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

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 I1-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 x 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 x 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. Wounding of plants is responsible for the regulation of a wide variety of gene products in plants (1, 3, 6, 13, 17, 26). The inducible characterized genetic systems include plant defences against microbes and herbivores (13, 17, 19). One of the best characterized examples of the induction of genes following wounding is that of the proteinase inhibitor genes of solanaceous plants (11, 12, 24, 31). In this system, two small gene families (termed proteinase inhibitor I and II), 'This work was supported by grants from the U.S. Department of Agriculture (87-CRCR1-2518), the State of Iowa, and the Iowa Biotechnology Council. which encode quantitative insect resistance factors, are induced from a quiescent to an active state by wounding. The complete mechanism by which these genes become induced is not known, but several steps in the process have been elucidated. Wounding of plant leaves releases intravacuolar glycosidases which interact specifically with adjacent cell walls to liberate low mol wt oligosaccharides (2, 6). These low mol wt oligosaccharides have been isolated and can be fed through the petiole of detached leaves to induce the accumulation of the proteinase inhibitors (24). The induction is regulated at the transcriptional level and the levels ofmRNA approach 0.7% of total poly(A+) mRNA following multiple wounds (1 1). The kinetics of induction for both families of proteinase inhibitors are quite long (4-6 h) following wounding before mRNAs begins to accumulate (11). This long preinduction period is unusual in inducible systems indeed even among other wound-inducible systems gene activation is quite rapid ( 17). The genes which code for these proteinase inhibitors have been isolated and characterized from both tomato and potato (5, 9, 16-18, 31). The wound-inducible nature of the pin22 genes was demonstrated independently by two separate groups (23, 31). This study utilizes whole plants and tissues transformed with the wound-inducible pin2-CAT gene to examine the biochemical mechanism ofwound-induction of plant defense