Identi�cation and Evolutionary Characteristic Analysis of STARD Gene Family, and Overexpression VvSTARD5 Responses to Salt Stress in Tomato

This study aimed to have a full understanding of the steroidogenic acute regulatory gene family member and evolutionary relationship in grape. 23 VvSTARD gene members were identi�ed and divided into �ve groups in different species. Analyses of the gene codon preference, selective pressure, and tandem duplication of the VvSTARD, AtSTARD, and OsSTARD genes indicated that synteny relationship occurred in grapes, Arabidopsis thaliana, and rice genomes. The 8 lipid transporter proteins were found in the tertiary structure of the STARD gene family in grape. Expression pro�les of the three species microarrays showed that the expression levels of the STARD genes in different organs and the response to abiotic stress in the same subgroup had similar characteristics. In addition, analysis of the VvSTARD genes expression levels was detected in response to different hormones and abiotic stresses by quantitative real-time polymerase chain reaction (qRT-PCR), and the results were the same as those predicted by the cis-elements and the expression pro�les. Meanwhile, VvSTARD5 gene was screened in high concentration NaCl treatment by qRT-PCR. Furthermore, the VvSTARD5 was located at the nucleus by subcellular location. Through the function analysis of salt tolerance in transgenic tomato, overexpression VvSTARD5 obviously improved tolerance to salt stress. Taken together, our �ndings Preliminary identify the functions of VvSTARD gene family and vertify STARD5 that be likely involved in regulating salt tolerance, which may have potential application molecular breeding in grape. family were identi�ed in grapes. In addition, Overexpression VvSTARD5 improved tolerance to salt stress in transgenic tomato. 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. VvSTARD chr6 (VvSTARD6/VvSTARD7) chr15 and chr16, another (VvSTARD9/VvSTARD11) was on chr4 and chr9. VvSTARD be manufactured and the primary driving force of the VvSTARD evolution was these duplication (gibberellin), and CGTCA/TGACG motif (MeJA responsive element) were identi�ed. 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 showed that six genes (VvSTARD1, VvSTARD2, VvSTARD3, VvSTARD5, VvSTARD6, and VvSTARD8) belonged to groups 1 and 2, whereas �ve genes (VvSTARD9, VvSTARD10, VvSTARD11, VvSTARD12, and VvSTARD13) belonged to group 3, and such genes were related to salt stress. The expression pro�les 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 in different groups, and two genes groups 1 and 2 VvSTARD8). expression patterns of various tissues and organs of the AtSTARD gene


Introduction
Salt stress is an important constraint factor on the crop quality and yield particularly in grape. Although many studies have reported on the mechanism of plant salt tolerance (Cheong et al. 2003;Shi et al. 2003;Cao et al. 2007), numerous genes have not been excavated and studied yet. The steroidogenic acute regulatory protein-related lipid transfer domain (STARD), which was rst discovered in mammals, has a 210-amino-acid conserved sequence, which forms an α/β helix-grip structure, thereby creating a hydrophobic cavity that binds to the ligand and small globular modules (Roderick et  The STARD protein family has been identi ed because many proteins contain the START domains in plants, and the homeodomain leucine zipper (HD-Zip III and HD-Zip IV subfamilies) transcription factor family is part of the STARD gene family (Nakamura et al. 2006). A total of 21 HD-Zip START domain transcription factors, which plays an important role in vascular bundle development, meristem formation, and polarity construction in Arabidopsis (Schrick et al. 2004). These factors include epidermal hair growth (GL2) (Szymanski et al. 1998 The V. vinifera "Pinot Noir" tube seedling was used as materials and cultured in the Fruit Tree Physiology and Biotechnology Laboratory of Gansu Agricultural University. The single-shoot stem segments of the test tube seedlings were attached to a solid GS (modi ed B5 solid medium) and cultured under white LED for 35 days. The grape seedings were treated with 0.2 mmol·l −1 of abscisic acid (ABA), 150 μmol·l −1 of methyl jasmonate (MeJA), 50 mg·l −1 of salicylic acid (SA), 100 μmol·l −1 of indole acetic acid (IAA), 50 mg·l −1 of gibberellin 3 (GA 3 ), 10% PEG6000, and 400 mmol·l −1 of NaCl at low temperature (4 °C) for 12 and 24 h. Three replicates were prepared for each treatment, and an equal volume of distilled water was used as control. All materials were collected, frozen in liquid N 2 , and stored at −80 °C for RNA extraction and qRT-PCR.
Cotyledons of "Micro Tom" tomato were used to transform the VvSTARD5 gene, and young seedings of three weeks were used for the salt treatment. The transgenic tomato was watered every 3 h with 400 mmol l −1 of NaCl, and the control was supplemented with the same volume of distilled water. Three biological replicates for each treatment and fresh sample leaves of tomato (0.1 g) were collected. The relative electrical conductivity, proline and malondialdehyde contents of tomato leaves were determined using the commercial ELISA kit (Jiangsu Keming Biotechnology Institute, Suzhou, China) in accordance with the manufacturer's protocol.

