Identification of the STARD gene family in grape
A total of 23 VvSTARD candidate genes were retrieved in the grape genome database (Table 1). VvSTARD1–VvSTARD23 were named on the basis of conserved domains and chromosomes sites. The second chromosome was retrieved 4 genes, and the 5th, 6th, 11th, 16th, and 17th chromosomes were only retrieved one gene among 12 chromosomes, respectively. The CDS coding sequences of VvSTART domain in grapes encoded 237–886 amino acids. The MW of VvSTARDs ranged from 26.77 kD (VvSTARD23) to 99.56 kD (VvSTARD7), showing large differences. VvSTARD proteins had hydrophilic values ranging from −0.466 to −0.077. The predicted pI values of the VvSTARD proteins ranged from 5.60 (VvSTARD5) to 9.66 (VvSTARD22). Furthermore, 20 VvSTARD proteins (86.95%) had an instability with index greater than 40.
Further analysis showed the VvSTARD proteins were predicted in the nucleus, chloroplast, and cytoplasm (Table S3). Except for VvSTARD19, VvSTARD21 and VvSTARD22, many proteins were predicated and located in the nucleus. Unlike other proteins, the VvSTARD1, VvSTARD2, VvSTARD8, VvSTARD14, and VvSTARD19 were not present in the chloroplast. A total of 13 (VvSTARD2, VvSTARD4, VvSTARD5, VvSTARD9, VvSTARD10, VvSTARD11, VvSTARD12, VvSTARD13, VvSTARD14, VvSTARD17, VvSTARD18, VvSTARD20, and VvSTARD23), 2 (VvSTARD16, and VvSTARD19), 1 (VvSTARD12), 4 (VvSTARD10, VvSTARD16, VvSTARD17, and VvSTARD22), 2 (VvSTARD17, and VvSTARD20), 1 (VvSTARD20) and 6 (VvSTARD9, VvSTARD10, VvSTARD11, VvSTARD13, VvSTARD18, and VvSTARD19) proteins were predicated and located in the cytoplasm, plasma membrane, cytoskeleton, mitochondria, extracellular matrix, golgi apparatus, and vacuole, respectively.
Phylogenetic and structural analyses of the START domain proteins
The phylogenetic tree was constructed using STARD protein sequences of grapes, Arabidopsis and rice (Fig. 1A). These START genes were mainly divided into five subgroups (groups 1–5). The members of 20 START domain proteins in group 1 (4, 8, and 8 members from grapes, rice, and Arabidopsis, respectively), which contained START and HD domains. 18 members were in group 2 (4, 4, and 10 members from grapes, rice, and Arabidopsis, respectively), which contained START and HD domains. 18 members in group 3 (5, 8, and 5 members from grape, rice and Arabidopsis, respectively), which contained the START, HD and MEKHLA domains. 13 members in group 4 (4, 2, and 7 members from grapes, rice, and Arabidopsis, respectively), which contained the structural START domain. 16 members in group 5 (6, 3, and 5 members from grapes, rice, and Arabidopsis, respectively), which contained the structural START, PH and DUF1336 domains (Fig. S1).
Further analysis showed that members from the same subgroups had similar exon/intron structures and motifs. As shown in Fig. 1B, the exon of VvSTARD gene members ranged from 5 to 22. Moreover, 6 conserved motifs (motifs 1, 2, 3, 4, 5, and 13) were shared by groups 1, 2, and 3 of the VvSTARD proteins family (Figs. 1C and S2). The 6 motifs (motifs 8, 9, 11, 15, 17, and 18) were shared by groups 1 and 2. The 4 motifs (motifs 6, 7, 16, and 19) were shared by group 3, and 3 motifs (motifs 10, 12, and 14) were shared by group 5. However, no system-conserved motif in the VvSTARD protein family was observed in group 4. In addition, the motif 16 was shared by groups 1 and 2. These results indicated that genes with very similar structures distributed in the same subgroups which might have similar biological functions, whereas the genes distributed in different subgroups likely have different biological functions.
