| Grapevine(Vitis vinifera L.) is one of the most widely grown and most economically fruit crops in the world. However, they are exposed to biotic and abiotic stresses throughout their life cycle, in which drought and salt stress are the major factors in reducing plant growth and productivity. Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes, and play crucial roles in various abiotic and biotic stresses, hormones, cell division and developmental processes in plants. Up to date, although a number of MAPK genes have been isolated from different plants, there is little information concerning MAPKKK, MAPKK and MAPK genes function in grapevine. Recently the grapevine genome has been sequenced, and it provides an opportunity to characterize the grapevine MAPKKK, MAPKK and MAPK gene families based on the genome sequence.In this study, we systematically identified MAPKKK, MAPKK and MAPK genes in the grapevine (Vitis vinifera, PN40024) genome for the first time. We analyzed the classification, protein motifs, gene structure, chromosomal distribution and evolutionary relationship of the family members. We also investigated expression patterns of these genes families in various grape organs, at different developmental stages and under various abiotic and biotic stresses and phytohormone treatments. We further studied the functions of VvMKK2, VvMKK4 and VvMPK6. The main results are as follows:We performed a systematic genome-wide survey and identified 45 MAPKKK genes from the grapevine genome. The phylogenetic analysis divided MAPKKK genes in grapevine into three subgroups, named MEKK, ZIK and RAF, which was strongly supported by the analysis of conserved motifs and exon-intron organizations. The microarray and qRT-PCR analysis also demonstrated that most MAPKKK genes had higher expression levels in certain organs, during the developmental stages of grapevine. Four genes(VvMAPKKK14,15,31,33) were highly expressed in stamen and pollen, VvMAPKKK18 was highly expressed in stem and root. The seedlings of grapevine were treated by E. necator (powdery mildew), drought, SA, ethylene (ETH) and hydrogen peroxide (H2O2), the analysis showed the differential expression profiles of 45 grape MAPKKK genes in response to biotic and abiotic stress, as well as hormone treatments which suggested that several MAPKKK genes might play an important role in biotic and abiotic responses in grapevine.We identified and cloned five MAPKK members from grapevine, nominated as VvMKK1-VvMKK5. The open reading frame (ORF) length ranged from 945 bp to 1557 bp, encoding polypeptides of 314 aa to 518 aa. Amino acids sequence alignment revealed that VvMAPKKs shared high identity with MAPKKs from Arabidopsis, and all proteins contain all the subdomains of the plant MAPKKs and the consensus sequence S/TxxxxxS/T, and five VvMKK genes are grouped into four groups with ten from Arabidopsis. The expression patterns of MAPKK genes in different grapevine tissue were analyzed by microarray and qRT-PCR analyses. The results indicated that the expression of VvMKK1, VvMKK2, VvMKK4 and VvMKK5 were up-regulated in mature leaf, young leaf and root, but the expression levels of these genes was relatively low in the petiole organs. When grapevine leaves were treated with E. necator, SA, ETH, H2O2 and drought, VvMKK2, VvMKK3 and VvMKK5 genes were differentially up-regulated, which showed that they may be involved in different signal pathways.We identified and annotated all 12 MAPK genes from the 12X V1 sequenced grapevine genome (nominated as VvMPK) and validated them through PCR cloning and RACE technology. The length of open reading frame (ORF) ranged from 1107 bp to 1842 bp, encoding polypeptides of 368 aa to 613 aa. Amino acids sequence alignment revealed that VvMPK genes shared high identity with MAPKs from Arabidopsis, and all the deduced protein contains 11 subdomains of MAPK and the phosphorylation activation motif, T-X-Y. These genes were renamed VvMPK1-VvMPK12. Gene structures and phylogeny showed that these VvMPK genes are classified into four subfamilies and genes within the same subfamily have similar exon/intron structures.The expression patterns of VvMPK genes in different grapevine tissue and developmental stages were analyzed by microarray and qRT-PCR. The results indicated that a much higher variability of gene expression was detected in VvMPK1 and VvMPK10. VvMPKl was highly expressed in stamen and pollen, and its transcript abundance progressively increases in berries during postharvest withering. The expression level of VvMPK10 was high in all flower organs and especially in pollen, but declined in the late stages of berry development and postharvest. A few other VvMPKs (VvMPK4,6 and 11) were expressed at a much lower level in certain organs or stages:VvMPK4 was poorly expressed in senescing leaves and winter buds, VvMPK6 was strongly expressed in stems but much less in flower organs, buds and seeds, while expression level of VvMPK11 decreased during berry maturation and postharvest and in senescing leaves. These results indicated that some MPK genes may have very important effects in grapevine organs and at different stages of development. The expression patterns of VvMPKs genes under powdery mildew, drought, SA, ETH and H2O2 treatments were analyzed by qRT-PCR. We found that VvMPK5 and VvMPK6 showed predominantly a reduced expression level, VvMPK1 and VvMPK10 genes showed the most prominent induction following E. necator infection, being expressed two-fold more than that in the control, at 48 h and 12 h post-inoculation, respectively, while VvMPK9 showed a lower but sustained increase over time during the experiment under powdery mildew treatment. Under drought treatments, all VvMPK genes were expressed at a level higher than that in mock controls. Under SA treatment, two genes that increased three-fold or more than in the control were VvMPK6 and VvMPK1 at 4 h and 12 h after treatment, respectively. Several other VvMPKs showed a significant although sometimes low increase in response to SA. After treatment with ETH, the majority of VvMPKs genes exhibited significant changes compared with those in the mock control. After treatment with H2O2, nearly all VvMPK genes were expressed at a level higher than that in the mock controls, especially VvMPKl which was expressed about 3.5-fold at 48 hpi and VvMPK8 which was expressed about 2.5-fold at least at one time point.To further test the roles of VvMKK2, VvMKK4 and VvMPK6 genes by over-expression in transgenic Arabidopsis under control of the CaMV 35S promoter. The transformed plants were verified by GUS staining and PCR. It was indicated that these genes had been recombined into Arabidopsis genome and transgenic Arabidopsis plants were obtained. Homozygous T3 seeds of transgenic Arabidopsis and wild-type lines were selected for the physiological analysis. The WT and over-expressing Arabidopsis showed no difference in germination on MS media without ABA treatment. However, ABA inhibited the germination of both WT and over-expressing seeds, and the inhibition effect of ABA on over-expressing seeds was stronger than that of ABA on WT seeds. Under salt stress, compared to WT plants, VvMKK2/VvMPK6 over-expressing lines showed a significantly higher germination rate, and seedling growth was also better than WT plants. Under drought stress, compared to WT plants, VvMKK2/VvMPK6 over-expressing lines showed a significantly higher germination and survival rates, and seedling growth was better than WT plants. Under salt and drought stress, the transgenic plants showed higher activities of antioxidant enzymes compared with WT plants, and less MDA content compared with WT lines. It was speculated that the enhanced antioxidant system in transgenic plants may contribute to the protection against ROS-mediated injury under stresses. These results indicate that over-expression of VvMKK2/VvMKK4/VvMPK6 in Arabidopsis can improve the toleration to salt and drought stresses. |
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