Identification and characterization of SlBES1 gene family
To identify BES1 gene family in tomato, the conserved BES1-type domain sequence collected from AtBES1 was used as a BLASTP query in Solanaceae Genomics Database. Totally 9 putative SlBES1 candidates were obtained with default parameters. Meanwhile, Phytozome database was also used to search SlBES1 gene family, and the same SlBES1 candidates were obtained. Then the presence of conserved BES1-type domain was confirmed by CD-Search and SMART. These 9 SlBES1 genes was subsequently named as SlBES1.1 to SlBES1.9 according to their genomic locus (Table 1). Particularly, two members (SlBES1.1 and SlBES1.7) were annotated as β-amylases whereas other were annotated as TFs. The annotated seven SlBES1 TFs showed relative less exon number ranged from 2 to 3, shorted protein length ranged from 180 to 333 amino acids (AA) and lighter predicted molecular weight ranged from 20389 to 35772.85 kDa. While those two annotated β-amylases had more exon number, longer protein length and bigger molecular weight (Table 1). Other detailed properties of SlBES1 genes like theoretical isoelectric point (pI) and BES1-type domain position were also provided in Table 1.
Table 1 Characteristics of SlBES1 genes and the encoded proteins identified in tomato
Gene name
|
Gene accession No.
|
Genomic locus
|
Exon number
|
AAa
|
MWb (kDa)
|
pIc
|
BES1-type domains position
|
SlBES1.1
|
Solyc01g094580
|
SL2.50ch01:85997496..86006359
|
11
|
695
|
77864.45
|
5.37
|
70-204
|
SlBES1.2
|
Solyc02g063010
|
SL2.50ch02:35030416..35032639
|
2
|
319
|
34474.87
|
9.38
|
38-119
|
SlBES1.3
|
Solyc02g071990
|
SL2.50ch02:41313401..41318179
|
3
|
324
|
34908.89
|
8.14
|
31-130
|
SlBES1.4
|
Solyc03g005990
|
SL2.50ch03:667344..672399
|
3
|
323
|
34696.65
|
8.18
|
31-132
|
SlBES1.5
|
Solyc04g079980
|
SL2.50ch04:64289859..64291884
|
2
|
328
|
35108.38
|
8.88
|
52-139
|
SlBES1.6
|
Solyc07g062260
|
SL2.50ch07:65038606..65041740
|
3
|
315
|
33827.99
|
9
|
3-99
|
SlBES1.7
|
Solyc08g005780
|
SL2.50ch08:604998..612717
|
10
|
666
|
75255.45
|
6.09
|
72-202
|
SlBES1.8
|
Solyc10g076390
|
SL2.50ch10:59363764..59364788
|
2
|
180
|
20389
|
8.68
|
37-122
|
SlBES1.9
|
Solyc12g089040
|
SL2.50ch12:64193208..64195373
|
2
|
333
|
35772.85
|
8.85
|
59-145
|
aAA Number of amino acids; bMW Molecular weight; cpI Theoretical Isoelectric point
Chromosomal distribution and conserved amino acid residues analysis of SlBES1 genes
SlBES1 gene family distributed on 8 chromosomes randomly, each SlBES1 gene located at one independent chromosome except chr.2 containing two SlBES1 genes, SlBES1.2 and SlBES1.3. Notably, most of SlBES1 genes positioned on distal ends of chromosomes, three of them distributed in a forward direction, while other six members distributed in a reverse direction (Fig. 1a).
The length of BES1-type domain was 86 to 135 amino acids in tomato. From the alignment of full length sequences, the comparative conserved sequences only showed in the N-terminal of BES1-type domain (Fig. 1b). We further analyzed the conservation of amino acids residues in this domain, similar to the analysis in A. thaliana, O. sativa and G. hirsutum [29], the amino acids residues in the N-terminal BES1-type domain remained conserved at most of loci, which was assumed to be required for DNA binding. Remarkably, an arginine bias region between amino acids 8 to 13 was also observed in SlBES1 family. The C-terminal sequence of BES1-type domain was less conserved, it harbored many serine-rich phosphorylation sites in contrast, which implied the potentially regulatory center of SlBES1 proteins (Fig. 1c) [30].
