SummaryThe tomato mutant notabilis has a wilty phenotype as a result of abscisic acid (ABA) de®ciency. The wild-type allele of notabilis, LeNCED1, encodes a putative 9-cis-epoxycarotenoid dioxygenase (NCED) with a potential regulatory role in ABA biosynthesis. We have created transgenic tobacco plants in which expression of the LeNCED1 coding region is under tetracycline-inducible control. When leaf explants from these plants were treated with tetracycline, NCED mRNA was induced and bulk leaf ABA content increased by up to 10-fold. Transgenic tomato plants were also produced containing the LeNCED1 coding region under the control of one of two strong constitutive promoters, either the doubly enhanced CaMV 35S promoter or the chimaeric`Super-Promoter'. Many of these plants were wilty, suggesting co-suppression of endogenous gene activity; however three transformants displayed a common, heritable phenotype that could be due to enhanced ABA biosynthesis, showing increased guttation and seed dormancy. Progeny from two of these transformants were further characterized, and it was shown that they also exhibited reduced stomatal conductance, increased NCED mRNA and elevated seed ABA content. Progeny of one transformant had signi®cantly higher bulk leaf ABA content compared to the wild type. The increased seed dormancy was reversed by addition of the carotenoid biosynthesis inhibitor nor¯urazon. These data provide strong evidence that NCED is indeed a key regulatory enzyme in ABA biosynthesis in leaves, and demonstrate for the ®rst time that plant ABA content can be increased through manipulating NCED.

SummaryThe 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 C 15 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.

The abscisic acid (ABA) biosynthetic pathway involves the formation of a 9-cis-epoxycarotenoid precursor. Oxidative cleavage then results in the formation of xanthoxin, which is subsequently converted to ABA. A number of steps in the pathway may control ABA synthesis, but particular attention has been given to the enzyme involved in the oxidative cleavage reaction, i.e. 9-cis-epoxycarotenoid dioxygenase (NCED). Cloning of a gene encoding this enzyme in maize was first reported in 1997. Mapping and DNA sequencing studies indicated that a wilty tomato mutant was due to a deletion in the gene encoding an enzyme with a very similar amino acid sequence to this maize NCED. The potential use of this gene in altering ABA content will be discussed together with other genes encoding ABA biosynthetic enzymes.

Overexpression of 9-cis-epoxycarotenoid dioxygenase (NCED) is known to cause abscisic acid (ABA) accumulation in leaves, seeds and whole plants. Here we investigated the manipulation of ABA biosynthesis in roots. Roots from whole tomato plants that constitutively overexpress LeNCED1 had a higher ABA content than wild-type (WT) roots. This could be explained by enhanced in situ ABA biosynthesis, rather than import of ABA from the shoot, because root cultures also had higher ABA content, and because tetracycline (Tc)-induced LeNCED1 expression caused ABA accumulation in isolated tobacco roots. However, the Tc-induced expression led to greater accumulation of ABA in leaves than in roots. This demonstrates for the first time that NCED is rate-limiting in root tissues, but suggests that other steps were also restrictive to pathway flux, more so in roots than in leaves. Dehydration and NCED overexpression acted synergistically in enhancing ABA accumulation in tomato root cultures. One explanation is that xanthophyll synthesis was increased during root dehydration, and, in support of this, dehydration treatments increased b-carotene hydroxylase mRNA levels. Whole plants overexpressing LeNCED1 exhibited greatly reduced stomatal conductance and grafting experiments from this study demonstrated that this was predominantly due to increased ABA biosynthesis in leaves rather than in roots. Genetic manipulation of both xanthophyll supply and epoxycarotenoid cleavage may be needed to enhance root ABA biosynthesis sufficiently to signal stomatal closure in the shoot.

The abscisic acid (ABA)-deficient tomato mutant notabilis (not) is believed to be a null mutation in the gene LeNCED1 , encoding a 9-cis -epoxycarotenoid dioxygenase involved in ABA biosynthesis. We have sequenced and analysed a 19 kb genomic clone containing LeNCED1 and 5.4 kb of its promoter. This clone was transferred to not homozygotes and several non-wilty transformed plants were obtained. The basal ABA content, water relations, shoot and root growth, adventitious rooting, ethylene evolution and ability to accumulate ABA under water stress are described for two of these lines, not comp.13 and not comp.1 . Partial complementation was observed for most parameters measured for not comp.1 . Full complementation was observed in not comp.13 for all parameters measured in whole plants under well-watered and water-stressed conditions. These data provide further evidence that LeNCED1 is the wild-type allele of the not mutant gene. However, not comp.13 was unable to accumulate the wild-type levels of ABA in rapidly dehydrated leaves, indicating that it too was only partially complemented. Since LeNCED1 is an environmentally regulated gene encoding a rate-limiting enzyme, precise levels and patterns of gene expression may be needed to fully recreate wild-type phenotype. The utility of partially complemented lines to study the role of ABA in plant responses to stress conditions, and in promoter analysis, is discussed.