Sucrose synthase gene family in Brassica juncea: genomic organization, evolutionary comparisons, and expression regulation

Sucrose synthase (SUS) plays an important role in sucrose metabolism and plant development. The SUS gene family has been identified in many plants, however, there is no definitive study of SUS gene in Brassica juncea. In this study, 14 SUS family genes were identified and comprehensively analyzed using bioinformatics tools. The analyzed parameters included their family member characteristics, chromosomal locations, gene structures and phylogenetic as well as transcript expression profiles. Phylogenetic analysis revealed that the 14 members could be allocated into three groups: SUS I, SUS II and SUS III. Comparisons of the exon/intron structure of the mustard SUS gene indicated that its structure is highly conserved. The conserved structure is attributed to purification selection during evolution. Expansion of the SUS gene family is associated with fragment and tandem duplications of the mustard SUS gene family. Collinearity analysis among species revealed that the SUS gene family could be lost or mutated to varying degrees after the genome was doubled, or when Brassica rapa and Brassica nigra hybridized to form Brassica juncea. The expression patterns of BjuSUSs vary among different stages of mustard stem swelling. Transcriptomics revealed that the BjuSUS01-04 expression levels were the most elevated. It has been hypothesized that they play an important role in sucrose metabolism during stem development. The expression levels of some BjuSUSs were significantly up-regulated when they were treated with plant hormones. However, when subjected to abiotic stress factors, their expression levels were suppressed. This study establishes SUS gene functions during mustard stem development and stress.


INTRODUCTION
Sucrose synthase (Sucrose Synthase, EC2. 4. 1. 13, SUS) is a tetramer composed of subunits with a molecular weight of approximately 83 kD to 100 kD. It is involved in sucrose metabolism (Tanase & Yamaki, 2000;Chalivendra et al., 2007). It catalyzes sucrose synthesis and decomposition. Although sucrose synthase is a reversible enzyme, it converts sucrose and UDP into UDP-glucose and fructose (Nguyen-Quoc & Foyer, 2001). During different stages of plant growth and development, sucrose, fructose and glucose levels are constantly altered. High glucose levels during the early stages are a source of carbon that is required during the rapid growth of plant tissues. This allows plant cells to maintain a high osmotic pressure to ensure adequate water absorption. The gradual increase in sucrose levels after water absorption enhances sugar accumulation in the plant tissues (Dai et al., 2011). Sucrose synthase is involved in sucrose synthesis and decomposition. Therefore, the biological activities and gene expression profiles of SUS vary among different stages of plant growth and development. Studies have reported that SUS is also important in the synthesis of starch (Heim et al., 1993) and fiber cell wall (Ruan, Llewellyn & Furbank, 2003), fruit development (Wang, Smith & Brenner, 1994) as well as in plant stress (Jha & Dubey, 2004).
Since the identification of the first SUS gene in wheat germs, it has been cloned in various crops such as potato, carrot, corn, citrus and Arabidopsis. The SUS gene family has been identified and reported in several plants. Five SUS genes have been identified in six in Arabidopsis, six in rice, three in maize, 15 in Populus and 14 in tobacco Baud, Vaultier & Rochat, 2004;Duncan, Hardin & Huber, 2006;Hirose, Scofield & Terao, 2008;Wang et al., 2015). The SUS gene family is involved in the regulation of multiple pathways throughout the growth cycle of the plant. It has been revealed that when the SUS4 gene is highly expressed during starch synthesis in potato tubers and the activity of SUS enzyme is significantly elevated, there is an increase in ADPG and UDPG levels and a nearly double starch yield (Baroja-Fernandez et al., 2009). Several SUS genes (GhSUS1A-GhSUS8) are involved in cellulose accumulation and secondary wall thickening of the tetraploid cotton xylem. They are important during fiber elongation at different stages of cotton growth (Ruan, Llewellyn & Furbank, 2003).
The tuber mustard is a stem variety of the mustard in which enlarged fleshy stems are widely consumed while fresh and used for mustard processing. During the growth and development of the tumorous stem, the base of the petiole is metamorphosed to form 1-5 tubercular bulges that laterally expand with stem swelling. The genetic analysis of the shape of the swollen stem showed that its shape was mainly controlled by two major genes and polygenes, and there were obvious additive, dominant and epistatic genetic effects (Liu et al., 2014). The tumor-like swelling of the tuber mustard is closely correlated with its yield and quality. Sugar production and transportation in the mustard accumulate in the stem, enhancing its expansion. Sucrose synthase gene functions during stem swelling have not been established. It is, therefore, necessary to determine the structure, expression patterns and evolutionary relationship of SUS gene family in tuber mustard.
Based on the published sequencing results of the mustard genome, we identified 14 SUS genes in the allotetraploid mustard. Bioinformatic tools were then used to identify and characterize the mustard SUS gene in the whole genome of the mustard. In addition, the expression patterns of each member of the mustard SUS gene family in the process of stem swelling and their potential roles in response to various hormones and abiotic stresses were determined. These results provide a basis for further research on the potential functions of each mustard SUS gene, especially during the development of mustard stems.