Phylogenetic and Syntenic analysis of SlBES1 genes
To understand the phylogenetic relationship of SlBES1 family genes, a total of 59 BES1 genes from S. lycopersicum (9), A. thaliana (8), C. annuum (9), G. max (16), O. sativa (6) and Z. mays (11), were used to construct Neighbor-Joining phylogenetic tree by MEGA X with default parameters. In keeping with the trees conducted by Liu et al. [29], Li et al. [31] and Song et al. [32], we grouped these 59 BES1 genes into five groups, named A to E, based on the bootstrap values and phylogenetic topology (Fig. 2a). Group A, B and E possessed the majority of BES1 genes and were further divided into 2 subgroups respectively. Subgroup A1 contained the key members BES1 and BZR1, which were the homologs of SlBES1.5 and SlBES1.9 respectively in tomato. As analyzed by Liu et al. [29], the corresponding group E was more ancient than other groups, and it was true that this group harbored BES1 genes from all of six species analyzed here. Additionally, genes in group E showed quite longer amino acids length and were annotated as β-amylases discriminatively. Group D contained quite less BES1 genes from three species, including one tomato BES1 gene, SlBES1.8, and group C specifically possessed two BES1 genes from G. max, this result showed the expansion and divergence of BES1 gene family in evolution.
To further understand the phylogenetic mechanisms of SlBES1 family, a comparative syntenic maps was conducted among three dicots (tomato, pepper and Arabidopsis) and one monocot (rice) (Fig. 2b). The results showed that the most tomato BES1 homologs presented in pepper, another solanaceae species, followed by Arabidopsis, and the monocot rice exhibited the fewest homologs. What’s more, all SlBES1 syntenic genes (9) could be found on pepper chromosome, and most of SlBES1 syntenic genes (7) could be found on Arabidopsis chromosome, while only two exhibited on rice chromosome. Taken together, the syntenic gene pairs of SlBES1 were more presented in dicot than in monocot. Meanwhile, as the solanaceae relative of tomato, pepper possessed superior synteny with tomato than Arabidopsis and rice. These results suggested that BES1 family may play important roles to plant evolution.
Gene structure and amino acids conserved motif of SlBES1 genes
With the evolution, genes tend to diverge their regulatory and/or coding regions based on the gene duplication. Thus amino acid-altering substitutions and/or alterations may occur, and function of genes could be changed to adapt different growth conditions [33]. A simpler Neighbor-Joining phylogenetic tree was constructed by using BES1 protein sequences from S. lycopersicum and A. thaliana to fully analyze the gene structure and conserved motif (Fig. 3a).
Structures of BES1 genes clustered in the same clade were very close, including number and position of exons and introns. For example, the annotated β-amylase genes contained much more exons (10 to 11) than those annotated TFs that obtained only 2 to 3 exons generally, and the third exon of those three tomato BES1 genes (SlBES1.3, SlBES1.4 and SlBES1.6) had only 4 nucleotides. Furthermore, most of introns of tomato BES1 genes appeared to be longer than their Arabidopsis homologs, which agreed with the fact that tomato had the bigger genome. Besides, the BES1-type domain of tomato BES1 genes was all located between exon1 and exon2 except SlBES1.1. Noticeably, a LxLxL type ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif, which was previously reported as a negative transcriptional regulatory motif [34], was observed in the C-terminal end of those BES1 genes annotated as TFs, implying a potential transcriptional inhibition function of these BES1 genes, while those annotated β-amylase genes didn’t contain this special motif (Fig. 3b).
Proteins containing highly consistent amino acid sequences particularly in functional domain tended to share similar biological functions, thus 10 conserved motifs of tomato and Arabidopsis BES1 proteins were explored by the MEME suite (Fig. S1). As shown in Fig. 3c, motif 1 was the most conserved motif exhibited in all BES1 proteins and it overlapped with BES1-type domain. The permutation and combination of these motifs were very closely related with their phylogenetic relationship. For example, group A and B shared the same motifs (motif 1, 5, 6, 7, 8 and 10) while exhibited an opposite order between motif 8 and 10, and the rest of motifs (motif 1, 2, 3, 4 and 9) were included into group E. Specially, group D exclusively contained the motif 1, suggesting a potential loss of function or functional differentiation of gene in this group.
Potential cis-element in SlBES1 gene promoters
To explore the potential cis-elements, 2kb upstream sequence of SlBES1 genes were submitted to PlantCARE database. The kind and position of cis-elements were marked as different icons (Fig. 4a), and their potential functions were annotated in Fig. 4b. All of these cis-elements detected could be mainly classified into three types: phytohormone responsive, plant development-related and stress responsive elements. Among these cis-elements, ABRE and STRE were conspicuous, which were involved in abscisic acid and stress responsiveness respectively, indicating that SlBES1 genes may be able to be induced or repressed by abiotic stress and subsequently participate in plant stress resistance. Besides, each of SlBES1 gene possessed different kinds and amount of cis-elements, we may assume that, under different growing status and environmental conditions, SlBES1 genes could function independently or synergistically to ensure plant normal growth and development.