Analysis of VvSTARD, AtSTARD, and OsSTARD genes codon preference
A total of 23 VvSTARD, 35 AtSTARD, and 25 OsSTARD gene families contained 15 989, 24 209, and 31 815 codons, respectively (including stop codons) (Fig. 2A). And the three species had RSCU > 1 codons of 9 916, 15 413, and 10 459, respectively (Fig. 2B). Among the RSCU > 1 codons, ending in A or U of coding STARD proteins had preferred codons in the grape and Arabidopsis. In grape, the total of 2 193, 4 674, and 3 049 codons ending in A, U, and G or C, respectively, accounting for 22.12%, 47.14%, and 30.74%, respectively, of the total number of codons with RSCU > 1. In Arabidopsis, codons ending with A, U and G or C accounted for 21.83%, 49.45%, and 28.72%, respectively, of the total codons in RSCU > 1. However, rice contained codons ending in G and C, accounting for 43.24% and 46.17%, respectively, of the total codons in RSCU > 1, whereas codons ending in A or U only accounted for 10.59% of the total codons in RSCU > 1 (Fig. 2B and Table S4).
The grape, Arabidopsis, and rice STARD genes had average CAI values of 0.193, 0.201, and 0.227, respectively; average CBI values of −0.063, −0.022, and 0.093, respectively; and average FOP values of 0.380, 0.405, and 0.469, respectively (Tables S5-1, S5-2 and S5-3). Grape, Arabidopsis, and rice had average Nc values of 54.45, 54.02, and 47.93, respectively; minimum values of 50.69 (VvSTARD14), 46.10 (AtSTARD35), and 31.76 (OsSTARD7), respectively; and maximum values of 57.51 (VvSTARD2), 61 (AtSTARD34), and 56.66 (OsSTARD21), respectively. Among the 23 and 35 STARD genes of grapes and Arabidopsis, respectively, none had an Nc value of less than 35. However, among the 25 OsSTARD genes, six (OsSTARD5, OsSTARD6, OsSTARD7, OsSTARD8, OsSTARD10, and OsSTARD25) showed an Nc value less than 35. The GC3 values in grapes ranged from 0.33 to 0.54, and the distribution was relatively concentrated. The GC3 values in Arabidopsis ranged from 0.29 to 0.49, and the distribution was relatively concentrated. The GC3 values in rice ranged from 0.37 to 0.94, and the distribution was relatively scattered. These findings showed that the codon usage preferences of the grape and AtSTARD gene families were strong and affected by selective pressure during evolution, whereas those of the VvSTARD gene family were weak and affected by the mutation pressure during evolution.
Correlation analysis revealed that the T3s had a negative correlation with C3s, G3s, GC3s, CBI, and Fop and that the C3s had a positive correlation with CBI, Fop, GC, and GC3s in grape, Arabidopsis, and rice (Fig. 2C, 2D). These correlations were highly consistent in grapes and Arabidopsis but quite different from those in rice (Fig. 2E). For instance, the T3s had a positive correlation with Nc in rice, but the T3s had a negative correlation with Nc in grape and Arabidopsis. Nc had a negative correlation with CAI, CBI, and Fop in rice, but Nc had a positive correlation with CAI, CBI, and Fop in grapes and Arabidopsis. Collectively, from the above-mentioned results, the genetic relationship between grapes and Arabidopsis was inferred to be close.
Chromosomal distribution and gene duplication analysis
As shown in Fig. 3A and Table S5-4, VvSTARD genes were unevenly distributed in four linkage groups (chr). The chr6/chr13 linkage group had two VvSTARD gene pairs. chr1, chr3, chr14, chr18, and chr19 had no synteny VvSTARD gene. In this study, tandem duplication genes, namely, VvSTARD14/VvSTARD15 and VvSTARD10/VvSTARD13, were discovered on chr6 and chr13, respectively. A pair of collinear genes (VvSTARD6/VvSTARD7) was observed on chr15 and chr16, and another pair (VvSTARD9/VvSTARD11) was found on chr4 and chr9. These results suggested that some VvSTARD genes might be manufactured via gene duplication, and the primary driving force of the VvSTARD evolution was these duplication events.
