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Article

Genome-Wide Analysis of the β-Amylase Gene Family in Brassica L. Crops and Expression Profiles of BnaBAM Genes in Response to Abiotic Stresses

by
Dan Luo
,
Ziqi Jia
,
Yong Cheng
,
Xiling Zou
and
Yan Lv
*
Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan 430062, China
*
Author to whom correspondence should be addressed.
Agronomy 2020, 10(12), 1855; https://doi.org/10.3390/agronomy10121855
Submission received: 28 October 2020 / Revised: 18 November 2020 / Accepted: 22 November 2020 / Published: 25 November 2020
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Abstract

:
The β-amylase (BAM) gene family, known for their property of catalytic ability to hydrolyze starch to maltose units, has been recognized to play critical roles in metabolism and gene regulation. To date, BAM genes have not been characterized in oil crops. In this study, the genome-wide survey revealed the identification of 30 BnaBAM genes in Brassica napus L. (B. napus L.), 11 BraBAM genes in Brassica rapa L. (B. rapa L.), and 20 BoBAM genes in Brassica oleracea L. (B. oleracea L.), which were divided into four subfamilies according to the sequence similarity and phylogenetic relationships. All the BAM genes identified in the allotetraploid genome of B. napus, as well as two parental-related species (B. rapa and B. oleracea), were analyzed for the gene structures, chromosomal distribution and collinearity. The sequence alignment of the core glucosyl-hydrolase domains was further applied, demonstrating six candidate β-amylase (BnaBAM1, BnaBAM3.1-3.4 and BnaBAM5) and 25 β-amylase-like proteins. The current results also showed that 30 BnaBAMs, 11 BraBAMs and 17 BoBAMs exhibited uneven distribution on chromosomes of Brassica L. crops. The similar structural compositions of BAM genes in the same subfamily suggested that they were relatively conserved. Abiotic stresses pose one of the significant constraints to plant growth and productivity worldwide. Thus, the responsiveness of BnaBAM genes under abiotic stresses was analyzed in B. napus. The expression patterns revealed a stress-responsive behaviour of all members, of which BnaBAM3s were more prominent. These differential expression patterns suggested an intricate regulation of BnaBAMs elicited by environmental stimuli. Altogether, the present study provides first insights into the BAM gene family of Brassica crops, which lays the foundation for investigating the roles of stress-responsive BnaBAM candidates in B. napus.

1. Background

Abiotic stresses reduce plant growth, productivity and quality, which leads to great economic loss for the farming community. To deal with abiotic stresses, plants have evolved adaptive mechanisms in their growth and development processes [1]. Starch is the major carbohydrate storage in plants, which is mainly produced in chloroplast during the day and mobilized during the following night to guarantee a steady supply of carbon and energy [2,3]. Previous studies have shown that starch metabolism is a key determinant in the stress response. For instance, the starch content of leaf is found to be decreased under drought [4,5,6,7], osmotic [8,9], extreme temperature [10,11], salt [12,13], and oxidative stress conditions [14]. These changes are accompanied by released sugars and other derived metabolites, that function as compatible solutes to protect against the damage caused by abiotic stress [11,15]. However, osmotic and cold stresses also trigger an increase in starch content, as well as the related soluble sugars [2,16,17,18], suggesting that starch may play different roles in plant species under stress conditions [3].
The general degradation pathway of starch involves α-amylase (AMY), β-amylase, limit dextrinase (PUL), β-glucosidase, and α-glucan phosphorylase (PHO) [19]. β-amylases (BAMs) have been recognized to be responsible for starch degradation and gene regulation [20,21]. The catalytic activity of BAM is found to elevate in a series of starch-deficient mutants of Arabidopsis (Arabidopsis thaliana (L.) Heynh.), these enzymes are subsequently cloned, including AtBAM1, AtBAM2, AtBAM3, AtBAM5 [22,23,24]. Only these four members (AtBAM1, AtBAM2, AtBAM3, AtBAM5) were catalytically active, which show highly conserved amino acid motifs known to be involved in catalysis [7]. AtBAM7 and AtBAM8 show to be catalytically inactive in vitro [25,26], which are localized in the nucleus and contain a BRASSINAZOLE RESISTANT1 (BZR1)-type DNA binding domain attached to the N-terminus of the BAM domain [27]. Deregulation of AtBAM7 and AtBAM8 (the bam7bam8 double mutant) causes altered leaf growth and development. These unique features suggest a regulatory role of AtBAM7 and AtBAM8 in controlling plant growth and development through crosstalk with brassinosteroid signaling and starch metabolism [28]. Besides the above, AtBAM4 has no catalytic capacity but facilitates starch breakdown independently of AtBAM1 and AtBAM3 [22]. Silencing of StBAM1 and collective silencing of StBAM1 and StBAM9 in potato (Solanum tuberosum L.) demonstrate decreased β-amylase activity in cold-stored tubers. Meanwhile, soluble starch content increases in the RNA interference (RNAi) line of StBAM1, but decreases in the RNAi line of StBAM9, suggesting that StBAM1 may regulate cold-induced sweetening in potato tubers by hydrolyzing soluble starch, whereas StBAM9 directly acts on starch granules [29]. These observations raise the possibility that BAMs perform regulatory roles within starch metabolic pathways.
In Arabidopsis, BAMs act at the non-reducing ends of α−1, 4-linked glucan chains to produce maltose, which are the main hydrolytic enzymes during the night when leaf starch is broken down [22]. Maltose is the central product of starch degradation [30] and is well documented for its protective role upon cold stress [11,31]. AtBAM1 could be transcriptionally induced by heat stress at 40 °C, while AtBAM3 could be influenced by cold stress at 5 °C, consistent with the known daytime role of BAM1 in the guard cell stroma or the nighttime role of AtBAM3 in the mesophyll cells, respectively [2]. These changes correlate with maltose accumulation; the in vitro assays demonstrate that maltose functions as a compatible-solute stabilizing factor to protect proteins, membranes, and the photosynthetic electron transport chain in the chloroplast stroma under acute temperature stresses [11]. Similarly, the RNAi line of AtBAM3 prevents maltose accumulation upon cold shock, which accordingly increases the sensitivity of PSII photochemical efficiency of Arabidopsis under freezing stress conditions [17]. Additionally, diurnal starch degradation is triggered by thioredoxin-regulated AtBAM1 in osmotically stressed mesophyll cells, and the produced osmolytes (maltose) reveals an active role in protection against osmotic stress [9]. Current studies on the upstream activators or repressors of BAMs are still limited. In rice (Oryza sativa L.), three OsBAMs function downstream of a negative regulator OsMYB30 under cold stress conditions, a further study on OsmiR528a has established a model that OsmiR528a targets OsMYB30-BAMs to enhance cold stress tolerance of rice [31,32]. PtrBAM1 is a chloroplast-localizing BAM gene of Poncirus trifoliate, which has been reported to function in cold tolerance by modulating starch degradation. The C-repeat binding factor (CBF), termed PtrCBF interacts with the promoter of PtrBAM1, and regulates PtrBAM1 in cold stress response by modulating soluble sugar levels [33]. Nevertheless, more regulation machinery of BAMs in stress signaling and response remains to be determined.
B. napus L. belongs to Brassica genus (B. genus), which is a major oil crop widely distributed worldwide. The allotetraploid B. napus (AACC, 2n = 38, http://www.genoscope.cns.fr) is generated by the crossing of Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18) [34]. Throughout the life cycle, B. napus experiences adverse environments, like extreme temperatures, drought, salinity or flood [35,36,37,38,39,40]. With the publishing of B. napus genome, genome-wide characterization of functional genes has been accelerated. The BAM gene family has been recognized across the plant kingdom, including 9 AtBAMs in Arabidopsis [41], 17 PbBAMs in pear (Pyrus bretschneideri Rehd.) [19], 10 OsBAMs in rice [42]. Nevertheless, the BAM members have not been reported in B. genus. The present study aims to characterize the BAM multi-gene family in B. napus, B. oleracea and B. rapa. The comprehensive analyses involving a phylogenetic tree, gene structure, chromosomal distribution, homologs and collinearity were conducted. Furthermore, the expression patterns under various abiotic stress conditions were evaluated to determine the function of BAM genes in B. napus. The information will provide a platform for future functional study of the molecular regulatory mechanisms of the BAM gene family in Brassica crops under abiotic stress conditions.

