Genome-wide Characterization, Expression Patterns And Functional Analysis Of The MAPK And MAPKK Gene Families In Watermelon

During their life span, plants often suffer from a variety of external environmental stresses, which severely affect the growth and development of plants. As sessile organisms, plants have developed complex signaling networks to sense environmental signals and adapt themselves to environmental stresses. Mitogen-activated protein kinase (MAPK) cascade, which consists of three functionally associated protein kinases, namely, MAPKKK, MAPKK and MAPK, is an universal module in signal transduction pathways of all eukaryotes. MAPK cascade have been deomonstrated to play important roles in the regulation of many physiological and biochemical processes in plants by progressively amplifying and transmitting the extracellular signals into cells. Watermelon(Citrullus lanatus) is a dicotyledonous flowering plants, which is drought-tolerant, thermophilic and widely planted worldwide, especially in China. Recently, the watermelon genome has been sequenced, which provides an unprecedented opportunity for genome-wide characterization and functional analysis of the MAPK cascades in watermelon. The main results are as follows:In this study, we identified15MAPK genes and6MAPKK genes in watermelon genome. We characterized them based on nomenclature, classification, phylogeny, sequence alignment, conserved domain, chromosomal location, expression pattern, their protein interaction relatioships and possible functions in disease resistance.(1) By homology searching using Arabidopsis MAPK and MAPKK protein sequences as queries, total of16MAPK and12MAPKK genes were identified from watermelon genome, respectively. Multiple sequence alignment, conserved domain/motif and phylogenetic tree analyses showed that the watermelon MAPK and MAPKK families could be classified into four groups corresponding to the group A, B, C and D in Arabidopsis. Chromosomal location analysis revealed that21ClMPKs and ClMKKs genes were distributed on10chromosomes and both tandem duplication and segmental duplication were involved in the evolution of these genes.(2) The expression patterns of21ClMPKs and ClMKKs genes in different watermelon tissues such as root, stem, leaf, in watermelon plants treated with various biotic and abiotic stresses (e.g. drought, salt, cold, heat stress, or pathogen infection) or with different hormons such as abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA) or1-aminocyclopropane-1-carboxylate (ACC) were analyzed. The results show that the expression level of three genes (ClMPK19, ClMPK7, ClMKK6) was significantly different in roots, stems and leaves; two genes (ClMPK4-2, ClMPK7) were highly expressed under drought treatment; while only one gene (ClMPK7) was affected by salt stress. Four genes (ClMPK7, ClMPK9-1, ClMPK9-2, ClMPK9-4) were sharply down-regulated by cold stress, whereas three genes (ClMPK7, ClMPK20-1, ClMPK9-4) were significantly up-regulated by heat stress. Espescially, the expression level of ClMPK3, ClMPK7and ClMKK5was9,13and6times, respectively, higher than mock9days after Fusarium. oxysporum f.sp. niveum inoculation. The expression level of ClMPK7in a variety of treatment was significantly higher than other genes, and was highly up-regulated in drought stress, heat stress and ABA induction, but significantly down-regulated in cold stress. These resultd illustrate that ClMPK7may participate in a number of different signaling pathways.(3)The protein-protein interaction relationships between8ClMPKs and5ClMKKs were examined using yeast two-hybrid assays. The results showed that ClMPK4-1not only interacted with ClMKK2-1, but also with ClMKK6and ClMKK9. Furthermore, ClMKK2-1could simultaneously interact with ClMPK4-1,ClMPK4-2, and ClMPK13. A MAPK may have interaction with multiple MAPKKs and vice versa. Therefore, we hypothesized that there is a complex cross-interaction between MAPK and MAPKK in watermelon, which reflected the diversity of MAPK signal transduction pathways.(4) Possbile involvement of14CIMPKs and CIMKKs genes in disease resistance response was explored by agrobacterium-mediated transient expression approach. The selected CIMPKs and ClMKKs genes were transiently expressed in Nicotiana benthamiana plants and then challenged by inoculation with Botrytis cinerea to examine their possible function in disease resistane. The results showed that transient expression of CIMPK7in N. benthamiana induced the expression of defense genes and accumulation of reactive oxygen species, and increased resistance against B. cinerea. In addition, transient expression of CIMPK7also induced hypersensitive reactions.