Three representative comparative systematic maps of Arabidopsis, grapes, and rice were constructed to further forecast the phylogenetic element of the VvSTARD family (Fig. 3B and Table S5-5). A total of 13, 14, and 9 STARD genes in grapes, Arabidopsis, and rice showed a collinearity relationship. Amongst these genes, 15 were homologous pairs of the STARD genes in grape and Arabidopsis, and 14 were homologous pairs of the STARD genes in grapes and rice. Some VvSTARD genes particularly the VvSTARD and AtSTARD genes were linked with three pairs of synonymous genes, such as VvSTARD7, which might play a critical role in the evolution of the STARD gene family. Some STARD collinear gene pairs between grapes and Arabidopsis were settled on highly conserved synonymous blocks. The phylogenetic relationship and codon preference analyses demonstrated that the evolutionary relationship between grapes and Arabidopsis might be close.
The modes of selection could be estimated using the ratio of the number of nonsynonymous substitutions per nonsynonymous site (Ka) to the number of synonymous substitutions per synonymous site (Ks). The Ka/Ks ratios of the STARD gene pairs of grapes, Arabidopsis, and rice were calculated to further understand the evolutionary relationship of the VvSTARD gene family (Fig. 4 and Table S5-6, S5-7, and S5-8). A total of 202 homologous gene pairs were found in the grape STARD gene family (Fig. 4A). A total of 79 pairs had Ka/Ks > 1, and 123 pairs had Ka/Ks < 1. A total of 382 homologous gene pairs were found in the AtSTARD gene family (Fig. 4B). A total of 161 pairs had Ka/Ks > 1, and 221 pairs had Ka/Ks < 1. A total of 260 homologous gene pairs were found in the OsSTARD gene family (Fig. 4C). A total of 70 pairs had Ka/Ks > 1. One pair (OsSTARD7/OsSTARD1) had Ka/Ks = 1, and 189 pairs had Ka/Ks < 1. These results showed that the VvSTARD, AtSTARD, and OsSTARD gene families might be dominated by purification selection during evolution.
Secondary and tertiary structure prediction of VvSTARD proteins
The results of the secondary structure analysis of the VvSTARD proteins family demonstrated that the secondary structures were α helix, β turn, and random coil (Table S6). The percentages of α helix, β turn and random coil were 30.52% (VvSTART17) to 44.11% (VvSTARD13), 3.24% (VvSTARD16) to 6.75% (VvSTARD23) and 32.91% (VvSTARD23) to 49.05% (VvSTARD17), respectively. The 3D structure analysis showed structures peculiar to several STARD proteins (Fig.S3 and Table S7). These proteins included thioesterase adipose-associated isoform brown fat-inducible thioesterase 2 (BFIT2; observed in VvSTARD14, VvSTARD16, and VvSTARD18), CERT (observed in VvSTARD2, VvSTARD3, VvSTARD4, VvSTARD5, VvSTARD6, VvSTARD7, VvSTARD8, VvSTARD15, VvSTARD17, VvSTARD20, and VvSTARD21), metastatic lymph node 64 (MLN64) protein (observed in VvSTARD9, VvSTARD10, VvSTARD11, and VvSTARD12), PCTP (observed in VvSTARD21 and VvSTARD22), START protein3 (observed in VvSTARD4 and VvSTARD7), cholesterol-regulated START protein4 (observed in VvSTARD11, VvSTARD13, and VvSTARD19), START protein5 (observed in VvSTARD10 and VvSTARD13) and START protein3 (observed in VvSTARD1–VvSTARD13 and VvSTARD20).