2. Materials and Methods

2.1. Identification of BAM Genes in B. napus, B.oleracea and B. rapa

The protein sequences of Arabidopsis BAM genes were obtained from Tair website (Tair10, http://www.arabidopsis.org) [43]. These AtBAMs were used as queries to search against the genomes of Brassica crops to characterize the candidate BAMs. The genome-related data from B. napus (Darmor-bzh), B. rapa (Chiifu), B. oleracea (var.capitata line 02–12) were derived from http://www.genoscope.cns.fr, http://brassicadb.org, https://plants.ensembl.org, respectively. The potential BAMs were checked in order to verify the existence of the conserved glycoside hydrolyase 14 domain (PF01373) by the Pfam (http://pfam.sanger.ac.uk) database [44]. The molecular weight and isoelectric point of each BAM protein were retrieved from the ExPASy program (http://web.expasy.org/protparam/).

2.2. Phylogenetic Analysis

The phylogenetic tree was constructed for BAM members of Brassica crops and Arabidopsis using the MEGA 7.0 software based on the Neighbour–Joining method. The evolutionary distances were evaluated using the Poisson correction method based on the units of the number of amino acid substitutions per site. The nodes of the tree were evaluated by bootstrap analysis with 1000 replicates [45]. Multiple alignments were carried out based on the full-length protein sequences of BAM gene family via ClustalX software (ver.1.83) with default settings, and Jalview [46].

2.3. Gene Structure, Chromosomal Location and Synteny Analysis

The exon/intron structural features of the corresponding BAM genes were explored using the Gene Structure Display Server (GSDS) database (http://gsds.cbi.pku.edu.cn). The start and stop locations of all BAM gene members were obtained from the Brassica database, MapChart software was used to visualize the distribution of each gene as described previously [47]. BLASTP program was employed to identify the tandem duplicated genes using BAM protein sequences of four dicots (i.e., Arabidopsis, B. napus, B. rapa, B. oleracea) with E-value cutoff  ≤1 × 10−10. These BAM proteins were employed to identify orthologous regions with the parameters (e =1 × 10−20). The syntenic analysis maps of four plant species were constructed using MCSCanX and linearized on the chromosomes using the Circos program [45].

2.4. Materials and Treatments

The semi-winter type of B. napus, named Zhongshuang 6 (ZS6), was used for gene expression analysis and physiological measurements under abiotic stress conditions. The germinated seeds of ZS6 were transplanted to 10 cm × 10 cm pots with uniform growth, each pot containing five seedlings. The ZS6 variety was planted in three pots as three biological repeats and grown in a chamber (MLR-35IH, Panasonic, Japan) with a 16-h-light/8-h-dark cycle at 21 °C, the light intensity was 250 μ mol m−2 s−1, and the humidity was controlled at 55% (±5%). To performed the short-term abiotic stress treatment, the four-leaf seedlings were treated with cold (continuous 4 °C in the chamber), dehydration (stopping water supply in the chamber), flooding (submerging completely with water in the chamber), salinity (irrigation with 100 mM/L NaCl solution in the chamber), heat (continuous 37 °C in the chamber). The leaves were sampled for three biological replications before stress treatments (denoted as 0 h), and 1 h, 6 h, 12 h as well as 24 h after stress treatments according to previous studies [11,19,33,39]. To perform the long-term cold stress treatment, the four-leaf seedlings were treated with a 16-h-light (8 °C)/8-h-dark (4 °C) cycle for 21 days. The leaves of seedlings were sampled for three biological replications before stress treatments (denoted as 0 d), and 1 d, 7 d, 14 d as well as 21 d after stress treatments.

