Genome-wide identification and expression analysis of the β-amylase genes strongly associated with fruit development, ripening, and abiotic stress response in two banana cultivars

β-amylase (BAM) is an important enzyme involved in conversion of starch to maltose in multiple biological processes in plants. However, there is currently insufficient information on the BAM gene family in the important fruit crop banana. This study identified 16 BAM genes in the banana genome. Phylogenetic analysis showed that MaBAMs were classified into four subfamilies. Most MaBAMs in each subfamily shared similar gene structures. Conserved motif analysis showed that all identified MaBAM proteins had the typical glyco hydro 14 domains. Comprehensive transcriptomic analysis of two banana genotypes revealed the expression patterns of MaBAMs in different tissues, at various stages of fruit development and ripening, and in responses to abiotic stresses. Most MaBAMs showed strong transcript accumulation changes during fruit development and late-stage ripening. Some MaBAMs showed significant changes under cold, salt, and osmotic stresses. This finding indicated that MaBAMs might be involved in regulating fruit development, ripening, and responses to abiotic stress. Analysis of five hormone-related and seven stressrelevant elements in the promoters of MaBAMs further revealed that BAMs participated in various biological processes. This systemic analysis provides new insights into the transcriptional characteristics of the BAM genes in banana and may serve as a basis for further functional studies of such genes.

BAM catalyzes the hydrolysis of α-1,4 linked oligo and poly glucans and is primarily responsible for hydrolysis of starch and degradation of transient starch to generate βmaltose as the predominant sugar exported from the chloroplast at night [8,15] . BAM is a member of glycosyl hydrolase family 14. It has typical glyco hydro 14 domains (PF01373) at the N terminus and a relatively central localization, is widely distributed in higher plants and microorganism and is encoded by a multigene family [7,8,15] . So far, genome-wide analyses have identified nine BAMs in Arabidopsis [5] , 10 in rice, 13 in corn, 11 in Brachypodium distachyon, 10 in sorghum, and 10 in millet [16] . Biochemical and genetic analyses have revealed that the function of BAM genes is related to plant germination, growth, development, and maturation. A BAM protein from fenugreek was reported to participate in seed germination [15] . Overexpression of BAM7 and BAM8 in Arabidopsis controlled shoot growth and development through crosstalk with brassinosteroid signaling [17] . Expression of Bmy1 gene in endosperm was involved in the development and maturation of barley grain [18] . In addition, expression analyses of the BAM gene family in various species indicated that its expression is altered in response to various abiotic stresses, such as cold, salt or drought [19][20][21] . Further studies support the suggestion that BAM genes play a role in plant response to abiotic stress. Mutant analysis indicated that Arabidopsis BAM1 protein decreases stomatal opening and improves drought tolerance, due to reduced starch breakdown in guard cells [20] . Arabidopsis BAM8 has been found to be involved in cold tolerance and maltose accumulation [22] while BAM proteins in barley has been identified as important regulators of salt tolerance [23] . Taken together, these studies have shown that the BAM gene family is involved in regulating plant germination, growth, development, maturation, and response to various stresses.
Banana (Musa acuminata) is not only the most popular high-starch fresh fruit (almost 20% in unripe banana fruit) but also an important staple food in some Africa and Latin American countries [24,25] . Starch degradation to sugar is very important in banana, since sugar concentration is a major component of fruit quality and economic value. Meanwhile as a large annual monocotyledonous herbaceous plant, banana production is frequently destroyed by various abiotic stresses during growth and development, such as cold, salt and drought [26][27][28] . Thus, investigations of key candidate genes involved in fruit development and responses to multiple stresses, based on complete genome sequences, are necessary to improve quality and enhance stresses tolerance [29,30] . However, few studies on identification and characterization of banana genes based on genome sequencing data have been reported [30] . To our knowledge, only one BAM gene has been reported to have been cloned in banana [31] . Considering the importance of the BAM gene family in plant growth and development, as well as response to abiotic stresses, we decided to conduct a genome-wide analysis of the banana genome.
In this study, we identified 16 MaBAM genes and analyzed their phylogenetic relationship, gene structure, protein motifs, and expression patterns in different tissues, different stages of fruit development and ripening, and the response to abiotic stresses (cold, salt, and drought) in two banana genotypes. Further, we analyzed the hormonerelated and stress-relevant cis-elements in the promoters of MaBAM genes. This comprehensive study has increased our understanding of BAM genes associated with fruit development processes and abiotic stresses responses, and will provide a crucial foundation for future studies of crop improvement medicated by BAMs.