The secondary and tertiary structure analyses showed that MLN64, PCTP, cholesterol-regulated START protein 4, and START protein 5 contained four α-helixes, of which two α helices (α2 and α3) formed an internal hydrophobic cavity that could hold a ligand molecule (Fig.S3). α4 was visible on the top of the hydrophobic channel, and α helix at the C-terminus formed the lid. In addition, START protein13 had two α-helices (α1 and α2), and the C-terminal α2 helix served as lid, thereby establishing an internal hydrophobic cavity. BFIT2, CERT and START protein3 contained six α-helixes. Further research found that START protein 5 contained only one 8-chain antiparallel β-sheet, whereas MLN64, PCTP, BFIT2, CERT, START protein 3, cholesterol-regulated START protein 4, and START protein 13 contained a 9-chain antiparallel β-sheet. The side view showed that the antiparallel β-sheets, that was β4, β5 and β6 at one end of the hydrophobic cavity formed a basket structure, whereas the β-sheets on the other side, that is β1, β2, β3, β7, β8, and β9, were formed another basket structure. These results suggested that VvSTARD proteins played a significant role in regulating plant lipid metabolism.
Cis -acting element and expression pattern of VvSTARD genes
Cis- acting elements related to the hormone and abiotic stress responses were speculated in the promoter region of the VvSTARD genes. There were 9 types cis-acting elements of hormone and stress-relation were presented in the promoters of STARD genes in grapes (Fig. 5A and Table S8-1). Three stress-related cis-acting elements, including TC-rich repeats (defense and stress), MBS (drought), and low-temperature responsive elements were annotated in grape genomic data. Six hormone-related cis- acting elements, including TGA element/AuxRR core (auxin), O2 site (zein metabolism), TCA element (salicylic acid), abscisic acid (ABA)-responsive element, GARE-motif/P-box/TATC-box (gibberellin), and CGTCA/TGACG motif (MeJA responsive element) were identified. All genes of VvSTARD contained cis- acting elements of abiotic stress or hormonal responses. Among of the VvSTARD genes, the promoter of 14 genes included ABA response element, and 14 genes were detected in the drought response element. In addition, the VvSTARD genes contained 14 auxins, 10 zeins, 9 GA3, 11 SA, and 13 MeJAresponsive elements. The results showed that the VvSTARD genes could regulate the metabolism of various hormones and abiotic stresses in response to different environmental factors. The expression mode and function of the STARD gene family in plants were not clear. Moreover, we analyzed the STARD gene expression data for organs/tissues and abiotic stress in grapes, rice, and Arabidopsis were downloaded from the BAR database.
The results of the analysis of the grape abiotic stress expression data (Fig. 5B and Table S8-3) showed that six genes (VvSTARD1, VvSTARD2, VvSTARD3, VvSTARD5, VvSTARD6, and VvSTARD8) belonged to groups 1 and 2, whereas five genes (VvSTARD9, VvSTARD10, VvSTARD11, VvSTARD12, and VvSTARD13) belonged to group 3, and such genes were related to salt stress. The expression profiles indicated that most VvSTARD genes were highly expressed at different times of NaCl, PEG and low temperature (5°C) treatments. Genes belonging to groups 5 (VvSTARD15, VvSTARD16, VvSTARD19, and VvSTARD23), 4 (VvSTARD20 and VvSTARD22) and 3 (VvSTARD9, VvSTARD10, VvSTARD11, and VvSTARD13) were related to drought stress. VvSTARD genes related to low-temperature stress were distributed in different groups, and two genes were found in groups 1 and 2 (VvSTARD6 and VvSTARD8).
The expression patterns of various tissues and organs of the AtSTARD gene family demonstrated that the expression of genes in different subfamilies had similarities (Fig. S4A and Table S8-4). Most STARD genes distributed in group 1, such as AtSTARD15, AtSTARD10, AtSTARD1, AtSTARD6, and AtSTARD9, were expressed in seeds. Two AtSTARD genes (AtSTARD5 and AtSTARD19) belonged to group 2, and such genes were expressed in seeds. Most AtSTARD genes in group 3, such as AtSTARD17, AtSTARD18, AtSTARD19, AtSTARD20, and AtSTARD21, were not expressed in the pollen but normally expressed in other tissues and organs. Two AtSTARD genes in group 5 (AtSTARD24 and AtSTARD25) were expressed in all organs and tissues. Except in seeds, AtSTARD22 belonged to group 5 and expressed in all tissues and organs. AtSTARD26 belonged to group 5, but it was expressed only in the roots and stamens. Most of the AtSTARD genes in group 4, such as AtSTARD28 and AtSTARD30, were not expressed in the pollen, seed, shoot and root but normally expressed in other tissues. AtSTARD27 and AtSTARD30 were not expressed in the shoot, and AtSTARD27 was not expressed in the root. Only AtSTARD31 could be expressed in various tissues and organs.