2.5. Expression Analysis

Total RNA was extracted using the TransZol Up Plus RNA Kit (TransGen Biotech Co., LTD, Beijing, China) according to the manual instructions. Three micrograms total RNA was synthesized to the first-strand cDNA using the EasyScript®One-Step cDNA Synthesis SuperMix (TransGen Biotech Co., LTD, Beijing, China) according to the manufacturer’s instructions. The Quantitative Real-time PCR (qRT-PCR) was performed using the SYBR®Green Premix kit according to the manufacturer’s instructions on a StepOnePlusReal-Time PCR System (Applied Biosystems, Waltham, MA, USA). The relative expression level was determined by the 2−ΔΔCt method based on three biological repetitions [48]. The primers used in this study are listed in an Additional file (Additional file 1: Table S1).

2.6. Starch Content and β-Amylase Activity Determination

To reflect the changes at the physiological level of B. napus L. under abiotic stress conditions, the starch and β-amylase activity of ZS6 seedlings were measured. The leaves of ZS6 were collected during the drought, flooding, heat, cold and salt treatments at a designed time and stored in −80 °C. The frozen leaves were grounded to powder in liquid nitrogen, and 0.2 g sample was added to 1 mL of 0.1 mol/L citric acid buffer, the extraction buffer was centrifuged for 15 min at 4 °C at 6000× g. The extraction was used to determine the β-amylase activity using a β-amylase kit (G0511W, Suzhou Grace Bio-technology Co., LTD, Suzhou, China) as previously described [29]. Meanwhile, the frozen leaves were grounded to powder in liquid nitrogen, and 0.1 g sample was added to 1 mL of 80% ethanol and extracted at 80 °C water bath for 30 min, the extraction buffer was centrifuged for 5 min at room temperature at 3000× g. The extraction was used to determine the starch content using the kit (G0507W, Suzhou Grace Bio-technology Co., LTD, Suzhou, China) [17].

2.7. Statistical Analysis

All experiments were performed with three biological replicates. Values are presented as mean ± SD. The significance of the data was evaluated using the Least-Significant Difference (LSD) test with Statistix 8.1 software. The significance level was expressed in lower case letters at p < 0.05.

3. Results

3.1. Characterization of BAM Genes in Brassica Crops

The full-length protein sequences of 9 AtBAMs in Arabidopsis were used as BLAST queries against the Brassica crops genomics database. A total of 30 BAM genes in B. napus, 11 BAM genes in B. rapa and 20 BAM genes in B. oleracea were obtained, respectively. The newly identified genes were named according to the closest homologs in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) (Table 1). In Table 1, “random” means that the gene was not assembled into the specific location of the genome. AtBAM1, AtBAM2, AtBAM6, AtBAM7, AtBAM8 presented the same number of homologs in the tetraploid Brassica napus L. (B. napus L.) with total homologs in Brassica rapa L. (B. rapa L.) and Brassica oleracea L. (B. oleracea L.). However, AtBAM3, AtBAM4 had more homologs in B. napus than that in the parental diploid species; AtBAM5, AtBAM9 had fewer homologs in B. napus than that in B. rapa and B. oleracea. The newly identified BAM genes exhibited coding potentials from 181 to 700 amino acids, and the protein molecular weights varied from 20.86 kDa to 79.06 kDa. Among the 30 B. napus genes, BnaBAM8.2 showed the longest open reading frame (ORF) of 2103 bp; BnaBAM3.6 showed the shortest ORF of 546 bp. To get insights into the evolutionary relationship of different BAM members, the phylogenetic tree was generated using the MEGA 7.0 software based on the BAM proteins of Arabidopsis (Arabidopsis thaliana (L.) Heynh.) and the Brassica L. species (Figure 1). BAM proteins were grouped into four clades, which was consistent with previous results [22]. Six BAM proteins of Brassica species (i.e., BnaBAM5.4, BraBAM9.2, BoBAM5.5, BoBAM5.6, BoBAM9.3, BoBAM9.4) differed highly from the other members and can not fall into any subfamilies. Subfamily IV contained the largest family members (18 BAM proteins) in three Brassica species, followed by 15 BAM proteins in Subfamily III, 12 BAM proteins in Subfamily II, and 10 BAM proteins in Subfamily I.

3.2. Identification of the Gene Structure and Conserved Regions of BAM Genes

To analyze the structural diversification of BnaBAMs genes, gene structure display server software was used to map the intron–exon structures. The evolutionary analysis revealed the diversity of the structural compositions of BnaBAMs genes (Figure 2), which was consistent with the phylogenetic analyses. It also showed that 30 BnaBAM genes have different exon number varying from 3 to 10. The maximum number of exons was found in BnaBAM4.1-4.5 with an exception of BnaBAM4.2. The structures and sizes were found to be well conserved among subfamily IV (BnaBAM1.1-1.4, BnaBAM3.1-3.4), subfamily I (BnaBAM7.1-7.2, BnaBAM8.1-8.2). However, subfamily II (BnaBAM5.1-5.4, BnaBAM6.1-6.2), and subfamily III (BnaBAM4.1-4.5, BnaBAM9.1-9.4) showed a very different intron–exon distribution.
The substrate-binding pocket and the active site of the active β-amylase GmBMY1 have been identified in soybean (Glycine max (L.) Merr.) [49,50]. The catalytic-related regions include two catalytic residues Glu-186 and Glu-380, the flexible loop, the inner loop [50,51]. In the present study, conserved amino acid residues in BnaBAM proteins were characterized via a multiple sequence alignment (Figure 3). Glu-186 was well conserved in 28 BnaBAM proteins, while notable divergence was observed within the other three catalytic regions. In the active enzyme AtBAM5, all of the 22 amino acids lining the active site were similar to those of GmBMY1. BnaBAM3.1-3.4 of subfamily IV presented the highest similarity to AtBAM5, and BnaBAM1.1-1.4 substituted at two places of the conserved active sites. Besides above, BnaBAM5.1 of subfamily II showed only one substitution. These observations were similar with the demonstrated enzyme activity of AtBAM1, AtBAM3 and AtBAM5, suggesting a potential catalytic role of BnaBAM1, BnaBAM3s and BnaBAM5, while the genes are encoding β-amylase-like proteins.

