He Evolutionary Analysis Of MAPK Cascade Gene Families In Plant And Functional Characterization Of SlMPK13 In Tomato

The mitogen-activated protein kinase (MAPK) signaling cascades are evolutionaly conserved in eukaryotes, including yeasts, animals, and plants. The classical MAPK signaling cascade is minimally composed of three kinases, namely, MAPK (MPK), MAPKK (MKK, MEK), and MAPKKK (MEKK). These kinases operate as sequential signal transducers that channel, integrate, and amplify information from the cellular environment to transcriptional and metabolic response centers via phosphorylation. In plants, MAPK cascades participate in plant growth and development, and defense responses to various biotic and abiotic stresses. In this study, a comprehensive genome-wide analysis of classification, conserved domain, gene structure, chromosomal localization, and expression profiles of MAPK cascade genes in tomato and cucumber was performed. Moreover, the distribution and evolutionary relationships of MAPK cascade family genes in plants were detected using bioinformatics analysis. Based on the identification of one tomato MAPK gene, SIMPK1S, preferentially expressed in stamens, we attempted to determine the function of MAPK genes in tomato growth and development. The subcellular localization, prokaryotic expression, phosphorylation activity in vitro, expression pattern analysis of the SIMPK13 gene was performed. Then the RNA interference (RNAi) technique was used to explore its roles in vegetative growth, floral development, and pollen development in tomato. At last, the possible regulation mechanism of SIMPK13 involved in pollen development was further investigated. The main results obtained are as follows.(1) Using BlastP and HMMER searches,89 MAPKKKs,5 MAPKKs and 1 novel MAPK in the tomato genome were identified, while 59 MAPKKKs,6 MAPKKs and 14 MAPKs were found in current cucumber genome database. Through multiple sequence alignment, conservative motif and the analysis of system evolution, MAPK and MAPKK gene family were grouped into four subgroups (A, B, C, and D), whereas MAPKKKs were divided into three subfamilies (MEKK, ZIK, and RAF). Quantitative real-time PCR (qRT-PCR) analysis showed that most MAPK cascade genes in Solanum lycopersicum and Cucumis sativus were expressed in more than one specific organs or tissues. Moreover, most of them could be induced by different biotic or abiotic stresses and hormone treatments.(2) The number of MAPKKK genes was greatly varied in different plants. The phylogenetic analysis suggested that MAPKKK genes evolved into three subfamilies before the evolution of algae. During the evolution of plants, MAPKKK family mainly underwent twice gene expansion. One happened during the evolution from algae to moss, the orther took place on the evolution of flowering plants from pteridophyte. Moreover, the expansion mainly led to the increasing number of genes belongs to RAF subfamily. The plant MAPKK genes belonging to A and B subfamilies appeared early in algae, however, the C and D subfamily genes appeared until the evolution of flowering plants. The rapid expansion of MAPK genes occurred during the process of evolution from pteridophytes to flowering plants, especially in D subfamily. MAPK genes in C and D subfamilies originated before the evolution of pteridophytes, while that in A and B subfamilies have emerged in bryophytes.(3) QRT-PCR and in situ hybridization analysis showed that SlMPK13 generally expressed in several organs or tissues as well as different development stages of stamens. However, SIMPK13 was preferentially expressed in stamens at anthesis, especially, in pollen grains during maturation. Prokaryotic expression showed that SlMPK13 encodes a hydrosoluble protein with approximately 44 kDa molecular weight. The phosphorylation activity was detected by the western blot experiment with the anti-pTyr antibody. Subcellular localization revealed that SlMPK13 is present in the plasma membrane, cytoplasm and nucleus.(4) The constitutive suppression of SIMPK13 was conducted via RNAi approach driven by the CaMV35S promotor. The transgenic plants were obtained via agrobacterium-mediated leaf disc method. The p35S::slmpkl3-RNAi transgenic plants is more dwarf with malformed leaves, the root development is inhibited, and floral organ morphology is abnormal, which suggested that SIMPK13 may play pleiotropy function in the process of tomato vegetative growth and reproductive development.(5) The suppression of SIMPK13 via RNAi approach with a pollen sppecific promoter LAT52 resulted in pollen development defects. These aborted pollen grains are malformed or collapsed and completely lacked viability. Cytological observations revealed that the anthers in pLAT52::slmpkl3-RNAi transgenic lines can develop normal binucleate microspores, but fail to form normal mature pollen grains. Transmission electron microscopy examination and DAPI staining further proved that the RNAi microspore developed abnormal vacuoles in the second bicellular vacuole stage and a portion of plasma membrane appeared to be irregularly intended and gradually degenerated during pollen maturation stage. Our results suggested that SIMPK13 has an essential role during tomato pollen development.(6) Transcriptome profiling of tomato stamenes using RNA-seq revealed that 1077 genes exhibited significantly altered expression between pLAT52::slmpkl 3-RNAi transgenic and control anthers at anthesis, including 846 up-regulated and 231 down-regulated genes. Gene ontology (GO) and pathway analysis suggested the majority of differentially expressed genes (DGEs) participate in several biological processes, such as the ’carbohydrate metabolic process’,’lipid metabolism’ ’cellular component organization’,’signal transduction’ and ’cell apoptosis metabolism’ categories. The results were consistent well with those from the subcellular localization of S1MPK13 and the cytology variation of the pLAT52::slmpkl3-RNAi transgenetic pollen grains. Taken together, we suggested that inhibition of SIMPK13 expression may promote the pollen cell apoptosis in late pollen stage, the plasma membrane and vacuoles developed abnormally, the metabolism process of carbohydrate and lipid is affected and finally the mature pollen development is disturbed in tomato.