Identification, expression analysis and characterization of defense and signaling genes in Vitis vinifera.

The reduction of phytochemicals applied to grapevine relies on the development of alternative strategies involving activation of the plant's own defense system. The aim of this work was to study the signaling of defense responses to pathogens in Vitis vinifera. We identified in V. vinifera cv. Chardonnay two putative regulatory elements, VvNHL1 and VvEDS1, with similarity to Arabidopsis defense regulators NDR1 and EDS1. Expression studies of these putative signaling genes together with other known grape defense genes show that they are differentially regulated by salicylic acid and jasmonate-ethylene treatments, as well as by inoculation with different types of pathogens. The expression of VvEDS1 was stimulated by salicylic acid treatment, Botrytis cinerea and Plasmopara viticola inoculation, whereas VvNHL1 was repressed by B. cinerea. VvNHL1 overexpression introduced in Arabidopsis ndr1 mutant did not complement the mutation in terms of sensitivity to avirulent Pseudomonas syringae pv. tomato. Moreover, we observed a weakened resistance to B. cinerea of ndr1 mutants overexpressing VvNHL1, which may be related to cell death enhancement. Together, our results identify two new pathogen-responsive regulatory elements in Vitis vinifera, with potential roles in pathogen defense.     

Cloning and characterization of a Dictyostelium gene encoding a small GTPase of the Rab11 family

Eukaryotic cells achieve complexity by compartmentalizing a subset of cellular functions into membrane-bound organelles. Maintaining this high level of cellular organization requires precise regulation of traffic between membranes. This task is accomplished, in part, by rab proteins. How these small GTPases regulate membrane traffic between cellular compartments is not clear. Here we report the characterization of a novel rab GTPase from the soil amoebae Dictyostelium discoideum. The predicted coding sequence of the new rab gene, Dictyostelium rab11b, encodes a protein of 25 kD containing all the structural hallmarks of a rab GTPase. Comparison of the sequence with the GenBank database and cladistic analysis demonstrated Dictyostelium rab11b to be a divergent member of the rab11 branch of rab proteins. Southern analysis revealed the presence of related genes in Dictyostelium. RNAse protection assays showed the Dictyostelium rab11b gene to be expressed at uniform levels throughout growth and development. Gene deletion experiments revealed that Dictyostelium rab11b was not essential for growth or development. Conceivably, the function of rab11b may be redundant with that of related genes in this organism. J. Cell. Biochem. 70:29–37, 1998. © 1998 Wiley-Liss, inc.     

Molecular and functional characterization of a unique Rab protein,RABRP1, containing the WDIAGQE sequence in a GTPase motif

Rab proteins of the small G-protein superfamily are known to be involved in intracellular vesicle transport. Here, we describe the unique characteristics of a novel Rab protein, RABRP1 (Rab-Related Protein 1). The Drosophila RabRP1 gene is mainly transcribed in the eyes and testes, where the 3-kb and 1.5-kb mRNAs, respectively, are the predominant gene products. The amino-acid sequence deduced from the longer cDNA indicated that the C-terminal 1/3 of the sequence shares homology with Rab proteins, whereas the rest of the peptide shows no significant homology with any other proteins. Immunoblot analysis using antiserum against the Rab-domain indicated that the multiple translates (94 k, 53 k, 30 k, 29 k and 27 k) were expressed in the eyes. In contrast, only smaller peptides (30 k, 29 k and 27 k) were identified in the testes. Molecular phylogenetic analysis revealed that RABRP1 forms a subgroup with Dictiostelium RabE and mammalian Rab29, Rab32, Rab38 proteins, whose functions have not been identified yet. RABRP1 and its relatives were characterized by the amino acid substitution occurring in the conserved GTP-binding motifs. Immunohistochemical studies demonstrated that RABRP1 was localized on the subrhabdomeric cisternae of photoreceptor cells and on the pigment granules in photoreceptor and pigment cells in the retina. The expression of the dominant negative RABRP1 caused the abnormal accumulation of autophagosome-like vesicles. These data suggest that RABRP1 is involved in the lysosomal vesicle transport pathway, including the biogenesis or degradation of pigment granules.     

Genome-wide and molecular evolution analysis of the subtilase gene family in Vitis vinifera

Background

Vitis vinifera (grape) is one of the most economically significant fruit crops in the world. The availability of the recently released grape genome sequence offers an opportunity to identify and analyze some important gene families in this species. Subtilases are a group of subtilisin-like serine proteases that are involved in many biological processes in plants. However, no comprehensive study incorporating phylogeny, chromosomal location and gene duplication, gene organization, functional divergence, selective pressure and expression profiling has been reported so far for the grape.

Results

In the present study, a comprehensive analysis of the subtilase gene family in V. vinifera was performed. Eighty subtilase genes were identified. Phylogenetic analyses indicated that these subtilase genes comprised eight groups. The gene organization is considerably conserved among the groups. Distribution of the subtilase genes is non-random across the chromosomes. A high proportion of these genes are preferentially clustered, indicating that tandem duplications may have contributed significantly to the expansion of the subtilase gene family. Analyses of divergence and adaptive evolution show that while purifying selection may have been the main force driving the evolution of grape subtilases, some of the critical sites responsible for the divergence may have been under positive selection. Further analyses of real-time PCR data suggested that many subtilase genes might be important in the stress response and functional development of plants.

