Identification of BGLU genes in the M. truncatula genome
A total of 51 candidate MtBGLU genes were obtained by homology search and domain confirmation, and they were designated as MtBGLU1 to MtBGLU51 based on their location on the chromosome, but four of them (MtBGLU48-51) were not assigned to any of the eight chromosomes (Table S2).
To avoid potential confusion, information on four previously published MtBGLUs are provided in Table S3. Physico-chemical properties of MtBGLU genes, including amino acid numbers, molecular weights, signal peptides, isoelectric points, GraVy, N-gly site and possible subcellular localization were listed in Table 1. The majority of MtBGLU proteins, except 9 MtBGLUs (MtBGLU5, 15, 16, 17, 18, 25, 45, 46 and 48), were predicted to have signal peptides ranging from 17 to 33 amino acids in length, which would target them to the secretary pathway. The lengths of the predicted precursor proteins varied from 315 aa (MtBGLU49) to 1030 aa (MtBGLU33), which correspond to protein molecular weights of 35.8 to 115.7 kDa. The length of mature polypeptides vary from 286 to 617 amino acids, corresponding to MW 32.3 to 70.3 kDa. All MtBGLUs contain one to five N-linked glycosylation sites. Isoelectric points of the predicted proteins ranged from 5.30 to 9.53. The GRAVY ranged from -0.578 to 0, indicating that these MtBGLU proteins are all hydrophilic proteins. The predicted subcellular locations showed that all BGLU proteins from M. truncatula were located in the vacuole or chloroplast, except MtBGLU33 that was located in the extracellular or Golgi.
Multiple sequence alignment, phylogenetic analysis, and classification of MtBGLU genes
The phylogenetic relationship of the MtBGLU proteins was examined by multiple sequence alignment analysis, which showed high conservation among each other (Fig. S1). All MtBGLU protein sequences contain the key amino acids involved in enzymatic catalysis: catalytic acid/base (WI/T/VTF/L/VNEP) and nucleophilic glutamate residues (ITENG), except for MtBGLU49 that either is a non-functional enzyme or has a catalytic mechanism different from all the other MtBGLUs. Sequence-based phylogenetic analysis between Medicago and Arabidopsis showed that these proteins were grouped into 7 distinct clusters. Among these 51 MtBGLU proteins, 25 belong to cluster I, 8 to cluster III and 6 to clusters II, IV and V, respectively (Fig. 1). Clusters I, III, IV and V contain members from Medicago and representatives from Arabidopsis, clusters AtI and AtII with members only from Arabidopsis, and cluster II with only members from Medicago.
Gene structure, conserved domain and motif pattern of MtBGLU genes
In order to better understand the evolution of the GH1 family members in M. truncatula, we conducted the exon/intron structures of all the identified MtBGLU genes. As shown in Fig. 3B, all MtBGLU genes possessed 6 to 17 exons (five with 10 or less exons, twelve with 11 to 12 exons, twenty five with 13 exons, and nine with 14 or more exons). For analysis of the BGLU domain, the shortest conserved amino acid length is 223 aa for MtBGLU49, the longest is 503 aa for MtBGLU47, and the average length of conserved amino acid among MtBGLUs is 457 amino acid (Fig. 2B, Fig. S2).
To identify the conservative structure of MtBGLU proteins, 10 motifs were constructed through the MEME motif analysis, it showed that the majority of the MtBGLU proteins (88.2%) contained all these 10 motifs (Fig. 2C). The lengths of these conserved motifs ranged from 13 to 50 amino acids (Fig. S3). However, six MtBGLU proteins lacked the complete combination of the conserved motifs, including MtBGLU25/MtBGLU46 (lacked only motif 4), MtBGLU2 (lacked motif 4 and 6), MtBGLU45 (lacked motif 2 and 4), MtBGLU30 (lacked motif 3, 8, 9 and 10), and MtBGLU49 (lacked motif 3, 6, 7, 8, 9 and 10). All these gene structure and conserved motif analyses demonstrated that the BGLUs of GH1 family in M. truncatula are highly conservative.
Chromosomal distribution and synteny analysis of MtBGLU genes
The MtBGLU genes were unevenly distributed on seven chromosomes (chr1-7) except chromosome 8 (Fig. 3). In addition, one gene (MtBGLU48) localized on scafford0108 and three (MtBGLU48~ MtBGLU51) on scafford0110 (Fig. 3). Around one third of them were present on chromosome 4, and only two or three genes located on chromosome 1, 5 and 6.