The results of abiotic stress expression analysis demonstrated that the AtSTARD genes clustered in the same group had similar resistance and different expression patterns (Fig. S4B and Table S8-5). In group 4, one gene (AtSTARD28) was highly expressed in the shoot and root under control, cold, salt, drought, wound, and heat stresses. Group 3 had three genes (AtSTARD18, AtSTARD19, and AtSTARD21) under the control, cold, salt, drought, wound, and heat stresses that were expressed higher in the root than in the shoot. In addition, under the control, cold, salt, drought, wound, and heat stresses, some genes showed a higher expression level in root than in shoot, with one gene belonging to group 5 (AtSTARD25) and another gene belonging to group 4 (AtSTARD31). Moreover, under the control, cold, salt, drought, wound, and heat stresses, the expression level in the shoot was higher than that in the root, and the genes were distributed in groups 1 (AtSTARD10 and AtSTARD12) and 4 (AtSTARD27, AtSTARD29, and AtSTARD30).
The expression patterns of the OsSTARD gene family in various tissues and organs showed that the expression of genes in different subfamilies had similarities (Fig. S4C and Table S8-6). Most of the STARD genes in groups 1 and 2, such as OsSTARD5, OsSTARD9, OsSTARD10, OsSTARD1, OsSTARD11, and OsSTARD6, were expressed in rice seeds, shoot apical meristem (SAM) and inflorescence. Some OsSTARD genes (OsSTARD15 and OsSTARD13) were placed in group 3 and expressed in SAM, in florescence and seedling root. Furthermore, OsSTARD14 and OsSTARD12 were expressed in SAM and inflorescence. Group 4 only contained one gene, that is, OsSTARD21, which was expressed in mature leaves, inflorescence P2, and seeds S2–S5. Group 5 contained three OsSTARD genes, namely, OsSTARD18, OsSTARD19, and OsSTARD20. OsSTARD19 was highly expressed in inflorescence P6 and seed S5. OsSTARD20 was highly expressed in SAM and young inflorescence. OsSTARD18 was highly expressed in mature and young leaves.
The analysis of rice abiotic stress expression data demonstrated that 17 genes were expressed in the normal growing shoot and root and evenly distributed in five subgroups (Fig. S4D and Table S8-7). Nine genes belonged to groups 1 (OsSTARD5, OsSTARD10, OsSTARD4, and OsSTARD2), 3 (OsSTARD16, OsSTARD13, and OsSTARD14), and 5 (OsSTARD19 and OsSTARD18), and such genes were highly expressed in the root and shoot under salt stress and evenly distributed amongst four subgroups. Groups 2, 1, 3, and 5 with 1 (OsSTARD24), 1 (OsSTARD7), 1 (OsSTARD2), 2 (OsSTARD12 and OsSTARD15), and 1 (OsSTARD20) gene were expressed in the root and shoot under cold stress and evenly distributed in six subgroups.