Conclusions

Tandem duplications as well as purifying and positive selections have contributed to the functional divergence of subtilase genes in V. vinifera. The data may contribute to a better understanding of the grape subtilase gene family.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1116) contains supplementary material, which is available to authorized users.     

葡萄Golden2-like转录因子家族的鉴定及其表达分析

本研究对葡萄（Vitis vinifera L.）的Golden2-like （GLK）转录因子家族进行了全基因组鉴定和表达模式分析,并利用品种‘玫瑰香’（V.vinifera cv.Muscat Hamburg）进一步验证其在低温胁迫下的响应。结果显示,葡萄Golden2-like家族共46个成员,分为5个亚族,同一亚族的保守结构域相似。46个VvGLK分别定位于细胞核、叶绿体、细胞质和过氧化物酶体中,其启动子区域含多种逆境应答顺式作用元件。基因芯片分析结果表明,22个Golden2-like基因在果实发育过程中变化显著。同时,有15、15和9个基因分别响应盐、干旱和低温胁迫。qRT-PCR分析发现26个基因参与低温应答。VvGLK41在所有胁迫处理中均下调表达。     

Structural and functional analysis of miraculin-like protein from Vitis vinifera

The so-called miraculin-like proteins (MLPs) are homologous to miraculin, a homodimeric protein with taste-modifying activity that converts sourness into sweetness. The identity between MLPs and miraculin generally ranges from 30% to 55%, and both MLPs and miraculin are categorized into the Kunitz-type soybean trypsin inhibitor (STI) family. MLP from grape (Vitis vinifera; vvMLP) exhibits significant homology to miraculin (61% identity), suggesting that vvMLP possesses miraculin-like properties. The results of size-exclusion chromatography and sensory analysis illustrated that vvMLP exists as a monomer in solution with no detectable taste-modifying activity. Crystal structure determination revealed that vvMLP exists as a β-trefoil fold, similarly as other MLPs and Kunitz-type protein inhibitors. The conformation of the loops, including the so-called reactive loop in the STI family, was substantially different between vvMLP and STI. Recombinant vvMLP had inhibitory activity against trypsin (K_i = 13.7 μM), indicating that the protein can act as a moderate trypsin inhibitor.     

葡萄Actin基因家族的鉴定及进化和表达分析

采用生物信息学方法从葡萄(Vitis vinifera Linn.)全基因组中鉴定Actin基因家族,并对各基因的染色体定位和结构特征,编码蛋白质的理化性质、亚细胞定位、二级结构、三级结构和系统进化,以及不同组织的基因表达进行研究.结果表明:葡萄Actin基因家族16个基因分布在12条染色体上.16个基因的结构特征及其编码蛋白质的理化性质差异较大.16个基因的长度及其内含子总长度的变化范围较大,编码序列(CDS)和外显子总长度的变化范围较小.除登录号GSVIVG01008254001和GSVIVG01014035001的基因外,其他14个基因的GC含量均低于其CDS的GC含量.除登录号GSVIVG01008254001的基因外,其他15个基因编码的蛋白质的理论相对分子质量为12534.54~82612.33,理论等电点为pI 4.92~pI 9.13.16个基因编码蛋白质的消光系数为14105~73645,脂肪族氨基酸指数为65.54~92.06,其中9个为稳定蛋白,7个为不稳定蛋白.除登录号GSVIVG01014035001的基因外,其他15个基因编码的蛋白质均为亲水性蛋白.登录号GSVIVG01016517001的基因编码的蛋白质定位于细胞质和细胞核,其他15个基因编码的蛋白质定位于细胞质.二级结构和三级结构显示:葡萄Actin基因家族16个基因编码的蛋白质均由α螺旋、无规则卷曲和延伸链构成,且总体以无规则卷曲为主.系统进化分析和不同组织的基因表达分析结果显示:与拟南芥〔Arabidopsis thaliana(Linn.)Heynh.〕相似,葡萄Actin基因家族16个基因编码的蛋白质分为3个亚家族,ClassⅡ亚家族(营养型)包括登录号GSVIVG01003099001和GSVIVG01026580001的基因编码的蛋白质,这2个基因在所有组织中的表达均较高;ClassⅢ亚家族(生殖型)包括登录号GSVIVG01033494001、GSVIVG01024980001和GSVIVG01016550001的基因编码的蛋白质,这3个基因在花粉、雄蕊和花中的表达均较高;ClassⅠ亚家族包括其他11个基因编码的蛋白质,这11个基因在各组织中的表达总体上较低.研究结果显示:葡萄Actin基因家族的表达具有组织特异性.     

