtPP2Cs and found that they shared similar gene characteristics. Following the analysis of gene features, we analyzed the transcript levels of clade A BjuPP2Cs under P. Brassicae , NaCl, low temperature and ABA treatment and different stages of stem swollen. The results showed that clade A BjuPP2Cs were induced by pathogen and abiotic stresses and induced in the stages of stem swollen, suggesting that clade A BjuPP2C family genes played crucial roles in plant response to abiotic stresses, pathogen P. Brassicae and regulation of stem swollen.

Materials and growth conditions
The tuber mustard cultivar Yong'an was used in this study. The seeds were sterilized and sowed in MS medium. The growth room was at 22°C and 6000 lx (long-day

Gene expression analysis
Total RNA of tuber mustard was extracted under pathogen and abiotic stresses treatment. qRT-PCR was performed using the cDNA. The transcript level was analysis by the comparative CT method, and BjuActin3 was used as the internal reference. The qRT-PCR experiments were carried out three times with three replicates each. The primers used in this study were listed in Table S1.

Statistical analysis
All data were analyzed using SigmaPlot 10.0 (Systat Software, Inc., Chicago, IL) and SPSS 16.0 software. The averages and standard deviations of all results were calculated, and for multiple groups of samples, the one-way ANOVA followed by the Dunnett test was used. The statically significant treatments were marked with '***' (P<0.001), '**' (0.001<P<0.01) and '*' (0.01<P<0.05). and B03 contained three genes ( Figure 1).

Phylogenic analysis and gene structures of clade A PP2C family genes
To analyze the evolutionary relationships between PP2C homologs in B. juncea var.
tumida and Arabidopsis thaliana, a phylogenetic tree was constructed by MEGA5 software using the neighbor-joining method. According to the phylogenic tree, twenty clade A BjuPP2Cs with nine AtPP2Cs were identified and clustered into nine clades.

Promoter cis-acting regulatory elements prediction of clade A BjuPP2C homologs
To further understand the potential roles of clade A BjuPP2Cs, the promoters of clade results showed that all BjuPP2Cs contained at least one hormone-related elements in the promoters such as ARFAT (TGTCTC, responsive to auxin) [20] and ASF1MOTIFCAMV (TGACG, responsive to auxin and salicylic acid) [21] (Figure 4).

Tissue specific expression pattern analysis of clade A BjuPP2Cs
To investigate the tissue specific expression patterns of clade A BjuPP2Cs, we analyzed the gene expression levels at different growth stages and tissues (root, stem, swollen stem, leaf, flower and pod) using qRT-PCR. According to the results, clade A BjuPP2Cs were expressed in multiple tissues. Interestingly, the expression levels of BjuABI2-1, BjuAHG3-1 and BjuHAB2 were inhibited ( Figure 6). brassicae tolerance (Figure 7). BjuPP2Cs in B. juncea var. tumida during stem

swelling stages
The swollen stem is the raw material of "Fuling Mustard", however, the regulation mechanism of stem swelling was still undiscovered. To further explore the roles of

Discussion
The swollen stem of B. juncea var. tumida is the raw material of Fuling mustard, which is an important economic vegetable in China and famous for its special flavour and nutritional value [17] . P. brassicae leads to the formation of clubroot in tuber mustard, which is one of the most serious stresses and results in the inhibition in plant growth and stem swollen, and leading to limitation in yield [18,31] . Clade A PP2C family genes play important roles in the regulation of plant development and tolerance to stress, however, the identity and expression patterns of B. juncea var. tumida clade A PP2C genes are unknown. This study is the first identification of clade A BjuPP2Cs in B. juncea var. tumida and adds a new layer to the function of clade A

BjuPP2Cs.
In this study, twenty clade A BjuPP2Cs genes were identified and located in 13 of 18 chromosomes based on the nine clade A AtPP2Cs sequences in Brassica Database ( Figure 1). ABI1 had five homologous genes located in A01, A03, B05, A08 and B03; ABI2 had two homologous genes located in B02 and A10; AHG1 had two homologous genes located in A03 and B08; AHG3 had three homologous genes located in A05, B07 and B01; HAB1 and HAB2 both had one homologous gene located in the same chromosome B03; HAI1 had two homologous genes located in A10 and B02; HAI2 had three homologous genes located in A09, B06 and A10 ( Figure 1, Table 1). The comparable homologous gene numbers in A sub-genome and B sub-genome suggested tuber mustard genome experienced co-linearity gene duplication. However, the homologous genes of HAB1, HAB2 and HAI3 were not duplicated or lost in tuber mustard, indicating that these genes existed a functional redundancy or divarication during the evolutionary process. The losses of genes during the genome duplication events also frequently exist in tuber mustard and other Brassica species, such as BjuPYLs family genes in tuber mustard [32] and chitinase family genes in B. rapa [33] . Gene duplication contained three evolutionary fates: subfunctionalization, neofunctionalization, or nonfunctionalization [14,34] . In our study, each of clade A BjuPP2Cs contained 1-5 copies with different gene structures and expression patterns. BjuABI1-2, BjuABI1-3 and BjuABI1-4 were induced by low temperature (Figure 6).
Gene expression analysis showed differences in clade A BjuPP2C genes expression pattern in tuber mustard. Our results indicated that clade A PP2C-based stem swollen regulation and P. brassicae response in tuber mustard has evolved distinctly.          stages. Data were normalized to the expression level of BjuActin3. The values are means ± standard error. Three independent biological repeats were performed.

Table S1
The primers used in this study.

Fig. S1
Multiple alignment of conserved motifs of the clade A PP2C family proteins.
The multiple alignment of conserved motifs was constructed using CLUSTALW. The clade A PP2C family proteins showed 11 conserved sequence motifs, named motif 1 to motif 11 along the top (blue font diaplay). Residues conserved of clade A PP2Cs highlighted in red (totally conservation) and yellow (high conservation).