Analysis of VvSTARD gene family tissues (Fig. 5C and Table S8-2) demonstrated that the tissue expression of the VvSTARD genes in the same group was similar, but the tissue expression sites differed because of evolutionary differences. VvSTARD4, VvSTARD5, VvSTARD6, and VvSTARD7 were members of the group 1, which contained the HD–START domain. Interestingly, VvSTARD4, VvSTARD5, and VvSTARD6 were expressed in the leaves, seedling, stems, flowers, buds, fruits, skin, seed, stamen, petals, pericarp, and carpel. However, the VvSTARD7 was only expressed in the leaves and seed-post fruits. VvSTARD1 and VvSTARD8, which were classified into group 2 and contained the HD–START domain, were expressed in the leaves, buds, flowers, pollens, and seeds. VvSTARD9, VvSTARD10, VvSTARD11, VvSTARD12, and VvSTARD13 belonged to group 3 and contained the HD–START–MEKHLA domain. VvSTARD10 and VvSTARD11 were not expressed in the pollen, seed, flesh, rachis, pericarp, and other tissues and organs. VvSTARD9 and VvSTARD12 were detected in the tendrils, leaves, seedling, stems, roots, flowers, buds, fruits, and carpels. Nevertheless, VvSTARD13 was extremely lowly expressed or not expressed in many tissues. VvSTARD20, VvSTARD21, VvSTARD22, and VvSTARD23, which were classified into group 4 and contained the START domain only, were expressed at different developmental stages of each organ and tissue. VvSTARD14, VvSTARD15, VvSTARD16, VvSTARD17, VvSTARD18, and VvSTARD19 belonged to group 5. VvSTARD14, VvSTARD15, and VvSTARD18 were expressed in other tissues except for seed, petal, seedling, and bud winter. The VvSTARD16 was expressed at different developmental stages of each organ and tissue, and VvSTARD17 was downregulated or not expressed in many organs. The VvSTARD19 was upregulated in the pollen, flesh midripening, flesh ripening, flesh, pericarp, and skin. VvSTARD23 was also upregulated in the tendrils, young leaves, seedlings, stalks, flowers, carpel, stamen, petals, pollen, seed veraison, flesh veraison, skin veraison, and pericarp veraison. Tissue expression analysis indicated that the expression levels of the VvSTARD genes in different tissues at different developmental stages of grapes had obvious difference.
qRT-PCR of the VvSTARD gene family
qRT-PCR was utilized to verify the expression profile data and further verify the physiological characteristics of the VvSTARD gene family. The results showed that most of the VvSTARD gene families could be expressed in grape leaves in response to hormones and abiotic stresses (Fig. 6). The expression levels of different hormones and abiotic stresses at 24 h were more evident than those at 12 h. A considerable degree of agreement was found among the predicted results. As shown in the chip expression profile, the VvSTARD gene family was expressed in grape leaves, which could respond to the exogenous hormone treatment and presented a high expression level. The expression levels of MeJA, SA, IAA, and GA3 were the same as those of VvSTARD1–VvSTARD4, VvSTARD14–VvSTARD15, VvSTARD7–VvSTARD10, VvSTARD16–VvSTARD21, VvSTARD10, VvSTARD13, and VvSTARD23. under the 400 mmol l−1 NaCl treatments for 24 h, expression levels of 17 genes (VvSTARD1–VvSTARD15, VvSTARD17, and VvSTARD19) were obvious upregulation compared with control. The members of VvSTARD gene family could obviously respond to high-salt stress conditions in grape.
Subcellular localization and the heterologous expression of STARD5
A fusion protein of VvSTARD5 and GFP were introduced into Arabidopsis protoplasts to determine expression site of VvSTARD5 (Fig. 7A). The VvSTARD5 gene was amplified and recombinational Agrobacterium identification (Fig.S5A-S5C). Confocal microscopy revealed that the 35S::VvSTARD5-EGFP fluorescence signal was localized at the nucleus (Figs. 7B). Transgenic tomato plants were obtained by Agrobacterium medicating leaf disc method and PCR identification (Figs.S6A-S6H). The overexpression recombine vector map and salt-tolerant phenotype of wild-type (WT) and transgenic tomatoes are shown in Figs. 7C and 7D. Combined with the result of the qRT-PCR analysis, VvSTARD5 showed a high level of expression under 24 h salt treatment (Fig. 7E). In addition, the relative electrical conductivity, malondialdehyde and proline contents of WT and transgenic tomato under salt stress were measured. The results showed that the relative electrical conductivity of transgenic tomatoes were significantly (p<0.01) lower than WT (Fig. 7F). Moreover, the contents of proline were significantly (p<0.01) higher than WT. However, the content of malondialdehyde were lower than WT (Fig. 7H). These results showed that the ectopic overexpression of STARD5 could significantly enhance the salt tolerance of tomatoes plants.