Cloning, molecular and functional characterization by overexpression in Arabidopsis of MAPKK genes from grapevine (Vitis vinifera).

BACKGROUND: The mitogen-activated protein kinases (MAPKs), as a part of the MAPKKK-MAPKK-MAPK cascade, play crucial roles in plant development as an intracellular signal transduction pathway to respond various environmental signals. However, few MAPKK have been functionally characterized in grapevine. RESULTS: In the study, five MAPKK (MKK) members were identified in grapevine (cultivar 'Pinot Noir'), cloned and designated as VvMKK1-VvMKK5. A phylogenetic analysis grouped them into four sub-families based on the similarity of their conserved motifs and gene structure to Arabidopsis MAPKK members. qRT-PCR results indicated that the expression of VvMKK1, VvMKK2, VvMKK4, and VvMKK5 were up-regulated in mature leaf and young blades, and roots, but exhibited low expression in leaf petioles. VvMKK2, VvMKK3, and VvMKK5 genes were differentially up-regulated when grapevine leaves were inoculated with spores of Erisyphe necator, or treated with salicylic acid (SA), ethylene (ETH), H2O2, or exposed to drought, indicating that these genes may be involved in a variety of signaling pathways. Over expression of VvMKK2 and VvMKK4 genes in transgenic Arabidopsis plants resulted in the production of seeds with a significantly higher germination and survival rate, and better seedling growth under stress conditions than wild-type plants. Overexpression of VvMKK2 in Arabidopsis improved salt and drought stress tolerance while overexpression of VvMKK4 only improved salt stress tolerance. CONCLUSIONS: Results of the present investigation provide a better understanding of the interaction and function of MAPKKK-MAPKK-MAPK genes at the transcriptional level in grapevine and led to the identification of candidate genes for drought and salt stress in grapes.

Genome-wide identification and analysis of mitogen activated protein kinase kinase kinase gene family in grapevine (Vitis vinifera).

BACKGROUND: Mitogen-activated protein kinase kinase kinases (MAPKKKs; MAP3Ks) are important components of MAPK cascades, which are highly conserved signal transduction pathways in animals, yeast and plants, play important roles in plant growth and development. MAPKKKs have been investigated on their evolution and expression patterns in limited plants including Arabidopsis, rice and maize. RESULTS: In this study, we performed a genome-wide survey and identified 45 MAPKKK genes in the grapevine genome. Chromosome location, phylogeny, gene structure and conserved protein motifs of MAPKKK family in grapevine have been analyzed to support the prediction of these genes. In the phylogenetic analysis, MAPKKK genes of grapevine have been classified into three subgroups as described for Arabidopsis, named MEKK, ZIK and RAF, also confirmed in grapevine by the analysis of conserved motifs and exon-intron organizations. By analyzing expression profiles of MAPKKK genes in grapevine microarray databases, we highlighted the modulation of different MAPKKKs in different organs and distinct developmental stages. Furthermore, we experimentally investigated the expression profiles of 45 grape MAPKKK genes in response to biotic (powdery mildew) and abiotic stress (drought), as well as to hormone (salicylic acid, ethylene) and hydrogen peroxide treatments, and identified several candidate MAPKKK genes that might play an important role in biotic and abiotic responses in grapevine, for further functional characterization. CONCLUSIONS: This is the first comprehensive experimental survey of the grapevine MAPKKK gene family, which provides insights into their potential roles in regulating responses to biotic and abiotic stresses, and the evolutionary expansion of MAPKKKs is associated with the diverse requirement in transducing external and internal signals into intracellular actions in MAPK cascade in grapevine.

Genome and transcriptome analysis of the grapevine (Vitis vinifera L.) WRKY gene family.

The plant WRKY gene family represents an ancient and complex class of zinc-finger transcription factors (TFs) that are involved in the regulation of various physiological processes, such as development and senescence, and in plant response to many biotic and abiotic stresses. Despite the growing number of studies on the genomic organisation of WRKY gene family in different species, little information is available about this family in grapevine (Vitis vinifera L.). In the present study, a total number of 59 putative grapevine WRKY transcription factors (VvWRKYs) were identified based on the analysis of various genomic and proteomic grapevine databases. According to their structural and phylogentic features, the identified grapevine WRKY transcription factors were classified into three main groups. In order to shed light into their regulatory roles in growth and development as well as in response to biotic and abiotic stress in grapevine, the VvWRKYs expression profiles were examined in publicly available microarray data. Bioinformatics analysis of these data revealed distinct temporal and spatial expression patterns of VvWRKYs in various tissues, organs and developmental stages, as well as in response to biotic and abiotic stresses. To also extend our analysis to situations not covered by the arrays and to validate our results, the expression profiles of selected VvWRKYs in response to drought stress, Erysiphe necator (powdery mildew) infection, and hormone treatments (salicilic acid and ethylene), were investigated by quantitative real-time reverse transcription PCR (qRT-PCR). The present study provides a foundation for further comparative genomics and functional studies of this important class of transcriptional regulators in grapevine.

Construction and validation of a gene co-expression network in grapevine (Vitis vinifera. L.).

Gene co-expression analysis has been widely used for predicting gene functions because genes within modules of a co-expression network may be involved in similar biological processes and exhibit similar biological functions. To detect gene relationships in the grapevine genome, we constructed a grapevine gene co-expression network (GGCN) by compiling a total of 374 publically available grapevine microarray datasets. The GGCN consisted of 557 modules containing a total of 3834 nodes with 13 479 edges. The functions of the subnetwork modules were inferred by Gene ontology (GO) enrichment analysis. In 127 of the 557 modules containing two or more GO terms, 38 modules exhibited the most significantly enriched GO terms, including 'protein catabolism process', 'photosynthesis', 'cell biosynthesis process', 'biosynthesis of plant cell wall', 'stress response' and other important biological processes. The 'response to heat' GO term was highly represented in module 17, which is composed of many heat shock proteins. To further determine the potential functions of genes in module 17, we performed a Pearson correlation coefficient test, analyzed orthologous relationships with Arabidopsis genes and established gene expression correlations with real-time quantitative reverse transcriptase PCR (qRT-PCR). Our results indicated that many genes in module 17 were upregulated during the heat shock and recovery processes and downregulated in response to low temperature. Furthermore, two putative genes, Vit_07s0185g00040 and Vit_02s0025g04060, were highly expressed in response to heat shock and recovery. This study provides insight into GGCN gene modules and offers important references for gene functions and the discovery of new genes at the module level.