3.3. Chromosomal Localization Analysis of the BAM Gene Family in Brassica Crops

In order to explore the chromosomal arrangement of 30 BnaBAM genes, each BnaBAM gene was mapped to B. napus chromosomes according to their physical locations (Figure 4A). The BnaBAM genes were widely distributed in 15 of 19 chromosomes of B. napus. However, the location of BnaBAM genes on chromosomes was relatively dispersed and uneven. The number of BnaBAM genes mapped on each chromosome varied from 1 to 6. B. napus was generated from the interspecific hybridization between A and C-genome ancestors (B.rapa and B. oleracea), which lead to genome duplication [52]. Therefore, the physical locations of BraBAM and BoBAM genes in the parental species was also presented (Figure 4B,C). BoBAM5.3, BoBAM5.5 and BoBAM9.3 were distributed to the unassembled genomic scaffolds, and could not be mapped to the chromosome. Overall, half of the BnaBAM genes were correlated with chromosomes inherited from parental-related species. A01, A05, A07 and A10 chromosomes of B. napus presented one or two BnaBAM genes, which was uniform with the distributions of BraBAMs in B. rapa. Similarly, the distributions of BnaBAMs on C01, C02, C04 chromosomes of B. napus were uniform with that in B. oleracea. Curiously, some BnaBAMs shared different distributions with that from B. rapa and B. oleracea. BnaBAM8.1, BnaBAM4.4 were clustered on certain fragments of A02 chromosomes and separated by approximately 3 Mb, while BraBAM4 and BraBAM8 were located in a wider interval of 10 Mb on C02 chromosomes of B. rapa. Additionally, 5 BnaBAM genes were densely located on C03 chromosome of B. napus while 5 BoBAM genes were distributing throughout the C03 chromosome of B. oleracea.

3.4. Collinearity Analysis of Detected BAM Genes

Arabidopsis genome has experienced two recent whole-genome duplications (WGD) of the crucifer (Brassicaceae Burnett) lineage, and a triplication event which is probably shared by most dicots [53]. To investigate the genetic divergence and gene duplications of BAM genes within Arabidopsis and Brassica species (B. napus, B. rapa, B. oleracea), the syntenic relationships of BAM genes were investigated (Figure 5). Overall, the distribution of collinear genes on chromosomes was relatively uneven. Among 30 BnaBAM genes, 21 members were found to be collinear with 9 AtBAM genes; 25 members were collinear with 10 BraBAM genes and 15 BoBAM genes (Additional file 2: Table S2). The results indicated that allotetraploid was the primary driving force for the rapid expansion of the BnaBAM gene family in B. napus. Because of the triplication event from their common ancestor with Arabidopsis, one Arabidopsis BAM should theoretically correspond to three orthologs in B. rapa and B. oleracea. However, only 9 orthologous gene pairs were obtained between Arabidopsis and B. rapa, followed by 12 orthologous gene pairs between Arabidopsis and B. oleracea (Additional file 2: Table S3). The synteny between BAM genes of parental-related species (B. rapa and B. oleracea) and their Arabidopsis homologs was less than expected, suggesting that duplicated genes might be lost during evolution. Eight AtBAM genes were found to be collinear with no fewer than one BAM gene of the three Brassica crops, indicating that these BAM genes may play vital roles during the evolution processes.

3.5. BnaBAM Genes Response to Various Abiotic Stresses

It has been well-documented that AtBAMs help plants adapt to unfavourable conditions [2], but how the environmental factors affect the BnaBAM gene family is still unclear. To further explore the potential roles of BnaBAM genes in response to different abiotic stresses, an extensive-expression analysis was performed in B. napus under drought, high temperature, low temperature, flooding and salt stress conditions. The results showed strong induction of BnaBAM genes at the transcript level, and the expression patterns of all BnaBAM genes varied greatly among different treatment groups. Sixteen BnaBAM genes were found to be continuously up-regulated by drought stress, whereas 14 genes were down-regulated (Figure 6A). Subfamily III included the homologs of AtBAM4 and AtBAM9, and showed a systemic up-regulation after drought treatment. On the contrary, subfamily II (homologs of AtBAM5 and AtBAM6), and subfamily I (homologs of AtBAM2, AtBAM7, AtBAM8) were generally down-regulated by drought stress. However, BnaBAM genes (homologs of AtBAM1 and AtBAM3) belonging to subfamily IV performed distinct expression patterns with each other. Nonetheless, flooding stress demonstrated a negative modulation on most BnaBAM genes, with exceptions of the slight induction during the initial period of flooding treatment. BnaBAM5.3 and BnaBAM3.6 differed from others, which were greatly up-regulated by flooding with time changing (Figure 6B).
BAM genes are widely known for their involvement in extreme temperatures, such as cold and heat stresses [2,11,17,29,31]. BnaBAM3.2, BnaBAM5.1 were significantly up-regulated by heat stress, whereas subfamily III was shown to be repressed (Figure 6C). When seedlings of B. napus were exposed to cold stimulation, subfamily IV including BnaBAM1.1-1.4, BnaBAM3.1-3.5 were highly induced, and the other three subfamilies with an exception of BnaBAM2 and BnaBAM5 exhibited down-regulated patterns (Figure 6D), which supported the critical roles of AtBAM1 and AtBAM3 in the response of temperature stresses [2]. Considering the well-described function for BAM genes in the cold adaption of plants, 16 BnaBAMs which significantly responded to cold shock stress, were selected to further evaluate the expression levels under lone-term cold stress (4–8 °C). As observed in Figure S1, BnaBAM3.2-3.3, BnaBAM2 markedly increased about 3- to 36- fold at 1 d-14 d of cold treatment, in contrast, the repressed expression pattern was observed for subfamily II and III, which exhibited agreement with the qRT-PCR investigations under cold shock stress condition (Figure 6D).
Unlike the responses mentioned above, BnaBAMs showed more dynamic expression patterns under salt stress conditions (Figure 6E). Specifically, BnaBAM3.1-3.6, BnaBAM9.1-9.4 were markedly induced at 1 h and 24 h after exposure to salt stress, but repressed at 6 h and 12 h after salt treatment. Meanwhile, BnaBAM4.1-4.5, BnaBAM1.2 were found to be increased over time under salt stress conditions. These differential expression patterns of BnaBAM genes under various abiotic stresses indicated that BnaBAM genes of B. napus may participate in stress response processes, which provides information for guiding future study on BnaBAM gene family in response to stress stimulus.

