Methyl jasmonate disrupts shoot formation in tobacco thin cell layers by over-inducing mitotic activity and cell expansion

Methyl jasmonate (MJ) has been shown to reduce the number of shoots formed at the end of culture in tobacco (Nicotiana tabacum L.) thin cell layers (TCLs, Biondi et al. – J Exp Bot 52:1-12, 2001). The principal aim of the present study was to determine early cyto-histological events related with this inhibition of shoot formation. To this end, 0.1, 1 and 10 M MJ were applied to TCLs cultured under shoot-forming conditions, and the explants were microscopically analysed during culture. The effect of MJ on the expression of two cell proliferation marker genes, ubi-CEP (ubiquitin carboxyl-extension protein) and top1 (topoisomerase 1), was also examined. Results show that MJ strongly stimulated mitotic activity early in culture (24-48 h) relative to untreated controls, both in superficial and deep layers. Enhanced proliferative growth was still present on days 5-7, in particular in the presence of the lower concentrations of MJ, and confirmed by both ubi-CEP and top1 transcript accumulation. The formation of meristematic cell clusters (MCC) on day 5 was also enhanced by 1 M MJ. Subsequent development of MCC into meristemoids (M) and shoot primordia (S) was, however, reduced by all MJ concentrations in a dose-dependent manner. Treatments with MJ also induced anomalous mitoses, whose presence was precociously (48 h) enhanced by the highest concentration, and later (day 9) by the lowest concentration. Cell expansion was stimulated by MJ, from 24 h up to day 12, especially around and within M and S. The expanded cells displayed thickened and suberized walls; cell wall thickness increased with increasing MJ concentration. Cell expansion and wall thickening/suberization caused alterations in the tunica and stem differentiation of the shoot dome. MJ also inhibited vascular differentiation, and promoted accumulation of phenolics in the vacuoles, especially in the presence of the highest concentration of MJ. All three concentrations of MJ stimulated ethylene biosynthesis, with the strongest effect being exerted by the intermediate one (1 M) at 30 - 48 h in culture. The apparently paradoxical role of MJ which deregulated shoot formation through a stimulation of growth events (i.e., mitotic activity and cell expansion) is discussed, also in relation to its early stimulation of ethylene biosynthesis. Keywords Cell expansion - Ethylene - Methyl jasmonate - Mitotic activity - Shoot formation - Tobacco thin cell layers