Expression, regulation and functional roles of beta-1,3-glucanase and chitinase in germinating tomato seeds

Weakening of the micropylar endosperm (endosperm cap), a physical barrier constraining the growth of the embryonic radicle, is a prerequisite for tomato (Lycopersicon esculentum Mill.) seed germination. Cell wall hydrolases are presumed to be involved in cell wall modification associated with endosperm weakening, facilitating radicle emergence. The possible contributions of β-1,3-glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) in endosperm cap weakening during tomato seed germination were investigated. β-1,3-glucanase and chitinase mRNA, protein, and enzyme activity are expressed specifically in the micropylar region at an early stage of tomato seed germination. Both enzymes are class I vacuolar basic isoforms encoded by the GluB and Chi9 genes, respectively. GluB mRNA is expressed exclusively in the endosperm cap tissue of tomato seeds prior to radicle emergence, whereas Chi9 transcript is located in both endosperm cap and radicle tip tissues. Neither gene is induced in gibberellin-deficient gib-1 tomato seeds unless gibberellic acid (GA) is provided, which also stimulates germination. However, abscisic acid (ABA), which delayed or prevented radicle emergence but not endosperm cap weakening in wild type seeds, suppressed GluB, but not Chi9, mRNA and protein accumulation. The hypothesis that these two enzymes are involved in endosperm cap weakening was tested by using puncture force experiments and by studying their ability to hydrolyze cell walls isolated from tomato micropylar endosperm. No appreciable hydrolysis of tomato endosperm cap cell walls by 1,3-glucanase or chitinase was detected. Therefore, it is unlikely that GluB or Chi9 directly participates in the endosperm weakening process in germinating tomato seeds.; Various elicitors of β-1,3-glucanases and chitinases were investigated to determine whether GluB and Chi9 mRNA and protein expression in gib-1 tomato seeds are induced directly by GA or indirectly in response to tissue wounding associated with endosperm cap weakening and radicle protrusion. Wounding and methyl jasmonate (MeJA) stimulated Chi9 (but not GluB) expression specifically in the micropylar endosperm, regardless of whether seeds were exposed uniformly to McJA, wounded at the chalazal end of the seed or punctured anywhere in the seed.{09}In contrast, ethylene, salicylate, fusicoccin, β-aminobutyric acid or ABA treatment had no effect on expression of either Chi9 or GluB in gib-1 seeds. Consistent with the effects of McJA treatment, Chi9, but not GluB , mRNA abundance was reduced significantly in germinating jasmonate-deficient defenseless-1 (def-1) tomato seeds and could be restored by application of McJA. The results support the hypothesis that Chi9 expression during germination may be associated with “wounding” from cell wall hydrolysis and weakening in the endosperm cap leading to radicle protrusion. Jasmonic acid may be involved in the signaling pathway for this response.