3.6. Abiotic Stresses Affect Starch Content and β-Amylase Activity in B. napus

Overall, most BnaBAM genes were responsive to abiotic stresses. Therefore, to determine how abiotic stresses affect starch metabolism of B. napus, the current study investigated the starch content and β-amylase activity in a widely cultivated variety ZS6, which was exposed to different abiotic stresses. It was found that total starch content was significantly reduced, and then increased to the original level after drought treatment, whereas starch content continuously declined after flood and cold treatment. A puzzling example was heat treatment, which resulted in increased starch content after 1 h of treatment, followed by a decrease during 6 h to 12 h, and finally a rise to the original level. However, salt stress had no significant effect on the starch content of rapeseed (Figure 7). Notably, there was no direct association between starch accumulation and β-amylase activity. The β-amylase activity showed approximately 20% to 80% reduction at different time points after drought, flood, heat, cold and salt treatments when compared with that under normal conditions (Figure 8). It was noted that salt stress exhibited severe influence on the enzyme activity than other treatments, and the β-amylase activity raised again at 24 h after salt treatment, and flooding caused a slight fluctuation in the β-amylase activity. These results reflected that abiotic stresses exhibited stimulatory impacts on β-amylase activity and the related starch content, however, the impacts on β-amylase activity were not correlated with the stress-induced alterations in starch metabolism.

4. Discussion

B. napus is the most productive and widely planted species among three oilseed rapes (B. napus, B. rapa, and B. juncea). To date, sets of genes in B. napus, including BnaCBF17, LEA3, BnSIP1-1, BnNAC485, BnCOL2 have been reported to be associated with responses of abiotic stresses [54,55,56,57,58]. BAM genes hold great potential for modulating the sugar homeostasis of plants under abiotic stress conditions [28,43]. Therefore, the current study systematically analyzed the BAM gene family of Brassica crops, especially in B. napus. The BAM genes of three Brassica species could be categorized into four subfamilies (Figure 1 and Figure 2) based on amino acid sequence alignments, which was in accordance with that of Arabidopsis, pear, banana (Musa balbisiana Colla), soybean and Triticeae Dumort. [19,59,60,61,62], suggesting that BAM genes were evolutionarily conserved among different plants. However, based on the conservation of intron positions in the BAM family in Arabidopsis, the recent studies have divided BAM genes into two subfamilies, which help to trace the origin of each Arabidopsis BAM gene back to the early land plant ancestors [23,43]. Most genome-wide replication events, including whole-genome duplication and whole-genome triplication (WGT), are accompanied by loss of genes [63]. The number of BAM genes in B. napus is almost the sum of that in B. rapa and B. oleracea with only one gene lost (Table 1), suggesting the gene loss might occur in the B. napus lineage process. A notable location was found on A03 and C03 chromosomes of B. napus, several BnaBAM genes were densely located on chromosomes and possessed higher accumulation, this suggested that tandem duplications and chromosomes segmental duplications might contribute to the expansion of the BAM gene family. In total, 25 of 30 BnaBAM genes exhibited the syntenic relationships between those in parental-related species (B. rapa and B. oleracea), followed by 24 BnaBAM genes that were found to be collinear with BAM genes of Arabidopsis. The above results may be contributable to the comparative study with the function-known BAM genes from Arabidopsis.
BAM genes have been reported to involve multiple abiotic stress responses of plants [3]. As expected, BnaBAMs generally responded to drought, flooding, heat, cold and salt treatments (Figure 6). A good example was the ubiquitous up-regulation of subfamily IV, AtBAM3 of Arabidopsis corresponded to 6 homologs in B. napus, most of BnaBAM3s were induced by flooding, cold, heat, and salt treatments; meanwhile, 4 homologs (BnaBAM1s) of AtBAM1 were up-regulated by drought stress. The greatest induction of BnaBAM1s and BnaBAM3s from subfamily IV was observed under cold stress conditions, which was consistent with the predominant role of AtBAM1 and AtBAM3 in cold tolerance [2,17]. In addition, the present study found evidence that two BnaBAM3s responded to long-term cold stress in B. napus (Additional file 3: Figure S1), therefore, BnaBAM3s were assumed to be responsible for cold stress adaption in B. napus. By contrast, subfamily I was widely down-regulated under drought, flooding, heat and cold stresses over time; and a puzzling result was found for subfamily II, BnaBAM5.1 and BAM6.1-6.2 showed opposite expression patterns under cold stress conditions, demonstrating distinct roles of BnaBAM2s, BnaBAM5s, BnaBAM6s, BnaBAM7s, BnaBAM8s in stress responses. Taken together, BnaBAMs may play a unique role in connecting starch metabolism and responses to abiotic stresses, while it is difficult to draw conclusions at this stage. However, the BnaBAM gene family would be a subject of considerable interest in understanding the genetic basis of stress resistance of B. napus.
Over the past few decades, progress has been achieved on the mechanisms of responding and adapting to environmental factors in plants, which composes of signal perception, signal transduction, transcriptional regulation, physiological and metabolic responses [64]. Precisely, plant cells sense abiotic signals, leading to the changes in membrane fluidity and cytoskeleton organization, which accordingly activate the second signal messengers (Ca2+, reactive oxygen species, etc.), thus the downstream signal transduction cascades (kinases, transcription factors, etc.) are activated, that finally trigger the intricate responsive reactions [65]. Osmotic adjustment is one of these mechanisms, which is generated by the accumulation of a large number of osmolytes or compatible constitutes. Starch, as well as the derived metabolites, functions as osmoprotectants to mitigate the negative effects on plants that are caused by challenging environments [8,66,67,68,69]. In the majority of current literature, starch content has been reported to be decreased during abiotic stress treatments [8,10,18,22,70,71]. Similarly, reduced starch content was observed under abiotic stress conditions in the present study (Figure 7), suggesting that starch degradation could be triggered by abiotic stresses in B. napus, and was independent of the analyzed plant species. However, β-amylase activity showed a decreased trend after exposure to abiotic stresses in B. napus, and it was not necessarily correlated with the alterations in starch content (Figure 8). The enzyme activity of tobacco (Nicotiana nudicaulis Watson) slightly accumulates at 1 d and then maintains decreasing until the end of cold treatment [33]. While the total β-amylase activity of leaves remains constant under heat and cold shock conditions in Arabidopsis and pea plants, even the accumulation of maltose is observed [11]. One possible explanation may be the intricate process of starch breakdown, which consists of multiple synergistic actions [21]. β-amylase acts as the major hydrolytic enzyme during the nighttime in the leaf chloroplasts [22], but the Arabidopsis genome encodes additional enzymes for starch degradation, such as phosphorylases, α-amylases and limits dextrinase [3]. For instance, AtBAM1 works synergistically with the α-amylase 3 (AtAMY3) to efficiently degrade starch in the guard cells [72]. Thus, there are likely to be more enzymes involved in stress-induced starch degradation than those identified so far [3]. To date, mechanisms controlling starch degradation are largely unknown in B. napus. Studying the knockout mutants of the key BnaBAMs may help to determine whether a correlation exists between starch content and β-amylase activity in B. napus.