Identi cation of STARD genes in grape
The AtSTARD sequences were downloaded from the Arabidopsis genome website (http://www.arabidopsis.org/). Grape and rice genome annotated information were downloaded from the phytozome website (https://phytozome.jgi.doe.gov/pz/portal.html) (Goodstein et al. 2012). The AtSTARD protein sequences (accession numbers: At1g05230, et al) were compared with the grape genome sequences, and the START conserved domain (PF01852) of all proteins were obtained (Table S1). The START conserved domain was used as queries to perform the BLASTP analysis (E < 10 −10 ). HMMER (https:// www.ebi.ac.uk/Tools/hmmer/), and Pfam (http://pfam.xfam.org/) (Potter et al. 2018; El-Gebali et al. 2019) were used to con rm the sequence accuracy, which contained START domain. Simultaneously, the STARD genes of Arabidopsis and rice were also named in the same way. The physicochemical properties of the VvSTARD proteins, such as molecular weight (MW), isoelectric point (pI), grand average of hydropathicity (GRAVY), aliphatic index and instability index, were obtained from the ExPASy (https://www.expasy.org/) (Wilkins et al. 1999).

Analysis of phylogenetic tree, gene structures and motifs
The multiple sequence alignment of the STARD genes of Arabidopsis, rice, and grapes was conducted using the ClustalX 2.0 (Conway Institute, University College Dublin, Dublin, UK) (Larkin et al. 2007). MEGA 7.0 (Pennsylvania State University, State College, PA, USA) was used to perform phylogenetic tree analysis (Kumar et al. 2016) with the NJ, and the "Poisson model" was adopted. The gap was set to "complete deletion", and the check parameter was bootstrap 1000 times with random seed. GSDS 2.0 (http://gsds.cbi.pku.edu.cn/) was used to analyze gene structures, namely, exon and intron (Hu et al. 2015). MEME online software (http://meme-suite.org/) was used to predict the conserved domain of the protein (Bailey et al. 2009), and the number of motifs in the conserved domain was set to 20.

Analysis of the STARD gene synteny and the Ka/Ks in grapes
For the collinearity analysis, the MCScanX was used to detect the collinearity of the STARD gene synteny (Wang et al. 2012), and the diagram was drawn via TBtools (Chen et al. 2018). The nonsynonymous/synonymous (Ka/Ks) values of duplicate gene pairs or triplicate gene groups (between any two genes in one triplicate gene group) were calculated through DnaSP 6.0, an application released by Universitat de Barcelona.

Codon usage bias analysis
The codon bias refers to the unequal use of synonymous codons for an amino acid (Hershberg et al. 2008;Larracuente et al. 2008;Plotkin et al. 2011;Wang et al. 2018). The coding sequences of the STARD were used to determine the codon adaptation index (CAI), codon bias index (CBI), frequency of optimal codons (FOPs), relative synonymous codon usage (RSCU), GC content and GC content at the third site of the synonymous codon (GC3s content) by using the online software CodonW 1.4.2 (http://codonw.sourceforge.net) (Wang et al. 2018). The R programming language was used to analyse the correlation amongst the T3s, C3s, A3s, G3s, GC, GC3s, L_sym, L_aa, GRAVY and Aromo.

Cis-acting element and expression analyzes of STARD gene in grapes
The promoter sequence of the 2,000 bp upstream of the coding region of VvSTARD genes was obtained from the website of grape genomes, and the PlantCARE was used to analyze the gene promoter elements (Lescot et al. 2002;Wang et al. 2016). The diagrams of cis-acting elements were constructed via GSDS2.0 (http://gsds.cbi.pku.edu.cn/) (Hu et al. 2015). Expression pro le data of different abiotic stresses was revitalized from GEO databases (Affymetrix GeneChip 16K Vitis vinifera Genome Array, accession number: GSE31594) (Wang et al. 2018). The expression data of VvSTARD genes was extracted from grape. The tissue expression data of grape, Arabidopsis and rice were retrieved from the Bio-Analytic Resource for Plant Biology (BAR, https://bar.utoronto.ca/) databases. In addition, stress expression data were retrieved from the BAR databases in Arabidopsis and rice. Heat maps were drawn in accordance with TBtools (Chen et al. 2018).