MATERIALS AND METHODS
Characterization and phylogenetic analysis of SUS genes in the B. juncea genome The B. juncea genome sequence data (V 1.5) used in our study are available within the Brassica database (http://brassicadb.org/brad/). Using BLAST (https://blast.ncbi.nlm.nih. gov/Blast.cgi) with the expectation value of E > 1e −10 , the amino acid sequences of SUS genes in Arabidopsis were used as query sequences to identify homologous genes. All the non redundant gene sequences encoding complete amino acid sequences were considered as BjuSUS genes. Sequence information of 14 BjuSUSs were submitted to NCBI website, with accession numbers from MW370524-MW370537.

Plant materials, phytohormone and abiotic treatments
The experimental samples used to analyze the expression patterns at the developmental stage (2, 4, 6 and 8 cm) were obtained from the four stages of swollen stems of the tuber mustard variety 'Fuza No.2'. All samples were grown in pots in a controlled growth environment. For hormonal and abiotic stress treatment, 4 week old plants with 5-6 true leaves were used in the experiments. The seedlings were separately sprayed with auxin (IAA) (10 mM), abscisic acid (ABA) (75 mM), salicylic acid (SA) (0.5 mM) and gibberellin (GA) (50 mM) and stored for 24 h. Abiotic stress was simulated by low temperature (4 C), drought treatment (10% PEG8000), NaCl treatment (400 mM) and high temperature treatment (38 C). All treatments were set to three biological replicates. After 12 h, the samples were obtained and immediately frozen in liquid nitrogen. They were then stored at −80 C.

RNA extraction and cDNA synthesis
Total RNA was extracted from stems at different growth stages by using the RNA prep Pure Plant kit (Tiangen, Beijing, China). The extracted RNA was spectrophotometrically quantified and electrophoresed on a 1.0% agarose gel to verify its integrity. First-strand cDNA was synthesized from 1.0 µg of total RNA using the PrimeScriptTM RT Reagent Kit with gDNA Eraser (TaKaRa, Tokyo, Japan). All cDNA samples were diluted 1:10 with RNase-free water.

Expression analysis and Quantitative real-time PCR verification
The data obtained from the transcriptome sequencing of the different developmental stages of the stem tumor mustard variety 'Fuza No.2' (2, 4, 6 and 8 cm in diameter, respectively) was submitted to NCBI under the accession number SRP151320. The FPKM value was used to determine the expression abundance of the SUS gene during different developmental stages of B. juncea. The gene expression heat map was plotted using TBtools.
Quantitative real-time PCR (qRT-PCR) was performed using the Bio-Rad CFX96TM real-time PCR System at the standard mode with the 2×T5 Fast qPCR Mix (SYBRGreenI) (TsingKe, Beijing, China). All experiments were performed in triplicate. To normalize gene expression, UBC was used as the internal control . The relative expression levels of BjuSUSs were calculated using the 2 −ΔΔCT method (Pfaffl, 2001). Primers were designed using Primer Premier 5.0 software. The primer sequences used in this study are listed in Table S1.