5. Conclusions

In conclusion, the data presented here identified 61 BAM genes in three Brassica species, including 30 BnaBAM genes, 11 BraBAM genes and 20 BoBAM genes. All BnaBAMs were highly conserved by the presence of the BAM domain, and clustered into four subfamilies based on gene evolution analysis. The exon–intron structures were prone to be similar within the same subfamily. A total of 25 BnaBAM genes demonstrated collinearity with 10 BraBAM genes and 15 BoBAM genes. The extensive examination at the transcript level and physiology level here provided a responsive overview of β-amylase under abiotic stress conditions, indicating their critical roles in diverse stress responses. The results presented in this report provide strong support for a functional study on the BAM gene family of Brassica crops, furthermore, facilitate our understanding of the molecular basis of the defenses against abiotic stresses in B. napus.

Supplementary Materials

The following are available online at https://www.mdpi.com/2073-4395/10/12/1855/s1, Figure S1: Expression profile of BnaBAM genes under long-term cold conditions. The transcripts of BnaBAM genes are investigated and depicted as a heat map. The color scale represents log2 (expression values), with red denoting high-level transcription and green denoting low-level transcription, Table S1: The primers used in this study, Table S2: Orthologous gene pairs of BAM-encoding genes between Arabidopsis and B. rapa, B. oleracea, B. napus, Table S3: Collinear gene pairs of BAM-encoding genes between B. napus and B. rapa, B. oleracea.

Author Contributions

D.L. and Y.L. conceived and designed the experiments, D.L., Z.J., X.Z., Y.C. performed the experiments and analyzed the data. D.L., Y.L. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by funds from the major science and technology project, Ministry of science and technology, China (2018ZX08020001), the Oil Crops Research Institution Basal Research Fund of the Chinese Academy of Agricultural Sciences (CAAS), China (1610172018010).

Conflicts of Interest

The authors declare that they have no competing interests.