By performing MCscan, we defined the tandem duplication and segmental duplication of MtBGLU genes in M. truncatula genome, which contribute to the formation of gene family during the process of evolution. Five MtBGLU gene pairs (MtBGLU8/9, MtBGLU15/16, MtBGLU33/34, MtBGLU43/44 and MtBGLU49/50/51) were identified as tandem duplication, and they were localized on chromosomes 2, 3, 4, 7 and scaffold 0110. In addition to tandem duplication events, two segmental duplication events with four MtBGLU genes (MtBGLU8/26 and MtBGLU32/35) located on chromosome 2, 4, 5 were also identified. All above results inferred that both tandem duplication and segmental duplication events played an important driving force for MtBGLU gene evolution, and the former played a predominant role.
Furthermore, three comparative syntenic maps of M. truncatula associated with two representative plant species Arabidopsis (dicot) and rice (monocot) were constructed to illustrate the evolution mechanism of MtBGLU gene family (Fig. 4). A total of 4 MtBGLU genes showed a syntenic relationship with those in Arabidopsis and rice, respectively (Additional file 1). Seven and five orthologous pairs were found between Arabidopsis and rice, respectively. MtBGLU1 and MtBGLU33 were found to be associated with two collinear gene pairs in Arabidopsis, which implied that these two genes may play an important role among GH1 gene family during evolution. Besides, another two syntenic pairs (MtBGLU1 and MtBGLU35) were identified between M. truncatula and A. thaliana/O. sativa, which indicated that these collinear pairs may have already existed before the divergence of ancestral genes.
In addition, Ka/Ks analysis of the MtBGLU gene pairs were analyzed in order to better understand the effect of evolutionary stress on the formation of MtBGLU gene family (Additional file 1). All tandem and segmental duplicated MtBGLU gene pairs, and the orthologous MtBGLU gene pairs had Ka/Ks value of less than 1, and these results clearly indicated that MtBGLU genes of the GH1 family might have undergone strong purifying selective pressure during evolution.
Analysis of cis-acting elements in the promoter regions of MtBGLU genes
To further explore potential regulatory mechanism of MtBGLU gene under hormone and stress responses, the 2.0-kbp upstream promoter regions of MtBGLU genes were submitted into PlantCARE to scan for the cis-acting elements. Two types of cis-acting elements, phytohormone responsive and abiotic and biotic stress-responsive elements, were detected and presented in Fig. 5. Firstly, ten hormone-responsive elements were widely presented in their promoter regions, including TGA-element, AuxRR-core (auxin responses), GARE, P-box, TATC-box (gibberellin responses), TGACG-motif, CGTCA-motif (MeJA responses), ABRE (Abscisic acid responses), ERE (ethylene responses) and TCA-element (salicylic acid responses). Seventy-five of these elements were ABRE, ERE, TGACG-motif and CGTCA-motifs, among the phytohormone responsive clusters. Secondly, six abiotic and biotic stress-responsive elements were detected, namely TC-rich repeats, W-box (stress responses), WUN motif (wound responses), MBS (drought inducible), LTR (low-temperature responses) and ARE (anaerobic induction) (Fig. 5). Additionally, many light-responsive regulatory elements and MYB binding sites were found in the promoter regions of majority of the MtBGLU genes (Additional file 2). Various cis-acting elements identified in the promoters of MtBGLU genes implied that they might be involved in the response to various stresses and hormone treatments by participating in distinct regulatory processes.
Analysis of the transcription levels of MtBGLU genes from microarray data
BGLUs are known to play an important role in plants’ response to environmental stresses [10, 11]. However, few BGLUs were documented to be involved in these stresses in M. truncatula . In the present study, seven genechip data sets of M. truncatula were retrieved from MtGEA Web Server, including salinity, drought, limit N, bacteria and fungus as well as YE, MeJA and NAA treatments. A total of 82 probes corresponding to 36 MtBGLU genes (70.6%) were identified. One representative probe for each gene was selected for expression analysis, and the expression level of the representative probes are relatively close to the average value of several probes (Additional file 3).