Functional divergence analysis
Diverge 3.0 was used to perform type I and type II functional disproportionation analysis by the phylogenetic tree of the SUS proteins. Both type I and type II functional divergence occurred after gene replication. Type I functional divergence leads to specific amino acid selective changes, that is, changes in evolutionary rates, and its coefficient θ I fluctuates between 0 and 1. This fluctuation reflects the functional divergence between gene categories from weak to strong. Type II functional divergence indicates that there are site-specific shifts of amino acid properties between subgroups (Gu, 2006). A high Qk (posterior probability) value indicates that the evolution rate or site-level physicochemical amino acid properties between the two clades are highly likely to be different. If Qk > 0.9, it is inferred that the amino acid position may exhibit functional divergence after SUS gene replication (Xun & Kent, 2002).

Analysis of non-synonymous mutation rate to synonymous mutation rate ratios
The homologous gene pair of the BjuSUS gene family was identified using the OrthoMCL software (https://orthomcl.org/orthomcl/). The non-synonymous mutation rate (Ka), synonymous mutation rate (Ks) and Ka/Ks ratio of each pair of homologous genes were calculated using the Ka/Ks Calculator software. If Ka/Ks > 1, the gene is said to have been subjected to positive selection pressure; Ka/Ks = 1 indicates neutral selection; Ka/Ks < 1 is considered to have purification selection.

Identification and analysis of BjuSUSs
Fourteen genes encoding BjuSUS proteins were identified and renamed BjuSUS01 through BjuSUS14. Detailed information on genomic positions, coding region lengths, exon numbers, subcellular localizations and the corresponding proteins are summarized in Table S2. The open reading frame (ORF) size of BjuSUSs ranged from 3,447 (BjuSUS04) to 9,068 bp (BjuSUS10). They coded for proteins with a length that ranged from 755 to 905. The molecular mass of the fourteen proteins ranged from 85.42 to 102.93 kD, whereas their theoretical PI was between 5.67 (BjuSUS01) and 6.36 (BjuSUS13). These findings show that the BjuSUS protein is a weakly acidic protein. Subcellular localization prediction revealed that, except for BjuSUS07 and BjuSUS08 that are located in the mitochondria, the remaining BjuSUSs are located in the cytoplasm.
Multiple sequence alignment revealed a high level of similarity between the amino acid (52.08-99.02%) sequences (Table S3). The highest percentage of amino acid sequence identity was found between BjuSUS09 and BjuSUS10 (99.02%). A high pairwise identity of amino acid sequences was found in BjuSUS01/02, BjuSUS01/04, BjuSUS07/08 and BjuSUS13/14, ranging from 97.01% to 98.64%. The most of the sequences showed less than 80% amino acid sequence identity. The lowest sequence identity was found between BjuSUS04 and BjuSUS09 with 52.08% amino acid sequences.
These 14 genes were widely dispersed on 10 of 18 B. juncea chromosomes and two contigs ( Fig. 1). The number of chromosomal gene distribution was similar in sub-genomes A and B. The BjuSUS13 and BjuSUS14 genes were shown to be accumulated on the same region of the chromosome. This proves the clustering phenomenon. Three BjuSUSs (BjuSUS12, 13 and 14) were located on the chrB03, while the other chromosomes only had one BjuSUS gene.

Phylogenetic relationships of BjuSUS genes
A phylogenetic tree of the mustard and eight other species of SUS proteins was constructed to determine the evolutionary relationships of the SUS gene family in B. juncea. As shown in Fig. 2A, the SUS genes were classified into three groups based on phylogenetic analysis: SUS I, SUS II and SUS III. The SUS I group was branched into the monocot and dicot subgroup while SUS II and SUS III groups were branched into the monocot subgroup only. Phylogenetic analysis revealed that the SUS family members of dicot and monocot plants were distributed in SUS I to SUS III. This implies that amplification of the SUS gene family occurs before differentiation of the dicot and monocot plants. Furthermore, all the SUSs in maize and Triticum aestivum occur in SUS I. Each SUS I and SUS II group contained four BjuSUSs while the SUS III family contained six BjuSUSs (Fig. 2B). The phylogenetic relationship diversity exhibited by the BjuSUSs show the discrete biological role for their paralogs, although multiple sequence alignments have proved that they have high sequence similarities.