Plant materials and treatments
In this study, two banana cultivars, BaXi Jiao (M. acuminata AAA group cv. Cavendish, BX) and Fen Jiao (M. AAB group Fenjiao, FJ), were selected to perform comparative analyses. BX is a triploid cultivar with high yield, high quality, and long-term storage, and is widely cultivated in tropical and subtropical regions in China. FJ is a triploid cultivar, but with a different genotype (AAB) compared with BX (AAA), and has the characteristics of good flavor, rapid ripening, and tolerance to abiotic stresses. It is widely cultivated in the Guangdong and Hainan Provinces of China. The two cultivars were obtained from the banana plantation at the Chinese Academy of Tropical Agricultural Sciences (Danzhou, Hainan, China). Roots, leaves, and fruits at 80 days after emergence from the pseudostem (DAF) were sampled for expression analysis in different tissues. For the banana fruit developmental process, fruits of 0, 20, and 80 DAF, which represent fruit developmental stages of budding, cutting flower, and harvest stages, were collected from both cultivars. For postharvest ripening, BX fruit were stored for 0, 8, and 14 d postharvest (DPH) and FJ 0, 3, and 6 DPH, which represented ripening stages of green, more green than yellow and full yellow according to Pua et al. [32] .  Whole banana (M. acuminata) protein sequences were downloaded from the banana genome database (http:// banana-genome.cirad.fr) [25] . BAM amino acid sequences from Arabidopsis and rice were obtained from the TAIR (http://www.arabidopsis.org) and RGAP (http://rice.plantbiology.msu.edu) databases, respectively. The Hidden Markov Model (HMM) profiles of the BAM typical conserved domains (glyco hydro 14 domains; PFAM: PF01373) (http://pfam.sanger.ac.uk) were used to query the predicted BAM proteins in the banana genome databases using HMMER software (http://hmmer.org) [33] . BLAST analysis was also used to identify the predicted banana BAMs with all BAMs from Arabidopsis and rice as queries. Conserved domain search of the potential banana BAMs was further validated using CDD (http://www.ncbi. nlm.nih.gov/cdd) and PFAM (http://pfam.sanger.ac.uk) databases. The accession number of all identified banana BAMs are given in Table S1 (Appendix A). Phylogenetic analysis of BAM amino acid sequences from banana, Arabidopsis, and rice was carried out using Clustal X 2.0 and MEGA 5.0 software with bootstrap values for 1000 replicates [34] .

Transcriptomic analysis
Total RNA from each sample was extracted using a plant RNA extraction kit (Tiangen, China) and used to construct cDNA libraries according to the protocols supplied by Illumina. The sequencing was performed with an Illumina GAII following the manufacturer's instructions with two replicates per sample. The sequencing depth was 5.34X on average. Adapter sequences in the raw sequence reads and low quality sequences were removed using FASTX-toolkit and FastQC, respectively, and clean reads were obtained and mapped to the DH-Pahang genome (M. acuminata, 2n = 22, A genome) [25] . Transcriptome assemblies were constructed with Cufflinks [35] . Gene expression levels were calculated as fragments per kilobase of exon per million fragments mapped (FPKM). DEGseq was used to identify differentially expressed genes.

Identification and phylogenetic analysis of banana BAM family members
To identify all banana BAM family members, HMM searches using the BAM typical conserved domains (glyco hydro 14 domain; PFAM: PF01373) as queries, as well as BLAST searches using Arabidopsis and rice BAM sequences as queries, were performed in the banana genome database. After validating the BAM domain using the CDD and PFAM database, a total of 16 non-redundant MaBAM genes were identified. The MaBAM genes were named according to their homologous genes in A. thaliana. The 16 predicted MaBAM proteins ranged from 224 (MaBAM5a) to 702 (MaBAM7a) amino acid residues and the relative molecular mass varied from 25.676 (MaBAM5a) to 77.438 (MaBAM7a) kDa, with isoelectric points in the range of 5.00-9.56 (Appendix A, Table S1), suggesting their potentially different roles in regulating biological processes under different conditions.
To study the evolutionary relationships of BAM family proteins, a phylogenetic tree was constructed by aligning 9, 10, and 16 BAM proteins from Arabidopsis, rice, and banana using Clustal X 2.0 and MEGA 5.0 software (Fig. 1). Based on the phylogenetic tree, all MaBAM proteins were grouped into four clades, Clades I, II, III, and IV. Clades I, II, and III are large with more than four MaBAM members, whereas Clade IV contains two MaBAM proteins, similar to those in the rice [5,16] . However, in banana, the number of MaBAM3 proteins in Clade I and MaBAM5 proteins in Clade II were obviously greater than in Arabidopsis and rice.

