Analysis of the NADP(+) malic enzyme gene from tomato (Lycopersicon esculentum Mill.) and its induction by wounding and chemical elicitors.

摘要

Malate Dehydrogenase NADP+ (decarboxylating) EC 1.1.1.40 (malic enzyme) oxidatively decarboxylates malate to yield pyruvate and CO 2 while reducing NADP+ to NADPH. In plants, the role of malic enzyme (NADP+ME) is well characterized in C4 photosynthesis, and may be important in fruit ripening and maintaining cellular pH. During plant defense, NADP+ME may provide metabolic energy for reactions occurring in response to wounding or pathogens. To elucidate this role, we analyzed expression of the cytosolic isoform of the tomato (Lycopersicon esculentum Mill. cv. VFNT Cherry) ME gene (LeME2) in response to wounding and chemical elicitors.;Northern analysis of tomato (cv. UC82B) RNA provided evidence that: (1) LeME2 expression is high in roots, moderate in stems and weak in leaves. (2) In leaves, LeME2 was induced by wounding within two hours both locally and systemically. (3) In leaves, chemical elicitors: cellulase, xylanase, methyl-jasmonate and possibly glutathione enhanced LeME2 expression.;A reporter gene (-1280LeME2::GUS) was constructed with 1280bp of the LeME2 promoter and untranslated leader sequence directing expression of beta-glucuronidase (GUS) and inserted into the tobacco genome (Nicotiana tabacum L. cv. Petit Havana SR1) via Agrobacterium. Expression of -1280LeME2::GUS in transformants was tissue-specific. Fluorimetry showed GUS expression was strongest in roots of soil-grown plants. Histochemical staining showed GUS expression was strong in the cortex and vascular cylinder of older stems, but barely detectable in young stems. In leaves, expression was weak and mainly associated with the vasculature. Expression in floral tissues was in the vasculature of sepals, petals and carpels, at the stigma and within anthers.;When leaves of transformed tobacco were wounded, -1280LeME2::GUS expression was induced within two hours, increased until eight hours, then continued to accumulate slowly until 48 hours. By 24 hours a 15-fold increase in expression was fluorimetrically detected in ten primary transformants. Chemical elicitors that induced GUS expression beyond levels induced by wounding include cellulase, abscisic acid, methyl jasmonate and possibly sulfhydryl reagents.;A Cauliflower Mosaic Virus 35S::GUS gene and a promoterless GUS gene were included as positive and negative controls, respectively. Expression of genes in these plants was neither tissue-specific nor inducible by wounding or chemical elicitors.