Structure and motif location analysis of BjuSUSs
The exon/intron structure analysis showed that the 14 BjuSUSs contained multiple exons and introns that ranged from 11 to 14 and 10 to 13, respectively (Fig. 3A). The organization of the exons/introns was highly conserved, comparable to the high-level similarity observed by the alignment of the 14 amino acid sequences. The coding sequences of BjuSUSs were interspersed by the introns. The BjuSUS10 gene had a long intron of approximately 5 kb sequence in length. It was the longest untranslated region. Fifteen conserved motifs were finally identified from the BjuSUS proteins. The 14 members shared at least 12 common conserved motif compositions and exhibited a coincidental order of motif arrangement ( Fig. 3B; Table S4). Motifs 6, 4 and 12 were missing in BjuSUS04, 09 and 11, respectively. In addition, the BjuSUS10 lacked motifs 13 and 9, while both BjuSUS13 and 14 genes had motif 15 at the end of the protein sequence. Motifs 1 and 5 were connected in each protein sequence, which was similar to motifs 2 and 4. However, the other motifs were separated.

Evolutionary analysis of the BjuSUS genes
Type-I and type-II functional difference analyses were performed among the three SUS groups. As shown in Table 1, the type-I functional divergence coefficient θ I was significantly greater than zero. It signified apparent type-I functional differences. This implied that amino acid site-specific selection may constraint the action in the different SUS groups and lead to specific evolution after diversification of different ethnic groups. The results from type-II functional divergence showed that the coefficient θ II of any two groups was greater than zero. This finding implied that the proteins encoded by the three SUS subtypes exhibited significant type-II functional differentiation. The proteins encoded by the sub-type SUS genes have different physicochemical properties at certain amino acid positions, causing significant disproportionation. In the type-I analysis, 0, 3 and 0 critical amino acid sites (CAASs) that are associated with functional divergence were found in SUS I/SUS II, SUS I/SUS III and SUS II/SUS III groups, respectively. Moreover, in type-II analysis, the corresponding CAASs numbers were 59, 80 and 64, respectively. Variations in critical amino acid numbers between the type-II and type-I groups suggested the promotion of functional divergence in the evolution of the SUS family is mainly attributed to the site-specific shift of amino acid properties, followed by changes in the evolution rate. The ratio of non-synonymous mutation frequency to synonymous mutation frequency for 15 homologous gene pairs in the SUS gene family was analyzed using the Ka/Ks calculation software. In the 15 paralogous gene pairs, all the Ka/Ks ratios were lower than 1, indicating that the 15 paralogous gene pairs were under a strong purifying selection during evolution (Table 2). Since deleterious mutations are usually eliminated by the purification selection process, SUS genes may share conserved functions in B. juncea species.

Synteny analysis of the SUS gene in B. juncea and Arabidopsis
To determine the evolutionary relationship and degree of homology between mustard and Arabidopsis, their comparative synteny at the genome level was analyzed to elucidate the origin of the BjuSUSs (Fig. 4A). There were 2,208 synteny blocks between the B. juncea and Arabidopsis genomes, of which 66.71% were collinear. In the largest linear region,  648 gene pairs were shared between the B. juncea chrA02 and Arabidopsis chr5. This implied that mustard and Arabidopsis have a high homology. Collinearity analysis of AtSUSs and BjuSUSs was used to identify homologous candidate genes for important traits such as morphological development, yield, quality and metabolism of the mustard (Fig. 5). Collinearity analysis revealed 29 SUS gene pairs in B. juncea and Arabidopsis. Among them, fifteen BjuSUSs pairs showed fragmental duplication. These findings revealed that most SUS genes are orthologous in both species. The results show that some BjuSUSs are co-linear with AtSUSs. BjuSUS01, 02, 03 and 04 were found to be homologous to AtSUS1. The evolutionary relationships of BjuSUS genes were further determined based on the genetic relationships between the Brassica crops (Fig. 4B). In the collinearity analysis of B. juncea, Brassica rapa and Brassica nigra, it was found that the synteny blocks between B. juncea and B. rapa was more than 70%. The same finding was established between B. juncea and B. nigra. The higher homology revealed that B. juncea was the offspring of B. rapa and B. nigra. Twelve BjuSUSs were collinear with the genes in B. rapa while 10 BjuSUSs were collinear with the genes in B. nigra. Most of the BjuSUS genes were homologous to the two genes in B. rapa and B. nigra, respectively.

