To identify genes involved in defense against heavy-metal stresses, a cDNA library originating from mercuric chloride-treated maize (Zea mays L. cv. INRA 258) leaves was constructed and analysed by differential screening using cDNAs derived from treated and untreated plants. Transcriptionally activated cDNA clones, designated CHEM (chemically-activated), were isolated and characterized. They represent various known proteins, such as glycine-rich proteins, pathogenesis-related proteins, chaperones and membrane proteins. The expression of the genes encoding these proteins was studied in maize subjected to other forms of abiotic stress. Expression of glycine-rich proteins was greatly enhanced by heat stress, and also stimulated by NaCl, polluted rainwater, wounding and cold stress. Pathogenesis-related proteins were strongly induced by ultraviolet light and to a lesser extent by NaCl, polluted rainwater and wounding. Heat-shock protein was mainly induced by heat and cold, and ubiquitin by wounding. Expression of the membrane channel protein was stimulated by heat stress, NaCl, polluted rainwater and ultraviolet-light irradiation.

Sterols become functional only after removal of the two methyl groups at C4 by a membrane-bound multienzyme complex including a 3b-hydroxysteroid-dehydrogenase/C4-decarboxylase (3bHSD/D). We recently identified Arabidopsis (Arabidopsis thaliana) 3bHSD/D as a bifunctional short-chain dehydrogenase/reductase protein. We made use of three-dimensional homology modeling to identify key amino acids involved in 4a-carboxy-sterol and NAD binding and catalysis. Key amino acids were subjected to site-directed mutagenesis, and the mutated enzymes were expressed and assayed both in vivo and in vitro in an erg26 yeast strain defective in 3bHSD/D. We show that tyrosine-159 and lysine-163, which are oriented near the 3b-hydroxyl group of the substrate in the model, are essential for the 3bHSD/D activity, consistent with their involvement in the initial dehydrogenation step of the reaction. The essential arginine-326 residue is predicted to form a salt bridge with the 4a-carboxyl group of the substrate, suggesting its involvement both in substrate binding and in the decarboxylation step. The essential aspartic acid-39 residue is in close contact with the hydroxyl groups of the adenosine-ribose ring of NAD + , in good agreement with the strong preference of 3bHSD/D for NAD + . Data obtained with serine-133 mutants suggest close proximity between the serine-133 residue and the C4b domain of the bound sterol. Based on these data, we propose a tentative mechanism for 3bHSD/D activity. This study provides, to our knowledge, the first data on the three-dimensional molecular interactions of an enzyme of the postoxidosqualene cyclase sterol biosynthesis pathway with its substrate. The implications of our findings for studying the roles of C4-alkylated sterol precursors in plant development are discussed.

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The Ext 1.2A gene of Nicotiana sylvestris L. encoding an extensin, a cell wall structural protein, was characterized. Ext 1.2A encodes a polypeptide of 311 amino acids having a highly repetitive structure and showing extensin features such as Ser-(Pro)(4) repeats and a high content of Tyr and Lys. The expression profile of the gene was demonstrated using the reporter GUS (beta-glucuronidase) fused to its promoter region (-630/+124, relative to the transcription start site) and by RNA gel blots. The results show that the (-630/+124) Ext 1.2A/GUS gene fusion is expressed in the root transition zone, where cells undergo an isodiametric growth but have not yet reached the rapid elongation phase, in stem inner and outer phloems and in cortical cells at the stem/petiole junction. The Ext 1.2A gene is also induced after wounding of stems, ribs, leaves or roots. The gene fusion is expressed in stem cortical cells, in ribs and at leaf edges upon wounding. These data suggest that the (-630/+124) promoter region contains regulatory elements responsible for expression in roots and stems, as well as for response to wounding in stems and leaves.