Simultaneous biochemical and molecular study of Methyl Jasmonate pathway on tolerant beans against Tetranychus urticae Koch.

Background and Aim: In order to study biochemical and molecular responses of Methyl jasmonate pathway in reproductive growth of bean under short-term attack of two-spotted spider mite (TSSM), two genotypes of common beans (۶۵-۰۶۲-۱۰۷ and ۶۵-۰۷۱-۴۰۰) were evaluated as the most tolerant and susceptible, respectively in a growth chamber condition in ۲۰۱۸. In addition, four levels of treatments were applied by mite on beans included ۱, ۳, ۵ interval days after infection, and control conditions. Simultaneously, both biochemical metabolites and gene expressions of lipoxygenase (LOX) and allene oxide synthase (AOS) as enzymes and genes on the jasmonic acid pathway were assessed on wounded leaves of common beans.Methods: Lipoxygenase enzyme activity (Kazemi Shahandashti et al., ۲۰۱۳) and AOS enzyme (Yang et al., ۲۰۱۱) were measured at ۲۳۴ nm for min-۱ mg-۱ protein. Gene expression was carried out after RNA extraction, cDNA synthase and designing specific primers by Real-Time PCR. Factorial experiments were performed using a completely randomized design with three replications. Data of enzyme activities were assessed using Duncan’s tests, and data of gene expression was supplied using REST software.Results: Lipoxygenase activity reached the highest level on the first day after infection and then started gently decreasing in the following days. Also, LOX enzyme was significantly lower in the tolerant genotype than the susceptible genotype as well. The pattern of changes in AOS activity was similar to the LOX trend too. The results of genes expression on Methyl jasmonate pathway showed that the jasmonic acid pathway activated by increasing LOX gene expression, its downstream gene AOS and a flavonoid called Ketol as well. Gene expression of LOX was highest in the tolerant genotype after ۲ days of mite attack (۲ fold) and then started decreasing. However, the trend of gene expression in susceptible genotypes continued to increase by the end of the fifth day (۱۵.۶ fold). In tolerance ones, AOS gene expression was at the highest level after ۳ days of infection (۳.۸ fold) and then it decreased slowly, while gene expression of AOS in susceptible genotype increased consequently (۸ fold) during TSSM attack.Conclusion: The results showed that lipoxygenase was considered a more efficient antioxidation system in controlling free radicals during the TSSM attack. Also, the activity of antioxidant enzymes of LOX and AOS increased at the TSSM attack in the tolerant genotype compared to the susceptible genotype because of hydrogen peroxide inhibitor synchronous with decreasing H۲O۲ accumulation. Therefore, lipoxygenase and allene oxide synthase enzymes were key genes in the Methyl jasmonate (JA) biosynthesis pathway in response to the wounding stress of TSSM in common bean. So that, the JA induction caused by wounds was regulated by the LOX and AOS genes at both transcriptional and enzymatic levels that induce defense responses. The study of genes expression on Methyl jasmonate pathway resulted that the jasmonic acid pathway activated by increasing LOX gene expression, its downstream gene AOS and a flavonoid called Ketols as well. In JA pathway, allene oxide synthase induced to synthase of downsteam genes of JA cycle and the Ketols as second metabolites. Furthermore, increasing the activity of antioxidant enzymes and genes (LOX and AOS) were observed more in tolerance ratio susceptible common beans during treatments.In conclusions, Lipoxygenase and allene oxide synthase in the jasmonic acid signaling pathway played important roles in defense response of common bean under biotic stress such as wounding by mite attack.

Keywords:

Tolerance common bean , Methyl jasmonate pathway , lipoxygenase and allene oxide synthase enzymes , LOX and AOS genes