Analysis of cis-regulatory elements and functional annotation of BjuSUS genes
All cis-regulatory elements in the promoter region of BjuSUSs were analyzed using the Plant-CARE database. Elements were classified into three groups based on their functional associations; plant growth and development (13 elements), phytohormone response (8 elements) as well as abiotic and biotic stress (7 elements) (Fig. 6). The results showed that the light-responsive elements (Box-4, G-box, GATA-motif and TCT-motif) appeared 29, 29, 18 and 16 times in the 14 BjuSUSs, respectively. These four cis-regulatory elements were widely distributed in the BjuSUSs promoter region and may be involved in the regulation of mustard growth and development. In particular, G-box appeared eight times in BjuSUS08 while Box 4 appeared six times in BjuSUS05, indicating that they may be significant in light responsiveness and photosynthetic product accumulation. Moreover, 26 abscisic acid responsive elements (ABRE) and 21 anaerobic induction elements (ARE) were detected in BjuSUSs promoters. GO annotation was performed to predict the functions of 14 BjuSUS genes. A total of 116 annotation results were obtained and further divided into 23 GO terms, which were in turn assigned into three ontology categories: biological process (BP), cellular component (CC), and molecular function (MF) ( Table S5). Biological process made up the majority of the GO annotations, followed by cellular component and molecular function. Among the annotations, all the BjuSUSs were involved in "sucrose metabolic process" and "sucrose synthase activity".

Expression levels of BjuSUS genes at different stem developmental stages
Analysis of the expression abundance of BjuSUSs in the transcriptome data at different developmental stages revealed that the expression of each BjuSUS gene has similarities and differences (Fig. 7A). Among them, BjuSUS01-04 maintained a high transcription level at each stage, especially in the first two stages. Its expression levels were highest in the second stage, with a significant decrease in the last two developmental stages. In addition, the expression of BjuSUS07 and 08 was low in the first stage, but was significantly elevated in the latter stage. BjuSUS05, 06 and 10 showed relatively low expression profiles throughout the stem development stages. As shown in Fig. 7B, most BjuSUSs exhibited a distinct but partially overlapping expression trend, that is, high expression in the early stages of stem swelling, and low expression levels in the latter stages of development. However, there were no detectable expression levels in the latter two stages of BjuSUS05 and 06. BjuSUS07 and 08 were highly expressed in the latter stages of development.
These findings were consistent with the results of the transcriptome analysis.

Expression patterns of BjuSUS genes in response to phytohormones
The expression patterns of BjuSUSs in response to abscisic acid (ABA), auxin (IAA), gibberellin (GA) and salicylic acid (SA) were determined (Fig. 8). The expression levels of BjuSUS04, 11, 13 and 14 were up-regulated after treatment with four phytohormones. The expression levels of BjuSUS13 were significantly elevated. In addition, most genes, especially BjuSUS03 and 07, exhibited elevated expression levels after ABA treatment.
BjuSUS02 and 07 exhibited up-regulated expression levels after IAA treatment, while the expression levels of BjuSUS03 and 07 were also up-regulated after SA treatment. Furthermore, the BjuSUS06, 08, 09 and 12 were all down-regulated with hormones.

Expression patterns of BjuSUS genes in response to abiotic stresses
To determine whether BjuSUSs are involved in abiotic stress, qRT-PCR was used to measure their expression levels under salt, drought, and high and low temperature stress. As shown in Fig. 9, the expression level of most BjuSUSs decreased under stress treatment compared to the control group, however, the expression patterns were different when subjected to different stresses. Most of the genes were down-regulated, with some expressions not being detected. The expression levels of BjuSUS03 were significantly

DISCUSSION
Comparative genomics approaches have been used to analyze SUS gene families in various plant species such as Arabidopsis, rice, maize, cotton, grape, peach and poplar (Xu et al., 2019). In this study, 14 SUS genes from B. juncea were identified. Based on their molecular structures, evolutionary models and expression profiles, the BjuSUSs play an important role in the development of B. juncea stems.