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Agronomy 10 01855 g001 550
Figure 1. Phylogenetic analysis of β-amylase (BAM) proteins in Arabidopsis, B. napus, B. rapa and B. oleracea. Bootstrap values are shown near the nodes. The different colored arcs represents different subfamilies of the BAM proteins. BAM proteins in Arabidopsis, B. napus, B. rapa and B. oleracea were colored in blue, black, red and green.
Figure 1. Phylogenetic analysis of β-amylase (BAM) proteins in Arabidopsis, B. napus, B. rapa and B. oleracea. Bootstrap values are shown near the nodes. The different colored arcs represents different subfamilies of the BAM proteins. BAM proteins in Arabidopsis, B. napus, B. rapa and B. oleracea were colored in blue, black, red and green.
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Agronomy 10 01855 g002 550
Figure 2. Gene structures of BnaBAM genes family. Gene structures of BnaBAMs based upon the number and position of exons (red boxes), introns (grey lines) and untranslated regions (blue lines).
Figure 2. Gene structures of BnaBAM genes family. Gene structures of BnaBAMs based upon the number and position of exons (red boxes), introns (grey lines) and untranslated regions (blue lines).
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Agronomy 10 01855 g003 550
Figure 3. Alignment of the B. napus BAM proteins and the AtBAM5 protein. The blue shading indicates highly conservative substitutions. Unshaded residues are not conserved. Black arrowheads indicate substrate-binding residues. Red arrowheads indicate the two catalytic residues. Red lines indicate the flexible loop structure. The alignment was made using ClustalX software program.
Figure 3. Alignment of the B. napus BAM proteins and the AtBAM5 protein. The blue shading indicates highly conservative substitutions. Unshaded residues are not conserved. Black arrowheads indicate substrate-binding residues. Red arrowheads indicate the two catalytic residues. Red lines indicate the flexible loop structure. The alignment was made using ClustalX software program.
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Agronomy 10 01855 g004 550
Figure 4. Chromosomal distribution of BAM gene family in B. napus, B. rapa and B. oleracea. The arrangement of 30 BnaBAM genes on 19 chromosomes of B. napus (A), 11 BraBAM genes on 10 chromosomes of B. rapa (B), and 17 BoBAM genes on 9 chromosomes of B. oleracea (C).
Figure 4. Chromosomal distribution of BAM gene family in B. napus, B. rapa and B. oleracea. The arrangement of 30 BnaBAM genes on 19 chromosomes of B. napus (A), 11 BraBAM genes on 10 chromosomes of B. rapa (B), and 17 BoBAM genes on 9 chromosomes of B. oleracea (C).
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Agronomy 10 01855 g005 550
Figure 5. Collinearity analysis of the BAM genes between Arabidopsis and three Brassica crops. The synteny relationship between each pair of BAM genes was detected using linear regression. Genes with the syntenic relationship are linked by lines of the same color. The inner-circle indicates the chromosome numbers and the outer circle indicates the location of AtBAMs, BnaBAMs, BraBAMs and BoBAMs on chromosomes of Arabidopsis, B. napus, B. rapa and B. oleracea.
Figure 5. Collinearity analysis of the BAM genes between Arabidopsis and three Brassica crops. The synteny relationship between each pair of BAM genes was detected using linear regression. Genes with the syntenic relationship are linked by lines of the same color. The inner-circle indicates the chromosome numbers and the outer circle indicates the location of AtBAMs, BnaBAMs, BraBAMs and BoBAMs on chromosomes of Arabidopsis, B. napus, B. rapa and B. oleracea.
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Agronomy 10 01855 g006 550
Figure 6. Expression profile of BnaBAM genes under different abiotic stress conditions. The transcripts of BnaBAM genes under drought (A), flooding (B), heat (C), cold (D) and salt (E) conditions are investigated and depicted as heat maps. The color scale represents log2 (expression values), with red denoting high level of transcription and green denoting a low level of transcription.
Figure 6. Expression profile of BnaBAM genes under different abiotic stress conditions. The transcripts of BnaBAM genes under drought (A), flooding (B), heat (C), cold (D) and salt (E) conditions are investigated and depicted as heat maps. The color scale represents log2 (expression values), with red denoting high level of transcription and green denoting a low level of transcription.
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Agronomy 10 01855 g007 550
Figure 7. The effect of various abiotic stresses on starch content of B. napus. Investigations of starch content in B. napus seedling under drought, flooding, heat, cold and salt conditions. Bars indicate the SE of three biological replicates. Statistical analysis is determined by least-squares difference (LSD) test (p < 0.05).
Figure 7. The effect of various abiotic stresses on starch content of B. napus. Investigations of starch content in B. napus seedling under drought, flooding, heat, cold and salt conditions. Bars indicate the SE of three biological replicates. Statistical analysis is determined by least-squares difference (LSD) test (p < 0.05).
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Agronomy 10 01855 g008 550
Figure 8. The effect of various abiotic stresses on β-amylase activity of B. napus. Investigations of β-amylase activity in B. napus seedling under drought, flooding, heat, cold and salt conditions. Bars indicate the SE of three biological replicates. Statistical analysis is determined by LSD test (p < 0.05).