RNA isolation and qRT-PCR
The plant total RNA isolation was performed through kit (Sigma, St. Louis, MO, USA). The M-MLV Reverse Transcriptase (RNase H − ) kit (Takara Bio, Inc., Japan) was utilized to synthesis reverse-strand complementary DNA (cDNA). The puri ed total RNA (1 μg) was reverse transcribed into the rst-strand cDNA and used for qRT-PCR. Subsequently, the TaKaRa SYBR Premix Ex Taq. II (Takara Bio, Inc., Japan) was used for qRT-PCR (Light Cycler 96 Real-Time PCR System, Roche, Basel, Switzerland). The cycling parameters were 95 °C for 30 s, 40 cycles at 95 °C for 5 s, and 60 °C for 30 s. For melting curve analysis, a program consisting of 95 °C for 15 s followed by a constant increase from 60 °C to 95 °C, was included following the PCR cycles. VvGAPDH (GenBank accession no. CB973647) and SlActin (GenBank accession no. NM_001330119) were used as control genes. The primer sequences are presented in Table S2.
The relative expression levels of the genes were calculated using the 2 −ΔΔCt method (Willems et al. 2008), and gures were drawn using the Origin 9.0 software.

Subcellular localization and function identi cation of VvSTARD5
The coding sequences of VvSTARD5 were ampli ed and inserted into pBI221-EGFP to clarify its expressing site in Arabidopsis protoplasts cell. And recombinant vector was constructed by using the NovoRec®PCR One Step Cloning Kit (Novoprotein Scienti c Inc., China). The GFP uorescence was detected using confocal laser-scanning microscopy (Olympus FV1000 Viewer, Tokyo, Japan). Arabidopsis protoplasts were transformed in accordance with the method of Yoo et al. (2007).
"Micro Tom" tomato was used for the transformation of the VvSTARD5. The complete coding regions of VvSTARD5 were inserted behind the 35S promoter and constructed pCAMBIA1300-VvSTARD5 recombinant plasmids that were introduced into the Agrobacterium strain GV3101. The Agrobacterium-mediated transformation of the "Micro Tom" leaves was performed as previously described (Ruf et al, 2001). The DNA of tomato plants was extracted using the TransDirect Plant Tissue PCR Kit (Beijing Quantising Biotechnology Co., Ltd.), and positive plants were detected using gene-speci c primers (35S-F: 5′-TGACGCACAATCCCACTATC-3′; STARD5-R: 5′-CGATGGTAGCGCTTCTTCTT-3′).

Statistical analysis
Statistical analysis was performed by one-way ANOVA using the IBM SPSS v.22 (IBM, Armonk, NY, USA). The p < 0.05 and < 0.01 indicated a signi cant difference and extreme signi cant difference, 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 ve 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). 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 ndings 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 puri cation selection during evolution.
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 signi cant role in regulating plant lipid metabolism.
Cis -acting element and expression pattern of VvSTARD genes Cisacting 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 cisacting elements, including TGA element/AuxRR core (auxin), O 2 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 identi ed. All genes of VvSTARD contained cisacting 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 ve genes (VvSTARD9, VvSTARD10, VvSTARD11, VvSTARD12, and VvSTARD13) belonged to group 3, and such genes were related to salt stress. The expression pro les 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 lowtemperature 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 in orescence. Some OsSTARD genes (OsSTARD15 and OsSTARD13) were placed in group 3 and expressed in SAM, in orescence and seedling root. Furthermore, OsSTARD14 and OsSTARD12 were expressed in SAM and in orescence. Group 4 only contained one gene, that is, OsSTARD21, which was expressed in mature leaves, in orescence P2, and seeds S2-S5. Group 5 contained three OsSTARD genes, namely, OsSTARD18, OsSTARD19, and OsSTARD20. OsSTARD19 was highly expressed in in orescence P6 and seed S5. OsSTARD20 was highly expressed in SAM and young in orescence. 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 ve 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, owers, 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 classi ed into group 2 and contained the HD-START domain, were expressed in the leaves, buds, owers, 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, esh, rachis, pericarp, and other tissues and organs. VvSTARD9 and VvSTARD12 were detected in the tendrils, leaves, seedling, stems, roots, owers, buds, fruits, and carpels. Nevertheless, VvSTARD13 was extremely lowly expressed or not expressed in many tissues. VvSTARD20, VvSTARD21, VvSTARD22, and VvSTARD23, which were classi ed 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, esh midripening, esh ripening, esh, pericarp, and skin. VvSTARD23 was also upregulated in the tendrils, young leaves, seedlings, stalks, owers, carpel, stamen, petals, pollen, seed veraison, esh 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 pro le 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 pro le, 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 ampli ed and recombinational Agrobacterium identi cation (Fig.S5A-S5C). Confocal microscopy revealed that the 35S::VvSTARD5-EGFP uorescence signal was localized at the nucleus (Figs. 7B). Transgenic tomato plants were obtained by Agrobacterium medicating leaf disc method and PCR identi cation  (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 signi cantly (p<0.01) lower than WT (Fig. 7F). Moreover, the contents of proline were signi cantly (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 signi cantly enhance the salt tolerance of tomatoes plants. For example, the expression of VvSTARD15 is 20-fold higher than that of the control when the plant is exposed to low temperature stress, whereas VvSTARD14 is not difference to low temperature stress. In addition, gene duplication, through either segmental or tandem duplication, played important roles in the expansion of new members during the evolution of a gene family (Holub 2001). Synteny analysis of the VvSTARD gene family reveals four pairs of tandem duplication genes distributed in a common subfamily, the results probably because certain fragments of the gene have been copied, exchanged, inverted, and changed during evolution and other events (Shen et al. 2014;). In addition, the synteny analysis of grapes and Arabidopsis shows that 14 pairs of synteny gene are distributed in same subfamily, and only one pair of genes (VvSTARD12/AtSTARD33) doesn't belong to the same group, VvSTARD12 belongs to group 3, and the AtSTARD33 belongs to group 2. Synteny analysis of grapes and rice has revealed nine pairs of synteny genes distributed in the same subgroup ( Fig. 3B and Table S6). The Ka/Ks analysis suggests that the evolution of the grapes, Arabidopsis, and rice STARD gene families is primarily a puri cation choice (Yang, 2007;Wang et al. 2018).

