SummaryUsing a highly synchronous in vitro tuberization system, in combination with an amplified restriction fragment polymorphism (AFLpt)-derived technique for RNA fingerprinting (cDNA-AFLPt), transcriptional changes at and around the time point of potato tuberization have been analyzed. The targeted expression analysis of a specific transcript coding for the major potato storage protein, patatin and a second transcript, coding for ADP-glucose pyrophosphorylase, a key gene in the starch biosynthetic pathway is described. This paper confirms that kinetics of expression revealed by cDNA-AFLP analysis are comparable to those found in Northern analysis. Furthermore, this paper reports the isolation and analysis of two tuberspecific transcript-derived fragments (TDFs) coding for the lipoxygenase enzyme, which are differentially induced around the time point of tuber formation. Analysis of the two Iox TDFs demonstrates that it is possible to dissect the expression modalities of individual transcripts, not independently detectable by Northern analysis. Finally, it is shown that using cDNA-AFLP, rapid and simple verification of band identity may be achieved. The results indicate that cDNA-AFLP is a broadly applicable technology for identifying developmentally regulated genes.

Segregation analysis between Lysopersicon esculentum (cultivated tomato) and L. hirsutum (wild form) in conjunction with positional verification by using near-isogenic lines demonstrated that biosynthesis of two structurally different classes of sesquiterpenes in these species is controlled by loci on two different chromosomes. A locus on chromosome 6, Sesquiterpene synthase1 ( Sst1 ), was identified for which the L. esculentum allele is associated with the biosynthesis of ␤ -caryophyllene and ␣ -humulene. At this same locus, the L. hirsutum allele is associated with biosynthesis of germacrene B, germacrene D, and an unidentified sesquiterpene. Genomic mapping, cDNA isolation, and heterologous expression of putative sesquiterpene synthases from both L. esculentum and L. hirsutum revealed that Sst1 is composed of two gene clusters 24 centimorgans apart, Sst1-A and Sst1-B , and that only the genes in the Sst1-A cluster are responsible for accumulation of chromosome 6-associated sesquiterpenes. At a second locus, Sst2 , on chromosome 8, the L. hirsutum allele specified accumulation of ␣ -santalene, ␣ -bergamotene, and ␤ -bergamotene. Surprisingly, the L. esculentum allele for Sst2 is not associated with the expression of any sesquiterpenes, which suggests that cultivated tomato may have a nonfunctional allele. Sesquiterpene synthase cDNA clones on chromosome 6 do not cross-hybridize on genomic DNA gel blots with putative sesquiterpene synthases on chromosome 8, an indication that the genes in Sst1 and Sst2 are highly diverged, each being responsible for the biosynthesis of structurally different sets of sesquiterpenes.

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A collection of 9,990 single-pass nuclear genomic sequences, corresponding to 5 Mb of tomato DNA, were obtained using methylation filtration (MF) strategy and reduced to 7,053 unique undermethylated genomic islands (UGIs) distributed as follows: (1) 59% non-coding sequences, (2) 28% coding sequences, (3) 12% transposons-96% of which are class I retroelements, and (4) 1% organellar sequences integrated into the nuclear genome over the past approximately 100 million years. A more detailed analysis of coding UGIs indicates that the unmethylated portion of tomato genes extends as far as 676 bp upstream and 766 bp downstream of coding regions with an average of 174 and 171 bp, respectively. Based on the analysis of the UGI copy distribution, the undermethylated portion of the tomato genome is determined to account for the majority of the unmethylated genes in the genome and is estimated to constitute 61+/-15 Mb of DNA (approximately 5% of the entire genome)--which is significantly less than the 220 Mb estimated for gene-rich euchromatic arms of the tomato genome. This result indicates that, while most genes reside in the euchromatin, a significant portion of euchromatin is methylated in the intergenic spacer regions. Implications of the results for sequencing the genome of tomato and other solanaceous species are discussed.

Short-and medium-chain-length fatty acids (FAs) are important constituents of a wide array of natural products. Branched and straight short-chain-length FAs originate from branched chain amino acid metabolism, and serve as primers for elongation in FA synthase-like reactions. However, a recent model proposes that the one-carbon extension reactions that utilize 2-oxo-3-methylbutyric acid in leucine biosynthesis also catalyze a repetitive one-carbon elongation of short-chain primers to medium-chain-length FAs. The existence of such a mechanism would require a novel form of regulation to control carbon flux between amino acid and FA biosynthesis. A critical re-analysis of the data used to support this pathway fails to support the hypothesis for FA elongation by onecarbon extension cycles of ␣-ketoacids. Therefore, we tested the hypothesis experimentally using criteria that distinguish between one-and two-carbon elongation mechanisms: (a) isotopomer patterns in terminal carbon atom pairs of branched and straight FAs resulting from differential labeling with The broad structural diversity of short-and mediumchain length fatty acids (scFAs and mcFAs, respectively) and their derivatives is incorporated into a wide array of biomolecules as components of antibiotics, insect pheromones, and plant storage lipids (Denoya et al., 1995;Laakel et al., 1994;Tang et al., 1994; Giblin-Davis et al., 1996;Schal et al., 1994;Pennanec' et al., 1991; Charlton and Roeloffs 1991;Knapp et al., 1991;Thompson et al., 1990; Hartman and Reimann, 1989). Understanding the biosynthesis of these compounds is critical both to understanding their regulation and designing strategies for their manipulation.scFAs and mcFAs are also found in sugar polyesters secreted by Solanaceous plants as defensive agents against a wide array of insect herbivores and pathogens (Gentile and Stoner, 1968; Gentile et al., 1968 Gentile et al., , 1969 Juvik et al., 1982 Juvik et al., , 1994 França et al., 1989). These polyesters are composed of either Glc or Suc to which as many as five or six FAs, respectively, may be esterified (Schumacher, 1970;Severson et al., 1985;King et al., 1986King et al., , 1990Shinozaki et al., 1991;Shapiro et al., 1994).The acyl substituents exhibit a remarkable degree of species-specific structural diversity: They range in length from 3:0 to 12:0, and include straight-chain, iso-branched, and anteiso-branched FAs with both odd and even numbers of carbon atoms.The biosynthesis of branched-chain FAs has been extensively investigated in bacteria (Oku and Kaneda, 1988;Kang et al., 1997aKang et al., , 1997bZelles, 1997). Iso-and anteisobranched FAs 14 to 17 carbon atoms long are derived from ␣-keto derivatives of Leu, Val, and Ile, which serve as short-chain primers for elongation. In this model, NAD ϩ -and CoA-dependent branched-chain ketoacid dehydrogenase provides acyl-CoA primers through oxidative decarboxylation of these ketoacid precursors. A FA synthase (FAS) system then elongates these three-to five-carbon primers utilizing malonyl...