Figure 8. The effect of various abiotic stresses on β-amylase activity of B. napus. Investigations of β-amylase activity in B. napus seedling under drought, flooding, heat, cold and salt conditions. Bars indicate the SE of three biological replicates. Statistical analysis is determined by LSD test (p < 0.05).
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Table
Table 1. List of identified BAM gene family of B. napus, B. rapa, B. oleracea exhibiting accession number, opening reading frame (ORF), intron number, genomic location, length of amino acid, molecular weight (MW), and iso-electric point (pI).
Table 1. List of identified BAM gene family of B. napus, B. rapa, B. oleracea exhibiting accession number, opening reading frame (ORF), intron number, genomic location, length of amino acid, molecular weight (MW), and iso-electric point (pI).
Serial No.Gene NameAccession NumbersORF (bp)Introns No.Genomic LocationProteinPI
Length (a.a)MW (kDa)
1BraBAM1.1Bra01502517133A07: 4200669-420342857063.385.31
2BraBAM1.2Bra00193716953A03: 19461784-1946394456462.995.68
3BraBAM3.1Bra02623016503A01: 10498540-1050042554961.726.98
4BraBAM3.2Bra01267616473A03: 22804184-2280608154861.456.28
5BraBAM4Bra03558615879A02: 7408259-741075752859.807.54
6BraBAM5Bra03808814976A08: 6619783-662221849856.185.07
7BraBAM6Bra00558117466A05: 6338859-634122458166.535.58
8BraBAM7Bra00494420198A05: 2587413-259043167275.085.64
9BraBAM8Bra02196213068A02: 17928394-1793129765373.635.60
10BraBAM9.1Bra00217816142A10: 11069000-1107086753758.595.82
11BraBAM9.2Bra00647321120A03: 3657746-365985770379.706.03
12BoBAM1.1Bo3g082110.116383C3: 30584504-3058692154561.165.62
13BoBAM1.2Bo7g034320.117133C7: 13225248-1322806057063.415.31
14BoBAM2Bo9g002380.116237C9: 245084-24724354061.175.20
15BoBAM3.1Bo1g052940.116473C1: 15029435-1503132654861.867.18
16BoBAM3.2Bo7g104960.116443C7: 40419711-4042160554761.376.28
17BoBAM4.1Bo3g021390.116569C3: 7167639-717038955162.657.91
18BoBAM4.2Bo2g037740.1191710C2: 10399330-1040260563871.785.76
19BoBAM5.1Bo8g040610.111794C8: 13860892-1386294339244.895.59
20BoBAM5.2Bo8g040630.19153C8: 13904477-1390592430434.184.77
21BoBAM5.3Bo02108s010.19153Unknown chromosome30434.184.77
22BoBAM5.4Bo3g185170.110054C3: 64572299-6457505433438.655.57
23BoBAM5.5Bo03100s010.14982Unknown chromosome16518.605.20
24BoBAM5.6Bo3g185180.16453C3:64580047-6458149521424.436.11
25BoBAM6Bo4g045870.118187C4: 10696860-1069939060569.455.66
26BoBAM7Bo4g025050.120379C4: 4131171-413447667875.885.62
27BoBAM8Bo2g127050.120319C2: 39136275-3913934667676.205.58
28BoBAM9.1Bo3g013270.115962C3: 4592661-459489953157.855.59
29BoBAM9.2Bo9g154650.117492C9: 46305431-4630744458264.256.34
30BoBAM9.3Bo08131s010.12430Unknown chromosome818.989.01
31BoBAM9.4Bo8g046130.16094C8: 15583635-1558500520223.048.45
32BnaBAM1.1BnaA03g37260D16683chrA03: 18437399-1843952055561.916.36
33BnaBAM1.2BnaC03g43570D16383chrC03: 28618604-2862102454561.165.88
34BnaBAM1.3BnaC09g21440D16953chrC09: 18644151-1864723756462.625.53
35BnaBAM1.4BnaA07g05790D17133chrA07: 6121654-612437357063.315.55
36BnaBAM2BnaA09g51890D16207chrA09_random: 126993-12914953961.125.22
37BnaBAM3.1BnaC01g21190D16473chrC01: 14819094-1482098854861.767.65
38BnaBAM3.2BnaA03g42940D16264chrA03: 21561950-2156383854160.726.46
39BnaBAM3.3BnaC07g34180D16473chrC07: 37099448-3710134854861.406.59
40BnaBAM3.4BnaA01g17940D16503chrA01: 9483862-948575154961.757.64
41BnaBAM3.5BnaA08g08230D7745chrA08: 8068950-807686925729.808.20
42BnaBAM3.6BnaC08g10900D5464chrC08: 16342627-1634363518120.868.73
43BnaBAM4.1BnaC03g14120D16029chrC03: 6768572-677133353360.498.74
44BnaBAM4.2BnaC03g71580D8974chrC03_random: 222415-22395929833.689.32
45BnaBAM4.3BnaA03g11260D15999chrA03: 5050886-505354753260.218.57
46BnaBAM4.4BnaA02g08900D15729chrA02: 4429617-443210552359.158.89
47BnaBAM4.5BnaC02g12830D15879chrC02: 8167152-816971352859.658.51
48BnaBAM5.1BnaA08g05660D14976chrA08: 5553480-555592449856.225.20
49BnaBAM5.2BnaA08g00480D13896chrA08: 296747-29920346253.35.84
50BnaBAM5.3BnaC03g78310D10054chrC03_random: 6484548-648730933438.655.57
51BnaBAM5.4BnaC03g78320D6453chrC03_random: 6492299-649374721424.436.11
52BnaBAM6.1BnaC04g12480D17496chrC04: 9717378-971976758266.785.77
53BnaBAM6.2BnaA05g10780D17466chrA05: 5901832-590417958166.756.20
54BnaBAM7.1BnaA05g05170D20318chrA05: 2697113-270013667675.575.86
55BnaBAM7.2BnaC04g04570D20199chrC04: 3393159-339624767275.195.71
56BnaBAM8.1BnaA02g36650D18999chrA02_random: 1168405-117186563271.065.62
57BnaBAM8.2BnaC02g31510D21038chrC02: 33718069-3372113070079.065.99
58BnaBAM9.1BnaA10g16290D16112chrA10: 12426993-1242885953658.526.03
59BnaBAM9.2BnaA03g07240D15932chrA03: 3214063-321644653057.835.73
60BnaBAM9.3BnaC03g09200D15962chrC03: 4394436-439668053157.955.76
61BnaBAM9.4BnaC09g39140D16142chrC09: 41822923-4182484053758.866.50
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Luo, D.; Jia, Z.; Cheng, Y.; Zou, X.; Lv, Y. Genome-Wide Analysis of the β-Amylase Gene Family in Brassica L. Crops and Expression Profiles of BnaBAM Genes in Response to Abiotic Stresses. Agronomy 2020, 10, 1855. https://doi.org/10.3390/agronomy10121855

AMA Style

Luo D, Jia Z, Cheng Y, Zou X, Lv Y. Genome-Wide Analysis of the β-Amylase Gene Family in Brassica L. Crops and Expression Profiles of BnaBAM Genes in Response to Abiotic Stresses. Agronomy. 2020; 10(12):1855. https://doi.org/10.3390/agronomy10121855

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Luo, Dan, Ziqi Jia, Yong Cheng, Xiling Zou, and Yan Lv. 2020. "Genome-Wide Analysis of the β-Amylase Gene Family in Brassica L. Crops and Expression Profiles of BnaBAM Genes in Response to Abiotic Stresses" Agronomy 10, no. 12: 1855. https://doi.org/10.3390/agronomy10121855

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