Induction of 1-aminocyclopropane-1-carboxylate oxidase mRNA by ethylene in mung bean roots: Possible involvement of Ca²⁺ and phosphoinositides in ethylene signalling

Ethylene markedly induces an increase in the mRNA level of 1-aminocyclopropane-1-carboxylate (ACC) oxidase, the final step of its biosynthetic pathway, in mung bean roots. To investigate the second messengers that possibly participate in ethylene signalling various pharmacological reagents known to affect the cytosolic calcium level and phosphoinositide (PI) metabolism were applied to mung bean roots, and then the induction pattern of ACC oxidase (VR-ACO1) by ethylene was monitored as a molecular paradigm for ethylene-dependent responses in this tissue. The ethylene-induced VR-ACO1 gene expression was effectively inhibited by ruthenium red, a putative inhibitor of intracellular Ca²⁺ fluxes, and to a lesser extent by ethyleneglycotetraacetic acid, a calcium chelator and GdCl₃, the plasma membrane calcium-channel inhibitor. However, LaCl₃ and verapamil, another type of plasma membrane calcium-channel blockers, did not prevent the ethylene action. The calmodulin antagonist W-7 significantly reduced the hormone-dependent expression of VR-ACO1. Treatment with neomycin or Li⁺, the antagonists of PI metabolism, almost completely blocked the ethylene-induced accumulation of VR-ACO1 transcript in a dose-dependent manner, whereas the addition of exogenous Ca²⁺ along with antagonists effectively restored the ethylene-induced VR-ACO1 gene activation. The results showed that the ACC oxidase enzyme activities and protein levels were in parallel with the abundance of transcript in response to various antagonists and exogenous Ca²⁺. These results are discussed in light of suggestion that the PI-derived second messenger and cytosolic calcium are necessarily involved in the ethylene-induced ACC oxidase gene activation in mung bean root tissue.