The Functions Of SlMPK1/2/3in Exogenous NO Induced Defense-Response Pathway Of Postharvest Tomato Fruit

Plants have developed sophisticated signaling networks to precisely regulate defense responses against pathogen attack. Mitogen-activated protein kinase (MAPK) cascades are common mechanisms to translate external stimuli into cellular responses in all eukaryotes, including higher plants. Some of MAPKs have been functionally characterized for their possible roles in regulating defense responses against biotic and abiotic stresses. Nitric oxide (NO) plays a crucial role in plant science, and plays multifunctional roles in diverse physiological processes, such as resistance to biotic and abiotic stress. However, whether MAPKs are involved in defense responses of tomato fruit, and wherher MAPKs are associated with NO-induced defense responses in tomato fruit remain to be studies. The mature green tomato fruit was used to investigate the influences of Botrytis cinerea (B. cinerea) inculation on the expressions of SIMPKs, the functions of SlMPK1/2/3on the innate immunity of tomato fuirt, and the effects of SIMPK1/2/3on NO-induced defense responses. The major results of this research were as follows:The relativite expressions of SIMPK1, SIMPK2, SIMPK3, SIMPK4, SIMPK5, and SIMPK16were higher in B. cinerea-inculated tomato fruit than that in control. The expression levels of SIMPK7, SIMPK9, SIMPK10, SIMPK11, and SIMPK12were inhibited in B. cinerea-inculated tomato fruit than that in control. The influences of B. cinerea inoculation on SIMPK6, SIMPK8, SIMPK13, SIMPK14, and SlMPK15changed with time. So we choose SIMPK1/2/3/4/5/16for the further study. SA induced exponentially SIMPK1/2/3/4gene expression, and induction of SIMPK5/16gene expression is not significant, so choose SIMPK1/2/3/4for the further study. Since the resistance of SlMPK4to B. cinerea has been reported in tomato, so ultimately determine SlMPK1/2/3for the further study.We nvestigated the effect of different concentrations of1,4-Diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene (U0126, a specific inhibitor of MAPKK) on the relative expressions of SIMPK1, SIMPK2, and SIMPK3, and found that10μM U0126significantly inhibited the expression of SlMPK1, SlMPK2and SIMPK3. Therefore,10μM U0126treatment was choosed for further study of the functions of SIMPK1/2/3in tomato disease resistance pathway. U0126(10μM) treatment promoted the gray mold rot of tomato fruit, enhanced Indole Acetic Acid (IAA), Abscisic Acid (ABA) and Gibberellic Acid (GA) concentration, and reduced the concentration of Methyl Jasmonate (MeJA). Moreover, U0126treatment promoted H2O2accumulation reduced the activities catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD). These results illustrated that plant hormones and reactive oxygen species (ROS) were involved in the disease signaling of SIMPK1/2/3.U0126treatment promoted the release of ethylene, elevated levels of1-aminocyclopropane-l-carboxylic acid (ACC), improved ACC synthase (ACS) activity and ACC oxidase (ACO) activity in most of the sampling point, induced the expression levels of SIACS1A, SIACS2, SlACS4, SlACO1, SlACO3, SlACO4and SlACO6. So we speculated that U0126treatment influence the biosynthesis pathway of ethylene, which can influence the ripening of tomato, and finally result in the disease resistance of tomato fruit against B. cinerea.We successfully cloned SIMPK1/2/3genes, and constructed the overexpression of pCAMBIA-2300-MAPK1/2/3vectors. Then the cotyledon of Micro-tom tomato was used as explants to obtain transgenic plants according to the method of agrobacturium-mediated leaf disk transformation. These are very important materials for further study about the functions of SIMPK1/2/3on NO-induced defense response. We successfully obtained SIMPK1, SIMPK3overexpression tomato plants and fruit. In SlMPK1overexpression tomato plants and fruit, the relative expression of SIMPK1was significantly increased; In SIMPK3overexpression tomato plants and fruit, the relative expression of SIMPK3was significantly increased.In SIMPK1overexpression tomato plant, gray mold prevention efficiency was35.06%; and in SIMPK3overexpression tomato plant, gray mold prevention efficiency was28.45%.To study (1) the effects NO on defense responses and the expression of SlMPK1/2/3,(2) the effects of SIMPK1/2/3NO-induced defense responses,0.2mM sodium nitroprusside (SNP), and SNP+U0126treatments were used. Results showed that NO could induce the disease resistance of tomato fruit, SNP treatment reduced the diseae incidence and lesion size after inculaotion of B. cinerea, and increased the acitvities of β-1,3-glucanases (GLU), chitinase (CHI), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL). However, U0126treatment significantly weakened the effects of NO-induced defense responses. These results illustrated that SIMPK1, SIMPK2and SIMPK3were associated with NO-induced defense response against in B. cinerea tomato fruit.To study (1) whether arginine (Arg) was involved in the disease resistance of tomato fruit and (2) the role of nitric oxide synthase (NOS) and MAPKs in the regulation of arginine-induced resistance responses, L-Arg and NG-nitro-L-arginine methyl ester hydrochloride (L-NAME, an inhibitor of NOS) were used. Treatment with L-Arg significantly increased NO content; enhanced the resistance of the tomato fruit against B. cinerea invasion and the activities of NOS, CHI, GLU, PPO and PAL. Moreover, the relative expression of SIMP K1, SIMPK2, and SIMPK3in the L-Arg-treated fruit was higher than that in the control fruit. Treatment with L-NAME significantly inhibited the activity of nitric oxide synthase and increased the incidence of disease and the size of lesion areas in the tomato fruit after being inoculated with B. cinerea. These results suggest that NOS and SIMPK1/2/3are involved in L-Arg-induced disease resistance of tomato fruit against B. cinerea.