Expression and characterization of Rab38, a new member of the Rab small G protein family

Rab38 is a new member of the Rab small G protein family that regulates intracellular vesicle trafficking. Rab38 is expressed in melanocytes and it has been clarified that a point mutation in the postulated GTP-binding domain of Rab38 is the gene responsible for oculocutaneous albinism in chocolate mice. However, basic information regarding recombinant protein production, intracellular location, and tissue-specific expression pattern has not yet been reported. We produced recombinant Rab38 using a baculovirus/insect cell-protein expression system. A combination of Triton X-114 phase separation and nickel-affinity chromatography yielded exclusively prenylated Rab38 that bound [alpha-32P]-GTP. The mRNA and the native protein were expressed in a tissue-specific manner, e.g., in the lung, skin, stomach, liver, and kidney. Freshly isolated rat alveolar type II cells were highly positive for the mRNA signal, but the signal was rapidly lost over time. Immunofluorescence staining demonstrated that expressed GST-tagged Rab38 was mainly co-localized with endoplasmic reticulum-resident protein and also partly with intermittent vesicles between the endoplasmic reticulum and the Golgi complex. These results indicate that Rab38 is expressed non-ubiquitously in specific tissues and regulates early vesicle transport relating to the endoplasmic reticulum, and hence suggest that Rab38 abnormality may cause multiple organ diseases as well as oculocutaneous albinism.     

Molecular linkage maps of Vitis vinifera L. and Vitis riparia Mchx

Two linkage maps for grape (Vitis spp.) have been developed based on 81 F(1) plants derived from an interspecific cross between the wine cultivar Moscato bianco (Vitis vinifera L.) and a Vitis riparia Mchx. accession, a donor of pathogen resistance traits. The double pseudotest-cross mapping strategy was applied using three types of molecular markers. The efficiency of SSRs to anchor homologous linkage groups from different Vitis maps and the usefulness of AFLPs in saturating molecular linkage maps were evaluated. Moreover, the SSCP technique was developed based on sequence information in public databases concerning genes involved in flavonoid and stilbene biosynthesis. For the maternal genetic map a total of 338 markers were assembled in 20 linkage groups covering 1,639 cM, whereas 429 loci defined the 19 linkage groups of the paternal map which covers 1,518 cM. The identification of 14 linkage groups common to both maps was possible based on 21 SSR and 19 AFLP loci. The position of SSR loci in the maps presented here was consistent with other published mapping experiments in Vitis.     

Whole genome identification and analysis of FK506-binding protein family genes in grapevine (Vitis vinifera L.)

In plant and animal species FK506-binding protein (FKBP) family genes are important conserved genes and it is defined as the receptors of FK506 and rapamycin, where they work as PPIase and protein folding chaperones. FKBP have been isolated from Arabidopsis thaliana, Oryza sativa, and Zea mays. In grape, twenty-three genes containing the FK506-binding domain (FKBP_C) were first time identified by HMMER and blast research, they were classified into three groups and 17 out of the 23 genes were located on 11 chromosomes (Chr1, 3, 5, 7, 8, 14, 15, 16, 17, 18, and 19). The predicted gene expression pattern and semi-quantitative RT-PCR results revealed that five VvFKBPs were expressed in all tissues, while seven VvFKBPs were expressed only in some of the tissues, and the remaining VvFKBPs were not expressed in leaf, stem, inflorescences, flowers, and a mixture of fruit tissues (small, medium and big-sized fruits). Most of the VvFKBPs in grapevine ‘Summer Black’ were similar to those predicted one in ‘Pinot Noir’ except for VvFKBP16-4 and VvFKBPa. VvFKBP12, FaFKBP12 and PpFKBP12 were cloned from ‘Summer Black’, ‘Sweet Charlie’ and ‘Xiahui 6’. Protein structure analysis confirmed that homologous genes have some differences during the process of protein structure construction. In this study, we characterized and verified 23 FKBP family genes in grapevine (Vitis vinifera L.) as well as their sub-cellular and chromosome location. The successful cloning of CDS regions and protein structural analysis of VvFKBP12, FaFKBP12, and PpFKBP12 can provide useful information for further study.     

Abiotic and oxidative stress-dependent regulation of expression of the thioredoxin h multigenic family in grape Vitis vinifera

Thioredoxins (Trxs) are small ubiquitous oxidoreductases that participate in dithiol-disulphide exchange reactions. They are present in all organisms from prokaryotes to higher eukaryotes and involved in a variety of cellular processes in higher plants. The expression pattern of the multigenic family of grape Trxs_h was analysed in different tissues by semiquantitative RT-PCR. The grape Trx_h genes were approximately expressed in all tissues, but with different expression patterns than each other. The in silico analysis of the promoter regions of grape Trxs _h demonstrated the presence of a number of potential cis-acting elements to respond to environmental signals, suggesting that Trxs_h may respond to a variety of environmental signals, including dehydration, salinity, heat, cold, heavy metals, light, pathogens, and plant hormones. The grape Trx h genes were also found to be differentially induced under abiotic stress conditions due to the presence of different putative regulatory elements in their promoters. Collectively, the distinct expression patterns and different responses to environmental stress conditions suggest that each isoform of the multigenic family of VvTrxh may have a specific and non-redundant function in grape.