Gene structure and conserved motifs analysis of banana MaBAM genes
The exon-intron structural evolution of the MaBAM genes was mapped using the GSDS software. Evolutionary analysis further confirmed the classification of the 16 banana MaBAMs into four groups (Fig. 2), which was consistent with the cluster analysis (Fig. 1). Gene structure analysis showed that the MaBAM genes contained four exons in Clade I, three to seven exons in Clade II, eight to nine exons in Clade III, and three exons in Clade IV. This suggests that the majority of MaBAM genes in the same group share similar exon-intron organization and the gene structure might reflect gene evolution.
To explore the conserved motifs and functional prediction from banana MaBAM sequences, a total of 15 conserved motifs in the MaBAMs were identified using MEME software and annotated with the InterPro database (Fig. 3). The results suggest that 11 motifs (motifs 1-7 and motifs 9-12) were annotated as glyco hydro 14 domains, which are basic characteristics of the BAM gene family. All 16 identified MaBAMs contained the typical glyco hydro 14 domain motifs. All MaBAMs in Clade I had the motifs 1-13. Most MaBAMs (except MaBAM5a) in Clade II shared motifs 1-5, 7-9, 11-13, and motif 14 at the C-terminal, whereas the MaBAMs in Clade III had motifs 1-7, 9-13, 15 and lacked motif 14 at the C-terminal. MaBAM9a and MaBAM9b in Clade IV contained motifs 2-11, 13 and lacked motifs 1, 12, and 14-15. These results suggest that most MaBAMs in the same group share conserved motif regions and further support the phylogenetic analyses of banana BAMs.

Expression patterns of MaBAM genes in different tissues of two banana cultivars
To investigate the role of MaBAM genes in banana growth and development, expression patterns of MaBAM genes in different tissues, including roots, leaves, and fruits, were tested in the two cultivars. Among the 16 MaBAM genes, 10 genes (excluding MaBAM2a, MaBAM3a, MaBAM3b, MaBAM5c, MaBAM5b, and MaBAM7b) showed expression in at least one tested tissue of two cultivars ( Fig. 4; Appendix A, Table S2).
Comparison of the tissue expression profiles of BX and FJ revealed that some MaBAM genes have similar expression patterns, including MaBAM1, MaBAM3c, MaBAM3d, MaBAM7a, and MaBAM9b. This indicates that these genes have similar functions in regulating tissues development in both cultivars. However, some genes exhibited differential expression patterns, e.g., MaBAM8 had abundant transcripts ( > 17.6) in roots and fruits of FJ, but low transcripts ( < 5.8) in roots and fruits of BX. MaBAM9a showed high expression levels ( > 41.5) in FJ roots, but low expression levels ( < 7.8) were detected in BX roots. This indicates that some MaBAM members have different roles during tissue development in the two cultivars. Additionally, the MaBAM9b gene showed high expression levels ( > 10) in all of the tested organs in both BX and FJ tissues, indicating a key role for the gene in   Comparison of the expression patterns of banana MaBAM genes at distinct phases of fruit development in both BX and FJ revealed that some MaBAM genes has similar high expression levels at 0 and 20 DAF in both BX and FJ, including MaBAM8, MaBAM9a, and MaBAM9b. This indicates that these genes have similar functions during early fruit developmental stages of both BX and FJ. However, the MaBAM3c had abundant transcripts ( > 18) at 0 DAF in BX, but low transcripts ( < 4) at 0 DAF in FJ. In contrast, MaBAM1 was highly expressed ( > 16) at 0 DAF in FJ, but had low expression levels ( < 8.2) in BX. This finding indicates that some MaBAM members have different transcriptional responses during early fruit development in both BX and FJ. In addition, MaBAM9a and MaBAM9b displayed high transcriptional abundance ( > 30) at all development phases in both BX and FJ, indicating key role for these two genes in regulating banana fruit development.

