Auxin Levels Regulate the Expression of a Wound-Inducible Proteinase Inhibitor II-Chloramphenicol Acetyl Transferase Gene Fusion in Vitro and in Vivo

Abstract

Proteinase inhibitor genes are expressed in solanaceous and leguminous plants following wounding of the foliage by mechanical methods. Previous studies have shown that a cloned proteinase inhibitor II-chloramphenicol acetyl transferase (pin2-CAT) chimeric gene is regulated in a wound-inducible manner in transgenic plants. In this study, we analyzed transgenic plant tissues for expression of the pin2-CAT gene in response to various plant hormones. We found that CAT activity was induced in tobacco (Nicotiana tabacum) callus incubated in the absence of any plant growth regulators. Addition of growth regulators to the medium thus permitted us to measure the effects of these substances on the activity of the pin2-CAT gene construction. Cytokinin (BAP) and ethylene (ethophon) even at low concentrations stimulated the expression of CAT activity by 25 to 50%. Abscisic acid at concentrations up to 4.4 × 10⁻⁵ molar had no effect upon CAT activity, but increasing auxin (naphthalene acetic acid) levels completely inhibited the synthesis of CAT protein. Gibberellic acid had little effect except at very high concentration (2.9 × 10⁵ molar). The kinetics of activation of the pin2-CAT gene were quite long (5 to 7 days) when unwounded calli were plated on media lacking auxin. This effect was documented for calli derived from several transformed plants, containing the full, chimeric pin2-CAT (pRT45) gene. In addition, calli from tissues transformed with wild-type vectors or from several plants transformed with pRT50 (a noninducible derivative of pRT45) were not induced by plating on media lacking auxin. Other naturally occurring and synthetic auxins had similar effects to naphthalene acetic acid in inhibiting the induction of the chimeric gene fusion. Finally, leaf discs from transformed plants were induced by incubation in MS liquid medium in the presence and absence of naphthalene acetic acid. NAA was also effective in down regulating the chimeric gene in whole plant tissues.