Evolutionary conservation among BjuSUS genes
Phylogenetic, structural and evolutionary pattern analyses are fundamental in predicting the possible genetic and evolutionary relationships as well as potential functions of gene families. Phylogenetic tree analysis showed that the mustard SUS genes were associated with dicotyledonous plants such as Arabidopsis. The BjuSUSs were found to be closely clustered in the subgroups. This emphasizes the diversity of SUS genes in monocot and dicot plants. Studies have documented that the protein structure, the length and positional characteristics of introns and exons of the SUS family genes are highly conserved among different species. This protein structure provides important information regarding evolutionary relationships between genes (Lecharny et al., 2003). We found that BjuSUSs have 10-13 introns, and most of the exons between SUS I, SUS II and SUS III groups are highly similar, which slightly differs from the 11-13 introns in the peach . In addition, the number of introns in PtrSUS in the Populus and MdSUS in the apple is between 10 and 14 (Tong et al., 2018;Zhang et al., 2011). These results indicate that the number of introns in the SUS gene family among different species is highly conserved. To determine the evolutionary relationships of the BjuSUS gene family and explore their possible functions, gene duplication events and homology analysis were performed. Tandem duplications (BjuSUS13 and BjuSUS14) were found in chromosomal mapping. Collinearity analysis of mustard and Arabidopsis revealed that there were more fragment duplications between BjuSUSs. Duplicated gene pairs were all from the same subgroup, with highly similar exon/intron structures and protein characteristics. This implies that the amplification methods of mustard SUS gene family include tandem and fragment duplication, which may increase copy numbers in the gene family, resulting in functional redundancy (Cannon et al., 2004). Furthermore, the Ka/Ks results showed that during evolution, BjuSUSs eliminated harmful mutations through purification and selection in order to maintain a conservative structure and functions. Homological analysis provides a basis for the evolutionary and functional characterization of the SUS gene family in mustard and Arabidopsis. Homology analysis revealed that 14 BjuSUSs exhibited a homology with 5 AtSUSs in Arabidopsis. The BjuSUS genes and their corresponding AtSUS genes have a close phylogenetic relationship, which strongly implies a certain degree of functional similarity. Brassica and Arabidopsis belong to the cruciferous family, but diverged about 20 million years ago (Vision, Brown & Tanksley, 2000). After being separated from the Arabidopsis, the Brassica species such as B. rapa and mustard have tripled at the genome level. Moreover, in the "U-triangle" theory, there are three basic subgenomes (A subgenome, B subgenome and C subgenome), and B. juncea (AABB, 2n = 36) is a heterotetraploid formed by interspecific hybridization of B. rapa (AA, 2n = 20) and B. nigra (BB, 2n = 16) (Nagaharu, 1935). Chromosomal rearrangements, translocations, duplications, and gene deletions during doubling greatly promoted the evolution of Brassica. Some SUS genes in mustard have paralogs, and they all exhibited a homology with certain genes (BjuSUS05/06 and BjuSUS01/02/03) in B. rapa and B. nigra. These findings confirm an evolutionary relationship. The mustard did not undergo a larger chromosomal doubling process after the triploid event (Song et al., 2020). However, some BjuSUS genes did not find their corresponding genes, indicating that the SUS family members may have been lost or mutated to varying degrees during evolution.