Expression profiles of MaBAM genes in different stages of fruit ripening in two banana cultivars
To investigate the transcriptional response of MaBAM genes in the banana fruit ripening process, the expression patterns of the MaBAM genes were measured in fruits sampled at 0, 8, and 14 DPH in BX, and 0, 3, and 6 DPH in FJ ( Fig. 6; Appendix A, Table S4). Among the 16 MaBAM genes, nine showed expression at different stages of banana fruit ripening.    Considerable evidence has indicated that BAM genes participate in plant response to various abiotic stresses, including cold, salt, and drought. To better understand the role of MaBAM genes in response to these 3 stresses, their expression in leaves of BX and FJ was examined under cold, salt, and osmotic treatments using data from RNAseq assays ( Fig. 7; Appendix A, Table S5). A total of 11 MaBAM genes showed transcriptional changes after abiotic stress in BX and FJ.
From these results, five genes (MaBAM1, MaBAM3c, MaBAM8, MaBAM9a, and MaBAM9b) were strongly induced by the three stress treatments, indicating that they have similar function in BX and FJ under cold, salt, and osmotic stress. However, two genes (MaBAM5d and MaBAM7a) showing differential expression patterns between BX and FJ under osmotic treatment. This finding indicates that some MaBAM members have different transcriptional responses to osmotic stress treatment in BX and FJ. 3.7 Hormone-related and stress-related cis-acting elements in the promoters of MaBAM genes A promoter is a molecular switch that initiates gene expression and studies of promoter cis-acting elements are very useful for investigating genes expression regulatory mechanism and potential functions [36] . BAM promoters have been documented as having roles in plant development and abiotic stresses in many species [5] . However, there are no such reports in banana. We chose banana MaBAMs for identification of potential promoter cis-acting elements to aid future understanding of the regulation of their expression and biological function. As shown in Table 1, five hormone-related (ABA, auxin, MeJA, ethylene, and gibberellin) and seven stress-related (anaerobic, fungal, heat, cold, drought, salicylic acid, and defense) elements were identified in the promoters of these MaBAM genes. No less than four hormone-and stressrelated elements were found in the 16 BAM promoters, suggesting that BAMs participate in the regulation of development, ripening, and stress response by hormoneand stress-related cis-acting elements.

Discussion
Despite the economic and social importance of banana, compared to other crops, research on banana has evolved slowly. To improve banana fruit quality and enhance stress tolerance, it is essential to explore the mechanisms underlying banana fruit development, ripening, and response to abiotic stresses. Plant BAM genes have various roles in plant growth, development and response to abiotic stresses, and are considered crucial regulators in multiple biological processes. However, limited information has been reported on the MaBAM gene family of banana. In this study, we performed genome-wide identification and characterization of expression of the MaBAM gene family during development, ripening, and response to cold, salt, and osmotic stresses in banana.