Subcellular localization of SlBES1 proteins
Subcellular localization implied the working position of a protein and was nonnegligible for gene functional research. To detect the subcellular localization of SlBES1 proteins, green fluorescent protein (GFP) fused with SlBES1 proteins was used to transiently express in tobacco (Nicotiana benthamiana) leaf. As shown in Fig. 5, seven BES1 proteins, SlBES1.2, SlBES1.3, SlBES1.4, SlBES1.5, SlBES1.6, SlBES1.8 and SlBES1.9, localized both in the nucleus and cytoplasm. This result was basically consistent with the fact that phosphorylated BES1 mainly distributed in the cytoplasm while dephosphorylated BES1 accumulated in the nucleus [15]. For those two annotated β-amylase proteins, SlBES1.1 and SlBES1.7, the green fluorescence pigment showed a non-nuclear shape, thus we further used DAPI to mark the nucleus of tobacco leaf cell, and the green fluorescence pigment was truly not overlapped with the nucleus (Fig. S2). The chlorophyll auto-fluorescence was also detected to analyze if these two proteins localized to the chloroplast, while clear distinction was observed between these two fluorescence pigments both in the size and position, indicating a non-chloroplastic localization (Fig. S2). Taken together, given the bigger size of the green fluorescence pigment than the nucleus, we assumed that these two annotated β-amylase genes localized in the endoplasmic reticulum.
Transactivation activity analysis of SlBES1 proteins
As most of SlBES1 genes were annotated as TFs, the transcriptional activation activity was necessary to be analyzed, hence the GAL4-responsive reporter system in yeast was used to detect the transactivation activity of SlBES1 proteins (Fig. 6a). After transformed the pGBKT7-SlBES1 fusion plasmids into yeast for 3 days, all yeast transformants grew well on SD/-Trp medium, while only those five yeast transformants containing pGBKT7-SlBES1.3, pGBKT7-SlBES1.4, pGBKT7-SlBES1.5, pGBKT7-SlBES1.6, pGBKT7-SlBES1.9 respectively and positive yeast transformant hydrolyzed X-α-Gal and showed the blue pigment and survived from Aureobasidin A (AbA) screening, indicating that these five SlBES1s had transactivation activity whereas other four SlBES1s, including SlBES1.1, SlBES1.2, SlBES1.7 and SlBES1.8, had no transactivation activity. According to the non-nuclear subcellular localization of SlBES1.1 and SlBES1.7 presented above, we assumed that these two SlBES1 proteins were not TFs (Fig. 5), consistent with this assumption, SlBES1.1 and SlBES1.7 truly didn’t have the transactivation activity (Fig. 6a). However, given the presence of EAR motif in the C-terminal end of those seven SlBES1 TFs (Fig. 3b), it was unexpected that five of them discovered to possess the transactivation activity (further discussed in Discussion). The rest of two SlBES1 proteins, SlBES1.2 and SlBES1.8, showing had no transactivation activity, were further to be ascertained if they acted as transcriptional repressor by dual-luciferase assay (Fig. 6b). Full length of coding sequences of these two genes were fused with GAL4 DNA binding domain as the effector. A strong transcriptional activator, VP16 [35], was used as a positive control. After co-expressed of effector and reporter in tobacco leaf, the LUC and REN value was measured. As anticipated, the relative LUC/REN ratios of pBD-SlBES1.2 and pBD-SlBES1.8 were pretty lower than the pBD alone. As a contrast, the VP16 transcriptional activator significantly increased the expression of the LUC reporter. Together with the transactivation activity analysis in yeast, we confirmed that SlBES1.2 and SlBES1.8 acted as the transcriptional repressor.
Tissue-specific and spatio-temporal expression profiles of SlBES1 genes
Development- and tissue-specific expression pattern could lead us to predict the potential function of a gene, thus the spatio-temporal expressions of SlBES1 genes were explored by quantitative real-time polymerase chain reaction (qRT-PCR). 22 templates of tomato tissue were selected for expression profile detection, including seedling at 12 days post germination (DPG), root, stem, leaf at 30 DPG, flower and floral organ (sepal, petal, stamen and ovary) at anthesis and 2 days before anthesis and fruit at different development stages (7 days and 15 days after anthesis, immature green, mature green, breaker, 2 days, 4 days and 7 days after breaker). In general, most of SlBES1 genes expressed ubiquitously in all organs detected except SlBES1.8 that principally expressed in flower organ, indicating a potential important function of SlBES1.8 during fruit set. Notably, SlBES1.1 and SlBES1.4 had the relative stable expression pattern, only a relative higher expression was observed in anthesis stamen and petal respectively, suggesting that SlBES1.1 and SlBES1.4 may function fundamentally to tomato plant development. What’s more, the expressions of SlBES1.2, SlBES1.5, SlBES1.6 and SlBES1.9 gradually increased with the development of fruit, reaching the highest level at IMG and MG stages, then decreased gradually with the fruit ripening (Fig. 7). Interestingly, these four genes possessed more close evolutionary relationship than other SlBES1 members (Fig. 3a), implying a potential functional redundancy or synergistic effect of these four SlBES1 genes to tomato fruit development.
