Chitosan is involved in elicitation of vestitol production in Lotus japonicus

Leaves of Lotus japonicus infected by powdery mildew accumulate isoflavonoids, especially vestitol and sativan as protective compounds, whereas their flavonol-glycosides or phenylpropanoic acids content were rather decreased. For a better understanding of the induction of phytoalexin production in L. japonicus, the influence of important signalling molecules, namely methyl jasmonate and salicylic acid, was tested by leaf application, up to 15 d. No effects after spraying the leaves with 0.5 mM salicylic acid were observed regarding the plant isoflavonoid content, whereas methyl jasmonate showed moderate effect on vestitol accumulation. Chitosan application led to a strong increase in vestitol content, sativan and vestitol derivative also increased. In a similar way to the effect of powdery mildew infection, the content of ferulic acid and kaempferol glycosides decreased, except for one compound, identified as kaempferol-6-deoxyhexose, that is the result of acetylation of kaempferol glycosides. The application of chitosan also led to an approximately 2.5- to 3.5-fold increase in hydrogen peroxide content, indicating the involvement of H2O2 in the chitosan signalling pathway. The activity of key enzyme of the phenolic biosynthesis, phenylalanine amonia lyase (PAL), was stimulated as well as the expression of 3 PAL-isogenes (LjPAL4, LjPAL6, and LjPAL9), whereas LjPAL8 decreased. Moreover, the expression of two key enzymes of vestitol and sativan biosythesis: pterocarpan reductase and isoflavone synthase were also strongly induced. The data suggest that chitosan acts as an elicitor of the fungal attack in Lotus japonicus that do not lead to a general stimulation of the metabolism of phenols, but rather to a specific induction of isoflavonoid-phytoalexine production, especially for production of vestitol.