GhMAP3K40, A Mitogen-activated Protein Kinase Kinase Kinase From Cotton, Is Involved In Response To Adverse Stress And Regulating Growth And Development

Sessile plants constantly suffer from diverse environmental stresses during their development. To survive, plants have developed complex and elaborate signalling networks to protect themselves from these attacks. The mitogen-activated protein kinase(MAPK) cascade is a conserved pathway involved in the response of plants to various stresses. Many researches revealed that MAPK pathways are involved in the signalling responding to drought,high salinity, reactive oxygen species, hormones and pathogen challenges in plants. So far,researches on MAPK pathways mainly focus on model plants, such as Arabidopsis thaliana and Nicotiana benthamiana. As the top members of the MAPK pathways, MAP3 Ks are of great significance. A large body of researches showed that MAP3 Ks not only play roles in response to biotic and abiotic stresses, but also in regulating plant growth and development.Cotton is an important fibre- and oil-producing crop worldwide. Therefore, in the present study, we selected the economically important species cotton(Gossypium hirsutum L.) as the experiment material, identified a Raf-like MAP3 K gene and named it GhMAP3K40. A series of studies have been carried out on its sequence analysis, expression patterns and part of the function identification, the main results are as follows:(1) Sequence analysis revealed that he full-length cDNA is 1459 bp long, containing an1188 bp open reading frame(ORF). The ORF encoded a 396-amino acid protein, with a predicted molecular mass of 43.921 kDa and an isoelectric point of 5.49. Multiple-alignment and cluster analysis revealed that the GhMAP3K40 is a typical plant Raf-like MAP3 K. Since the gene we obtained shared a high similarity with the MAP3K40 in Gossypium raimondii,we named the novel gene GhMAP3K40.(2) Expression patterns at transcriptional level of GhMAPK3K40 indicated that it could be induced by abiotic stresses such as low temperature, high salinity, drought, oxidative stress,wounding and biotic stress such as R. solanacearum. Signalling molecules like SA, ET, GA3 and MeJA also induce its expression. These signalling molecules are involved in multiple processes of response to adverse stress and development in plants. The results provided a clue that GhMAP3K40 may play roles in responding to adverse stress and regulating growth and development.(3) Plants overexpressing GhMAP3K40 showed an enhanced resistance to drought and high salinity at germination stage. However, during the seedling stage, overexpression of GhMAP3K40 reduced the resistance to drought, Ralstonia solanacearum and Rhizoctonia solani infection, and the transgenic plants suffered more severe oxidative damage. The results suggested that the overexpression of GhMAP3K40 changed the resistance of plants to adverse stress. While on the other hand, the overexpression of GhMAP3K40 activated the defence-related genes and antioxidant system in transgenic plants which indicated that GhMAP3K40 play positive roles in responding to adverse stress.(4) The root system of the vector control plants were stronger compared with the transgenic plant, and the detached leaves from transgenic plants were less able to prevent pathogenic invasion, which indicated that GhMAP3K40 may negatively regulate growth and development. GhMAP3K40 was specifically expressed in the cell and plants; the expression of GhMAP3K40 was induced drastically and persistantly after GA3 treatment; the growth status of the transgenic germen was worse than the control group under normal condition.All of the results indicated that GhMAP3K40 are involved in the process of plant growth and development. So we inferred that GhMAP3K40 play positive roles in response to adverse stress but negative roles in growth and development. The overexpresion of GhMAP3K40 changed the physical structure of plants, so the transgenic plants showed a reduced resistance to adverse stress.(5) It was verified by yeast two-hybrid system that GhMAP3K40 could be interacted with GhMPKK4, and GhMPKK4 could be interacted with GhMPK4. The expression patterns of GhMPKK4 and GhMPK4 revealed that these interacting proteins could be all induced by drought, SA and ET, which indicated that these proteins may be involved in multiple physical processes and signaling pathways.