Validation by isolation and expression analyses of the mitogen-activated protein kinase gene family in the grapevine (Vitis vinifera L.)

Abstract

Background and Aims:Mitogen-activated protein kinases have been found to play essential roles in mediating biotic and abiotic stress responses in plants. Investigation of their possible involvement in grapevine resistance to biotic or abiotic stresses and of their development will be possible only through future functional genomics experiments of gain/loss of function in the grapevine. Methods and Results: We identified and re-annotated all 12 mitogen-activated protein kinases genes from the 12X V1 sequenced grapevine genome and re-nominated them according to international standards as VvMPK. All were validated by cloning their cDNA sequences through polymerase chain reaction amplification. Expression analysis of VvMPK genes using microarray analysis and quantitative real-time polymerase chain reaction demonstrated that all VvMPK genes are expressed during grapevine growth and development. Based on expression analysis of grapevine tissues and organs at several developmental stages, and of leaf tissues treated with Erysiphe necator (powdery mildew), salicylic acid, ethylene, hydrogen peroxide and drought, we identified for further functional characterisation several VvMPK candidate genes which might be involved in grapevine growth and development and in biotic and abiotic responses. Conclusions: We identified several grapevine MPK genes modulated at the transcriptional level in several stages of grapevine growth and development and during the response to development and environmental stresses. Significance of the Study: This is the first comprehensive experimental survey of the grapevine MPK gene family, which provides insights into their potential roles in regulating responses to biotic and abiotic stresses. Ongoing functional characterisation of important candidate VvMPK genes will assist unravelling their biological roles in grapevine.

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

“…In this study, we identified 5 MAPKK genes from grapevine, which were unevenly distributed in 4 chromosomes and were classified into 4 clades, which were identical to the Arabidopsis MAPKK genes. ) also was investigated by qRT-qPCR as previously described [7,33]. Tissue-specific expression was analyzed in five tissue-types obtained from in vitro (tissue culture) plants.…”

“…The primer pairs designed to amplify ORFs and 3′ untranslated region sequences are listed in Table S1 and Table S2, respectively. The PCR protocol used was as previously described [7]. After confirming the accuracy of the obtained full-length sequences, multiple sequence alignments of predicted VvMKKs sequences with cloned VvMKK coding sequences was conducted using the ClustalW program at the nucleotide level.…”

“…Plant MAPKs have been well documented to regulate various physiological responses by interacting with upstream and downstream protein components in a cascade manner MAPKs, MAPKKs and MAPKKKs [47]. To date, we have identified12 VvMAPKs, 5 VvMKKs and 45 VvMAPKKKs in grapevine [7,33], further studies are needed to elucidate the mechanisms involved in stress tolerance in grapevine and the pair-wise interactions of MAPKKK-MAPKK-MAPK genes. It is well known that the modules of AtMKK1/AtMKK2-AtMAPK4 negatively regulates immunity in Arabidopsis [23].…”

“…First strand cDNA synthesis was carried out using 1 µg total RNA and a PrimeScritpt RT reagent Kit (TaKaRa, Japan) following the manufacturer's instructions. Reverse transcriptionquantitative PCR (qRT-PCR) was performed as previously described [7,33]. The gene-specific primers used in the qRT-PCR analysis of MKK genes were designed using Beacon Designer 7.0 software (Premier Biosoft International, USA) and targeted the 3′ UTR of each of the genes and designed to generate approximately 200 bp products (Table S3).…”

“…Although MAPK cascades are involved in transducing multiple defense and stress signals, the role of MAPK cascades in grapevine in response to biotic and abiotic stresses has not been elucidated. In our previous studies, MAPK and MAPKKK gene families in grapevine were identified and their expression in different organs and under different stress conditions was analyzed [7,33]. More recently, using the V. vinifera genome (http://genomes.cribi.unipd.it/grape/) [34], members of the MAPKK gene family in grapevine were identified and subjected to a phylogenetic analysis, however, no experimental evidence on their expression and function was provided [3].…”