Expression profiles of SlBES1 genes in response to plant hormone
It has been widely studied in the past century that plant hormones played vitally important roles in the regulation of plant growth and development. Understanding of the responsiveness of a gene to plant hormone especially for those TFs could provide us the clue in the research of gene function. In this study, nine major kinds of plant hormones or their analogues, including indole-3-acetic acid (IAA), 6-Benzylaminopurine (6-BA), Gibberellin A3 (GA3), Abscisic Acid (ABA), ethephon, epi-brassinolide (EBL), salicylic acid (SA), methyl jasmonate (MeJA) and strigolactone (GR24), were used to treat the tomato seedling at 12 DPG.
First of all, the efficient effects of plant hormone treatment were validated by the reference genes that were reported previously had responsiveness to plant hormone (Fig. S3). ARF5 [36], TAS14 [37], E4 [38], PR1 [39], WRKY37 [40] and D27 [41] could be induced by IAA, ABA, Ethephon, SA, MeJA and GR24 respectively, while CLAU [42], GA20ox1 [43] and CPD [44] could be repressed by 6-BA, GA3 and EBL respectively. And expectedly, the expression of these genes under corresponding hormone treatment were basically in line with the reports published before, for example, TAS14 and PR1 were greatly induced over hundreds times by ABA and SA respectively, suggesting the effective treatment of plant hormone.
The responsiveness of SlBES1 genes to these hormones was investigated by qRT-PCR (Fig. 8). The fold change of expression level more than 2 times was regarded as having responsiveness to the plant hormone. According to this, the responsiveness of SlBES1 genes to these nine kinds of plant hormones was summarized in Table S1 and the presence of responsiveness was marked as “Y”. In general, all SlBES1 genes could response to at least one kind of plant hormone, while the responsiveness to different plant hormone was distinguishing. For example, SlBES1.6 could response to 8 kinds of plant hormones while SlBES1.9 could only response to one, i.e. GR24. On the other hand, GR24 could affect the maximum number of SlBES1 gene, up to 8 members, indicating that SlBES1 genes may have potential connection with strigolactone signaling. However, ethephon could only influence the expression of SlBES1.2, suggesting that SlBES1 genes probably had no contribution to tomato fruit ripening. Besides, SlBES1 genes showed an identical trend in response to some plant hormones, in this case, SlBES1 genes were generally induced by IAA while repressed by GR24. On the contrary, SlBES1 genes could also be affected by some plant hormone with an opposite trend, for instance, ABA induced the expression of SlBES1.6 and SlBES1.8 while repressed SlBES1.3 and SlBES1.5. Taken together, the variational expression of SlBES1 genes under different plant hormone treatment implied that this gene family involved in multiple hormonal signals in a complicated way. The detailed role of this gene family in the crosstalk of plant hormones thus was worth to studying and may provide us the new insight in the field.
Expression profiles of SlBES1 genes in response to stresses
To further explore the potential responsiveness of SlBES1 genes to biotic and abiotic stresses, we analyzed their expression profiles to drought, osmosis, salt, oxidization, dehydration and wound stress (Fig. 9). The presence of responsiveness to these stresses was summarized in Table S2 and marked as “Y”. Overall, SlBES1 gene family could be affected by multiple stresses, which principally exhibited the downregulated trend in response to all of these six stresses. This indicated that SlBES1 gene family may play the negative roles in tomato stress tolerance. In detail, four members (SlBES1.2, SlBES1.3, SlBES1.4, SlBES1.5) were hyperresponsive to all treatments analyzed here. Besides, at least four treatments can repress or induce the other five members. Notably, the strongest responsiveness of SlBES1 family genes was detected after the wound treatment. In contrast, the relative mild responsiveness was observed in salt stress. The extensive involvement of SlBES1 genes in response to these stresses implied the potential important functions of them.