SummaryControlled expression of transgenes in plants is key to the characterization of gene function and the regulated manipulation of growth and development. The alc gene-expression system, derived from the ®lamentous fungus Aspergillus nidulans, has previously been used successfully in both tobacco and potato, and has potential for use in agriculture. Its value to fundamental research is largely dependent on its utility in Arabidopsis thaliana. We have undertaken a detailed function analysis of the alc regulon in A. thaliana. By linking the alcA promoter to b-glucuronidase (GUS), luciferase (LUC) and green uorescent protein (GFP) genes, we demonstrate that alcR-mediated expression occurs throughout the plant in a highly responsive manner. Induction occurs within one hour and is dose-dependent, with negligible activity in the absence of the exogenous inducer for soil-grown plants. Direct application of ethanol or exposure of whole plants to ethanol vapour are equally effective means of induction. Maximal expression using soil-grown plants occurred after 5 days of induction. In the majority of transgenics, expression is tightly regulated and reversible. We describe optimal strategies for utilizing the alc system in A. thaliana.

Many transgenic plant studies use constitutive promoters to express transgenes. For certain genes, deleterious effects arise from constant expression in all tissues throughout development. We describe a chemically inducible plant gene expression system, with negligible background activity, that obviates this problem. We demonstrate its potential by showing inducible manipulation of carbon metabolism in transgenic plants. Upon rapid induction of yeast cytosolic invertase, a marked phenotype appears in developing leaves that is absent from leaves that developed before induction or after it has ceased.

Onion (Allium cepa), garlic (A. sativum) and other Alliums are important because of the culinary value of their flavours and odours. These are characteristic of each species and are created by chemical transformation of a series of volatile sulphur compounds generated by cleavage of relatively stable, odourless, S-alk(en)yl cysteine sulphoxide flavour precursors by the enzymes alliinase and lachrymatory-factor synthase. These secondary metabolites are S-methyl cysteine sulphoxide (MCSO, methiin; present in most Alliums, some Brassicaceae), S-allyl cysteine sulphoxide (ACSO, alliin; characteristic of garlic), S-trans-prop-1-enyl cysteine sulphoxide (PECSO, isoalliin; characteristic of onion), and S-propyl cysteine sulphoxide (PCSO, propiin; in onion and related species). Information from studies of the transformation of putative biosynthetic intermediates, radiolabelling, and from measurements of sulphur compounds within onion and garlic have provided information to suggest a biosynthetic pathway. This may involve alk(en)ylation of the cysteine in glutathione, followed by cleavage and oxidation to form the alk(en)yl cysteine sulphoxide flavour precursors. There is also evidence that synthesis of the flavour precursors may involve (thio)alk(en)ylation of cysteine or a precursor such as O-acetyl serine. Both routes may occur depending on the physiological state of the tissue. There are indications from the effects of environmental factors, such as the availability of sulphur, that control of the biosynthesis of each flavour precursor may be different. Cysteine and glutathione metabolism are discussed to indicate parallels with Allium flavour precursor biosynthesis. Finally, possible avenues for exploration to determine the origin in planta of the alk(en)yl groups are suggested.

SummaryWe describe a chemically induced gene control mechanism for plants based on the ALCR transcription factor and alcA promoter of Aspergillus nidulans, which we have called the alc system. Ethanol, the chemical inducer, is not toxic at levels required for induction, and can be applied to the plants by spraying, root drenching and addition to liquid growth media. The alc system is very sensitive to ethanol and the induction is rapid; 0.01% (1.7 mM) ethanol in liquid growth media initiates chloramphenicol acetyl transferase (CAT) reporter gene expression within 4 h, with maximal expression occurring after 4 days. In the complete absence of ethanol, there is no detectable expression of CAT, nor do we observe induction in plants subjected to wound, cold or drought stress, or following treatment with either salicylic acid or methyl jasmonate. However, extreme anoxia resulting in elevated levels of alcohol dehydrogenase activity in both roots and leaves gave substantial induction of CAT in leaves but not in roots. We believe that the alc system will have broad utility in the exogenous control of plant gene expression in pure science and that it also has considerable potential in agriculture.

In response to excess metal, higher plants produce metal-binding peptides ([yEC].G) whose biosynthesis is believed to be mediated by enzymes involved in glutathione (yECG) metabolism. In contrast, animals synthesize metallothioneins, gene-encoded low molecular weight cysteine-rich metal-binding proteins. In an investigation of copper-regulated genes in the copper-tolerant flowering plant Mimulus guttutus, we have isolated a series of cDNA clones identifying two genes which encode a protein with class I metallothionein domains. This represents the first description of a metallothionein gene in a flowering plant.