RESUMO
Two genes encoding enzymes in the abscisic acid (ABA) biosynthesis pathway, zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED), have previously been cloned by transposon tagging in Nicotiana plumbaginifolia and maize respectively. We demonstrate that antisense down-regulation of the tomato gene LeZEP1 causes accumulation of zeaxanthin in leaves, suggesting that this gene also encodes ZEP. LeNCED1 is known to encode NCED from characterization of a null mutation (notabilis) in tomato. We have used LeZEP1 and LeNCED1 as probes to study gene expression in leaves and roots of whole plants given drought treatments, during light/dark cycles, and during dehydration of detached leaves. During drought stress, NCED mRNA increased in both leaves and roots, whereas ZEP mRNA increased in roots but not leaves. When detached leaves were dehydrated, NCED mRNA responded rapidly to small reductions in water content. Using a detached leaf system with ABA-deficient mutants and ABA feeding, we investigated the possibility that NCED mRNA is regulated by the end product of the pathway, ABA, but found no evidence that this is the case. We also describe strong diurnal expression patterns for both ZEP and NCED, with the two genes displaying distinctly different patterns. ZEP mRNA oscillated with a phase very similar to light-harvesting complex II (LHCII) mRNA, and oscillations continued in a 48 h dark period. NCED mRNA oscillated with a different phase and remained low during a 48 h dark period. Implications for regulation of water stress-induced ABA biosynthesis are discussed.
Assuntos
Ácido Abscísico/biossíntese , Solanum lycopersicum/metabolismo , Ácido Abscísico/farmacologia , Northern Blotting , Ritmo Circadiano , DNA Antissenso/genética , DNA Complementar , Escuridão , Dioxigenases , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Oxirredutases/genética , Oxigenases/genética , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transformação Genética , Água/farmacologia , Xantofilas , Zeaxantinas , beta Caroteno/análogos & derivados , beta Caroteno/metabolismoRESUMO
The notabilis (not) mutant of tomato has a wilty phenotype due to a deficiency in the levels of the plant hormone abscisic acid (ABA). The mutant appears to have a defect in a key control step in ABA biosynthesis--the oxidative cleavage of a 9-cis xanthophyll precursor to form the C15 intermediate, xanthoxin. A maize mutant, viviparous 14 (vp14) was recently obtained by transposon mutagenesis. This maize genetic lesion also affects the oxidative cleavage step in ABA synthesis. Degenerate primers for PCR, based on the VP14 predicted amino acid sequence, have been used to provide probes for screening a wilt-related tomato cDNA library. A full-length cDNA clone was identified which is specific to the not gene locus. The ORFs of the tomato cDNA and maize Vp14 are very similar, apart from parts of their N-terminal sequences. The not mutation has been characterized at the DNA level. A specific A/T base pair deletion of the coding sequence has resulted in a frameshift mutation, indicating that not is a null mutant. This observation is discussed in connection with the relatively mild phenotype exhibited by not mutant homozygotes.