Our expression analyses indicated that many MtBGLU genes were induced in response to these stresses, especially under NaCl, PEG and NAA treatments (Fig. 6A, B, H and Additional file 3). However, minor activation effects on MtBGLUs were detected by limit N, bacteria and fungus treatments (Fig.6 C, D, E and Additional file 3). In order to further screen the responsive genes under different stress treatments, the average induction times per treatment period (total induction times/number of treatment periods) of MtBGLU genes were calculated under different stress treatments (Fig. 7B and Additional file 3). It showed that, among MtBGLU family members, the expression levels of six genes (MtBGLU14, 21, 22, 26, 28 and 30, in the red box) is greatly up-regulated under these treatments compared with the remaining MtBGLUs. Furthermore, we also counted the number of treatments for each MtBGLUs that were up-regulated by more than two fold, which is consistent with the above results for the six genes (Fig. 7).
Because of the close relationship between gene expression level and gene function, the expression profiles of MtBGLUs in eight tissues (root, shoot, leaf, vegetative bud, stem, petiole, 20-day-old seed, flower and pod) from microarray were investigated (Fig. 8A, B and Additional file 3). It showed that MtBGLU genes showed different transcription levels in various tissues. Most MtBGLU genes in cluster I had specifically low transcription level in roots; instead, genes in cluster II were highly expressed in all other tissues except roots. MtBGLUs in other three clusters (III, VI and V) had distinct expression patterns in different tissues, without obvious consistency. In general, the expression profiling of MtBGLU genes varies greatly, suggesting they might be involve in different functions.
Validation of the expression profile of MtBGLU genes by qPCR
Among all 51 MtBGLU genes, ten of them (MtBGLU14, 16, 18, 19, 21, 22, 26, 28, 30 and 34) were highly induced by various stresses, and two of them (MtBGLU43 and MtBGLU44) were relatively highly expressed in various tissues (Fig. 7, 8B), based on available microarray dataset. To valid this result, these 12 representative MtBGLU genes were further validated by qPCR analysis.
The expression levels of these 12 representative MtBGLU genes were carried out with six another set of tissues: stems, roots, leaves, flowers, pods and seeds (20-day-old). It was revealed that MtBGLU28, MtBGLU30 and MtBGLU34 were expressed differently in all tissues. MtBGLU21 and MtBGLU22 were preferentially expressed in pods, and MtBGLU16, 18, 19 and 26 showed the highest transcript abundances in roots. Instead, MtBGLU43 and MtBGLU44 were expressed in all tissues except roots.
To confirm the expression changes of these 12 MtBGLU genes under various abiotic stresses and hormones, two representative stresses (NaCl and PEG) and four hormones (IAA, ABA, SA, GA3) treatments were mined (Fig. 9, 10 and Fig. S4). Overall, most MtBGLU genes were strongly induced by multiple treatments. Under NaCl and PEG stresses, MtBGLU14, 19, 21, 22, 26, 28 and 30 were significantly up-regulated to a certain level, which is consistent with the above microarray data (Fig. 7). MtBGLU18 and MtBGLU44 were obviously down-regulated during the late treatment stages. However, MtBGLU16, MtBGLU34 and MtBGLU43 seemed to have no significant response under these stresses before 72 h of NaCl treatment (Fig. 9).
In IAA treatment, the expression levels of six MtBGLUs were greatly activated, including MtBGLU14, 19, 21, 22, 28 and 30. Interestingly, the expression levels of four genes (MtBGLU16, 18, 34 and 43) were only up-regulated after 3 h. However, MtBGLU26 and MtBGLU44 experienced no significant increment in response to IAA supplement (Fig. 10). For ABA treatment, the expression levels of MtBGLU19, 21, 22, 26, 28, 30 and 34 genes were markedly promoted, whereas ABA showed no evident effect on the expression levels of MtBGLU14, 16, 43 or 44 genes. Additionally, MtBGLU18 was remarkably diminished after 24 h ABA treatment. Under SA treatment, MtBGLU14, 19, 21, 22, 28 and 30 genes were dramatically activated, which were opposite to other six genes (MtBGLU16, 18, 26, 34, 43 and 44) that were significantly suppressed by SA. With regard to GA3, the expression levels of MtBGLU19, 21, 22, 28 and 30 genes were evidently increased, while MtBGLU16 and MtBGLU18 genes were inhibited by GA3 treatment to some extent. Besides, GA3 had no significant activating effect on the expression levels of MtBGLU14, 34, 43 or 44 genes.
Collectively, qPCR analyses of 12 representatives MtBGLU genes during various treatments were strongly paralleled with their expression pattern obtained from genechip data (Fig.7, Fig. S4). qPCRs analyses also confirmed that four MtBGLU genes (MtBGLU21, 22, 28 and 30) were frequently and strongly activated by various treatments, and they are most likely key BGLU genes involved in response to stress and hormone stimuli in M. truncatula.