Discussion
Previous studies showed that the In this study, the VvSTARD5 (HDZ20) from the HD-Zip IV subfamily plays key roles in salt stress. In addition, the present study has described the functional characterization of the PH-START protein AtAPO1 (Arabidopsis APOSTART1), indicating that the AtAPO1 is involved in the control of seed germination (Resentini et al. 2014), whereas plants withstand drought and low temperature conditions. However, in the present study, the expression of PH-START proteins VvSTARD14 and VvSTARD15 are upregulated under salt and cold stresses, and HD-START proteins can also exhibit high expression levels under high-salt stress conditions. For instance, the HD-Zip IV subfamily member VvSTARD5 has high expression level under salt stress . Moreover, members with only one START domain have low or even no expression under high-salt stress conditions (Fig. 6). The relative electrolyte leakage serves as an indicator for the damage caused by salt stress (Cao et al. 2007), and the proline and the malondialdehyde contents can change under the salt stress in plants (Fedina et al. 2002). Therefore, the relative electrolyte leakage, proline and malondialdehyde contents are determined from tomato leaves of overexpression VvSTARD5, the results showed that the relative electrolyte leakage and malondialdehyde were lower in transgenic tomato plants than WT, but the content of proline signi cant increase. These results demonstrated that the overexpression of VvSTARD5 can reduce salinity leading to cell membrane damage of leaves, and increasing transgenic tomato plants salt tolerance. The data from the present study strongly indicates the important functions of VvSTARD genes in response to salt stress.

Conclusion
In this study, 23 VvSTARD genes are identi ed in grape. Subsequently, these genes are divided into ve subgroups and disseminated broadly on 12 chromosomes of grape genomes. Different expression pattern in the function of STARD genes are found amongst grape, Arabidopsis and rice. and the majority of VvSTARD genes can response salt stress. In addition, the VvSTARD5 can increase salt tolerance in transgenic tomato. Therefore, the VvSTARD genes were identi ed and further explore its function for genetic improvement of agronomic traits of grapes. Declarations HHH, SXL, and JM designed the experiments, coordinated, and organized the whole research activities. HMG, QZ, XJC, PW, SXL, ZHM, participated in most of the experiments and data collection. HMG, QZ, XJC, PW, SXL, ZHM, provided technical assistance to HHH. HHH wrote the manuscript with contributions from all the authors. BHC and JM revised the manuscript. All authors read, reviewed, and approved the nal manuscript.

Con ict of Interest Statement
The authors have no con icts of interest to declare. Table   Table 1. The characteristic of START domain-encoding genes  Synonymous codon preference and correlation analysis of VvSTARD, AtSTARD and OsSTARD genes. A The number of synonymous codon preference in grapes, Arabidopsis and rice. B The relative synonymous codon usage in grapes, Arabidopsis and rice. C Blue represents positive correlation; red represents negative correlation, and white represents no correlation. The larger circle with darker color is stronger correlation, and vice versa. Inter chromosomal relationships of grape and synteny analysis of STARD genes between grapes and two representative plant species. A Chromosomal distribution and inter chromosomal relationships of VvSTARD genes. Gray lines indicate all synteny blocks in the grape genome, and the red, green, blue, and yellow lines indicate duplicated STARD gene pairs. The chromosome number is indicated at the bottom of each chromosome. B Synteny analysis of STARD genes among Arabidopsis, grapes, and rice. Gray lines in the background indicate the collinear blocks within Arabidopsis, grapes, and rice genomes, whereas the purple line highlights the syntenic STARD gene pairs in grapes and Arabidopsis,, and the red line highlights the syntenic STARD gene pairs in grapes and rice.

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