Divergence in BjuSUS gene expression patterns
BjuSUS genes involved in mustard growth and development Plant growth and development is a complex process. A majority of the cis-regulatory elements associated with plant growth and development are found in the promoter regions. In this study, four genes (BjuSUS01, 02, 03 and 04) were highly expressed during growth and development. The expression levels exhibited an overall decreasing trend, implying that these genes are more important in the early stages of stem development. However, their high expression levels in stems indicate that they are involved in the entire developmental stage of the stem and sucrose metabolism. Similar results have also been reported in other studies. Overexpressing the bamboo BeSUS5 gene increased the stem cellulose content, cell wall thickness and fiber quality in transgenic poplar (Huang et al., 2020). Also, the TcSUS3 gene in cacao pods exhibited a downward expression trend in the developmental stage of the pods . Similar findings were observed in the expression of the SBSS2 gene in sugarbeet root tissues (Haagenson, Klotz & McGrath, 2006).
The four BjuSUSs were found to be structurally similar and belonged to the SUS I group. This implied that they exhibit the same functions during stem development. Studies have shown that the transcription level of the AtSUS1 gene in A. thaliana is highest in the stem while the expression levels of OsSUS1, OsSUS2 and OsSUS3 genes in rice are highest during the early caryopsis development stages (Bieniawska et al., 2007;Hirose, Scofield & Terao, 2008). These genes belong to the SUS I group and exhibit a high level of redundancy, where some genes with similar expression patterns have similar functions. The six AtSUS genes also exhibit distinct but partially overlapping expression patterns (Baud, Vaultier & Rochat, 2004). Except for BjuSUS01-04, the expression levels of BjuSUS genes was low or not expressed, while BjuSUS07 and 08 were shown to be significantly up-regulated in the middle and late stages of stem swelling. During cucumber fruit development, the expression of CsSUS4 in the latter stages has been shown to be significantly high when compared to the early stages. This indicates that they may play an important role in mustard development, thereby, affecting the size and weight of the stem (Fan et al., 2019). Understanding the structure, phylogeny, and homology of the SUS gene is essential in the functional analysis of the SUS gene family. Nucleotide and amino acid sequence similarities of the four BjuSUSs were all found to be above 90%, indicating that they have a high sequence homology. This finding lays credence to their structural and functional similarities.

Phytohormones induce BjuSUSs expression in mustard
When subjected to phytohormonal pressure, the expression levels of some genes in the mustard SUS family were altered. After being subjected to ABA, the expression levels of most genes were shown to be up-regulated. It has been previously reported that ABA elevates the expression levels of grape SUS gene in grape berry calli . BjuSUSs are up-regulated when exposed to GA while GhSusA1 was shown to be upregulated in fiber culture after exogenous administration of gibberellin (Bai et al., 2014). Furthermore, the expression levels of BjuSUS03, 07, 11 and 13 after phytohormonal treatment were found to be significantly different from those of the control group. Therefore, we hypothesized that BjuSUSs are involved in responses to a variety of phytohormone signals and regulate the associated physiological processes.

Abiotic stress-induced BjuSUSs expression in mustard
During plant growth and development, many abiotic stresses are encountered. The SUS gene family has been shown to be involved in regulating stress responses. For example, it can induce AtSS1 gene expression in A. thaliana when the plant is exposed to cold, drought or hypoxic stress factors (Baud, Vaultier & Rochat, 2004). Members of the mustard SUS gene family showed different expression patterns in leaves when exposed to different abiotic stresses. When exposed to salt, drought, heat, and cold stress, the expression levels of BjuSUS03 were significantly up-regulated, indicating that abiotic stress induces its overexpression to enhance the ability of the mustard to resist stress.
The overexpression of OsSUS3, which belongs to the SUS I group, suppresses the damage associated with high temperatures to rice yield and quality (Takehara et al., 2018). Furthermore, the expression of BjuSUS08 was found to be significantly up-regulated in cold stress conditions and down-regulated in the presence of other abiotic stressors. The expression level of tobacco Sus5 was also shown to be significantly elevated after low-temperature treatment (Wang et al., 2015). The expression levels of the other BjuSUSs were significantly down-regulated after exposure to the different abiotic stress factors. These findings are in tandem with those documented in grapes (Zhu et al., 2017) and rubber trees .

CONCLUSIONS
Fourteen SUS genes were identified in mustard. Collinearity analysis revealed that the SUS gene family may be lost or mutated to varying degrees after genome doubling or hybridization of B. rapa and B. nigra to form mustard. The expression patterns of BjuSUSs in different stages of mustard stem development indicate that BjuSUS01-04 have an important role in the biological processes associated with development. Plant hormones and abiotic stress induced the expression of BjuSUSs, indicating that this gene family is involved in their regulation.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This work was supported by the National Natural Science Foundation of China (No. 32002027), the Shuangzhi project of Sichuan Agricultural University (03573134) and the Undergraduate Research Interest Cultivation Project of Sichuan Agricultural University (No. 2019295). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures
The following grant information was disclosed by the authors: National Natural Science Foundation of China: 32002027. Shuangzhi project of Sichuan Agricultural University: 03573134. Undergraduate Research Interest Cultivation Project of Sichuan Agricultural University: 2019295.