A protein factor, identified in nuclear ex-

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Phytoene synthase (Psy) and phytoene desaturase (Pds) are the first dedicated enzymes of the plant carotenoid biosynthesis pathway. We report here the organ-specific and temporal expression of PDS and PSY in tomato plants. Light increases the carotenoid content of seedlings but has little effect on PDS and PSY expression. Expression of both genes is induced in seedlings of the phytoene-accumulating mutant ghost and in wild-type seedlings treated with the Pds inhibitor norflurazon. Roots, which contain the lowest levels of carotenoids in the plant, have also the lowest levels of PDS and PSY expression. In flowers, expression of both genes and carotenoid content are higher in petals and anthers than in sepals and carpels. During flower development, expression of both PDS and PSY increases more than 10-fold immediately before anthesis. During fruit development, PSY expression increases more than 20-fold, but PDS expression increases less than threefold. We concluded that PSY and PDS are differentially regulated by stress and developmental mechanisms that control carotenoid biosynthesis in leaves, flowers, and fruits. We also report that PDS maps to chromosome 3, and thus it does not correspond to the GHOST locus, which maps to chromosome 11.

A population of Arabidopsis thaliana recombinant inbred lines was constructed and used to develop a high-density genetic linkage map containing 252 random amplified polymorphic DNA markers and 60 previously mapped restriction fragment length polymorphisms. Linkage groups were correlated to the classical genetic map by inclusion of nine phenotypic markers in the mapping cross. We also applied a technique for local mapping that allows targeting of markers to a selected genome region by pooling DNA from recombinant inbred lines based on their genotype. We conclude that random amplified polymorphic DNAs, used in coglunction with a recombinant inbred population, can facilitate the genetic and physical characterization of the Arabidopsis genome and that this method is generally applicable to other organisms for which appropriate populations either are available or can be developed.The crucifer Arabidopsis thaliana is a useful system for basic studies in plant molecular genetics due to its relatively small genome size, small amounts of dispersed repetitive DNA, and rapid generation time (1). These attributes have made Arabidopsis an attractive model system for the analysis of genome organization and the development and use of technology to clone genes known only through their genetic map position.High-density genetic maps based upon DNA markers can provide starting points for chromosome walking experiments. Markers closely linked to a mutation of interest can reduce the amounts of DNA to be cloned and help establish the direction of the chromosome walk. Restriction fragment length polymorphisms (RFLPs) have been used as markers to construct genetic maps (2) and as starting points for chromosome walking (3). To date, two different RFLP maps have been reported in Arabidopsis (4, 5).Recently another class of genetic markers [random amplified polymorphic DNAs (RAPDs)] has been described (6, 7), which relies on the observation that a single oligonucleotide primer, of arbitrary nucleotide sequence, will direct the amplification of discrete loci (for a more detailed description of this method, see ref. 8). We report here the use of RAPD markers to construct a genetic map of A. thaliana. This map has been constructed with unprecedented speed by using RAPD markers and a recombinant inbred (RI) population. For many mapping purposes RI populations are superior to F2 or backcross populations because they constitute a permanent population in which segregation is fixed (9). Additional markers scored on the same RI population are automatically integrated with the existing map, making map information cumulative (9).Near-isogenic lines have been used to target RFLP (10) or RAPD (11) markers to specific segments of a genome. However, construction of near-isogenic lines is time consuming, and unlinked portions of the donor genome remain even after several crosses to the recurrent parent (12). Pooling DNA based on phenotype has been used as a means of either identifying additional RFLP loci (13) or mapping existing RFLP loci (14) ...

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Tomato (Lycopersicon esculentum) fruit ripening is a devel

Tomato (Lycopersicon esculantum) ASR1 (abscisic acid stress ripening protein), a small plant-specific protein whose cellular mode of action defies deduction based on its sequence or homology analyses, is one of numerous plant gene products with unknown biological roles that become over-expressed under water- and salt-stress conditions. Steady-state cellular levels of tomato ASR1 mRNA and protein are transiently increased following exposure of plants to poly(ethylene glycol), NaCl or abscisic acid. Western blot and indirect immunofluorescence analysis with anti-ASR1 antibodies demonstrated that ASR1 is present both in the cytoplasmic and nuclear subcellular compartments; approx. one-third of the total ASR1 protein could be detected in the nucleus. Nuclear ASR1 is a chromatin-bound protein, and can be extracted with 1 M NaCl, but not with 0.5% Triton X-100. ASR1, overexpressed in Escherichia coli and purified to homogeneity, possesses zinc-dependent DNA-binding activity. Competitive-binding experiments and SELEX (systematic evolution of ligands by exponential enrichment) analysis suggest that ASR1 binds at a preferred DNA sequence.

Diurnal oscillations in steady‐state mRNA levels and transcription rates were measured for seven transcripts (five of which encode chloroplast‐localized proteins) in tomato seedlings: photosystem I and photosystem II chlorophyll a/b binding proteins (CAB/I and CAB/II), small subunit of RuBisCO (RBCS), actin, subunit II of the photosystem I reaction center (PSAD), subunit I of the photosystem II oxygen‐evolving enzyme (OEE1), and a biotin‐binding protein of unknown function. CAB/II mRNA levels were found to oscillate greater than 20‐fold, showing a peak at noon, while only marginal diurnal oscillations are seen in RBCS transcripts. The oscillations are at least partially controlled at the transcriptional level. Transcription rates of both CAB/II and RBCS, measured by nuclear run‐on experiments, were found to oscillate, with a peak around 8 a.m. Transcription rates of the ‘biotin’ clone also oscillated, with a peak around noon. Transfer of plants to constant darkness or constant light conditions alters the amplitude of the transcriptional oscillation, but does not abolish it, suggesting that it is at least partially controlled by a circadian clock. The oscillations are still visible after three days in complete darkness, and have a period very close to 24 h. The oscillator phase can be reset by out‐of‐phase light treatment.