Identification and evolutionary analysis of banana MaBAM genes
In this study, a total of 16 MaBAM family genes were identified in the banana A genome (M. acuminata, 2n = 22). This finding indicates that the banana MaBAM gene family has expanded compared to Arabidopsis [5] and rice [16] . Based on phylogenetic analysis, these genes were classified into four subfamilies, which was consistent with the classification of those in the Arabidopsis and rice [5,16] . The phylogenetic results were also supported by the gene structure, which indicated that MaBAM genes contain three to nine exons, and each subfamily shared a similar exonintron organization (Fig. 2), which was also found in Arabidopsis, rice, and barley [5,16,37] . However, the number of exons in banana BAM genes is greater than in barley (7) [37] and sweet potato (2) [38] , implying that the banana MaBAM genes have changed greatly during evolution. Conserved motif analysis indicated that all MaBAMs have the typical glyco hydro 14 domains, and each subfamily shares similar motifs, further supporting the classification of MaBAMs. Together, the identification and classification of the banana BAM gene family was supported by Banana fruit yield and quality are directly affected by fruit developmental processes. Thus, it is necessary to understand the mechanisms involved in banana fruit development. The BAM family has been reported to participate in the plant development of many species such as apple [39] , Arabidopsis [17] , barley [18] , and rye [40] . In apple, the amount of BAM protein was markedly greater during fruit development [39] . Two BAM genes in Arabidopsis regulate shoot development [17] . The expression of a distinct transcript, from the barley Bmy1 gene, suggested its potential involvement in grain development [18] . In rye, expression of two BAM genes changed significantly during seed development [40] . However, whether BAMs participate in fruit development of banana remains unclear. In this study, we found that most banana BAM genes showed increased expression during fruit development. Moreover, many genes showed high transcriptional abundance ( > 10) at all developmental stages of both BX and FJ (Fig. 5).
Based on the evidence above, BAM genes are likely to be involved in fruit development in banana. Banana postharvest ripening is crucial to improving fruit quality and extending fruit shelf life [41,42] . In this study, the number of BAM genes with high transcript abundance ( > 15) at 14 DPH in BX and 6 DPH in FJ was greater than that at other stages (Fig. 6), suggesting that BAM genes are crucial in the late stages of banana fruit ripening. Previous evidence from other fruits has revealed the importance of BAM genes during the late stages of ripening [18,43,44] . In tomato, BAM gene transcript accumulation and enzyme activity were elevated during the late stages of fruit ripening and had an important role in starch depletion and ripening process [43] . The barley Bmy2 protein was increased markedly in mature grain and decreased in developing grain [18] . In mango, BAM protein was involved in starch degradation during fruit ripening [44] . Overall, these results suggest the importance of BAM genes in regulating fruit ripening.
Increasing evidence has suggested that BAM genes could transcriptionally respond to multiple abiotic stresses in various plant species [2,[19][20][21]36] . Further biochemical and genetic evidence has demonstrated that plant BAM proteins, acting as signal regulators, positively regulate plants response to drought [20] , cold [22] , and salt stresses [23] . In this study, many BAM genes showed significant changes after cold, salt, and osmotic treatment in the two banana cultivars, suggesting their potential role in the banana abiotic stress responses. Comparison of the expression patterns of BAMs in BX and FJ under abiotic stress showed that BAM genes similar expression patterns (Fig. 7), indicating that they function similarly in BX and FJ under the cold, salt, and osmotic treatments. Collectively, it is concluded that BAM genes are potentially important in contributing to abiotic stress responses in two banana cultivars. 4.3 Cis-elements analysis in the promoters of BAM genes It has been reported that BAM promoters participate in multiple developmental processes and abiotic stress responses in many species. The Arabidopsis Ct-Bmy promoter is involved in the development of chloroplastlocalized transitory leaf starch [45] . Expression of two Arabidopsis BAM promoters in leaves, sepals, siliques or root tips suggested their potential control of starch breakdown in different tissues [19] . In sweet potato, activity of BAM promoter was exclusively detected in the sugar metabolism process [46] . In Poncirus trifoliata, BAM1 contains a CBF-recognizing element and is important in cold tolerance by modulating soluble sugar levels [36] . Promoter activity analysis of Arabidopsis BAM1 suggested that thioredoxin-regulated BAM1 activates starch degradation in illuminated mesophyll cells under osmotic stress [5] . Additionally, gibberellic acid-and abscisic acidresponsive elements in the promoter of AMY genes have been identified in barley [47] and wheat [48] . In rice, the function of the AMY promoter is in the growth or development of mature leaves, stems, sheaths, roots, and seeds [49] . However, no study has reported hormone-related elements of the BAM promoter. In this study, five hormone-related and seven stress-relevant elements were analyzed in the promoters of 16 MaBAM genes ( Table 1), indicating that BAMs participate in the regulation of multiple development process and stress responses by hormone-related and stress-related cis-acting elements. These findings have provided a solid foundation for further studies of the BAM protein-mediated development and abiotic stresses signal response in banana.

Conclusions
This study identified 16 BAM genes from the banana genome and established their classification and evolutionary relationship using phylogenetic, gene structure, and conserved protein motif analyses. Expression analyses demonstrated that banana BAM genes are involved in regulating fruit development, the late stage of ripening, and abiotic stress responses. Furthermore, hormone-related and stress-related cis-acting elements analyses of MaBAM promoters indicated that they are potentially involved in responding to developmental, ripening, and abiotic stresses signaling. These results will advance the understanding of the functional characterization of BAM genes in the banana developmental and ripening processes, and in responses to abiotic and biotic stresses, and provide a solid foundation for further genetic improvement of banana quality and resistance to various stresses.