Genetic map length and gene number in eukaryotes vary considerably less than genome size, giving rise to the hypothesis that recombination is restricted to genes. The complex genome of maize contains a large fraction of repetitive DNA, composed principally of retrotransposons arranged in clusters. Here, we assess directly the contribution of retrotransposon clusters and genes to genetic length. We first measured recombination across adjacent homozygous genetic intervals on either side of the bronze (bz) locus. We then isolated and characterized two bacterial artificial chromosome clones containing those intervals. Recombination was almost 2 orders of magnitude higher in the distal side, which is gene-dense and lacks retrotransposons, than in the proximal side, which is gene-poor and contains a large cluster of methylated retrotransposons. We conclude that the repetitive retrotransposon DNA in maize, which constitutes the bulk of the genome, most likely contributes little if any to genetic length.A lthough the amount of nuclear DNA in eukaryotes varies greatly, the total length of their genetic maps does not. This can be considered the genetic version of the C-value paradox, which states that the size of eukaryotic genomes bears little relationship to organismic complexity or number of genes (1). To account for the discrepancy between the sizes of genomes and genetic maps, Thuriaux hypothesized that meiotic recombination may be largely restricted to genes (2). Maize, with its large amount of repetitive DNA (3), is a particularly suitable organism for testing this hypothesis. One prediction of the hypothesis is that recombination within genes, expressed as the ratio of genetic to physical length (cM͞kb), should be much higher than the genome's average (4). Data from intragenic recombination experiments support that prediction (5-9). Recombination within the bronze (bz) locus, for example, is at least 100 times higher than the maize genome's average (5). A second prediction of the hypothesis is that repetitive DNA should not contribute significantly to genetic length, i.e., recombination across repetitive DNA should be much lower than across genic DNA. Here we analyze how this prediction holds around the bz locus, one of the regions of the maize genome where recombination has been studied in greatest detail (5, 10).Approximately 80% of the 2,500-megabase maize nuclear genome (11) is present in more than 100 copies (12). As in other plants with large genomes, much of this repetitive DNA consists of different families of retrotransposons (13-15). In maize, at least 50% of the repetitive DNA is made up of retrotransposon blocks dispersed throughout the gene-containing regions of the genome (16). We recently isolated a 240-kb bacterial artificial chromosome (BAC) contig centered around Bz-McC, one of the bz alleles used in the earlier intragenic recombination studies, and established that the bz gene is located in an unusually gene-dense region flanked at either end by retrotransposon blocks (17, 18). Thus, the bz geno...

The bronze (bz) locus exhibits the highest rate of recombination of any gene in higher plants. To investigate the possible basis of this high rate of recombination, we have analyzed the physical organization of the region around the bz locus. Two adjacent bacterial artificial chromosome clones, comprising a 240-kb contig centered around the Bz-McC allele, were isolated, and 60 kb of contiguous DNA spanning the two bacterial artificial chromosome clones was sequenced. We find that the bz locus lies in an unusually gene-rich region of the maize genome. Ten genes, at least eight of which are shown to be transcribed, are contained in a 32-kb stretch of DNA that is uninterrupted by retrotransposons. We have isolated nearly full length cDNAs corresponding to the five proximal genes in the cluster. The average intertranscript distance between them is just 1 kb, revealing a surprisingly compact packaging of adjacent genes in this part of the genome. At least 11 small insertions, including several previously described miniature inverted repeat transposable elements, were detected in the introns and 3 untranslated regions of genes and between genes. The gene-rich region is flanked at the proximal and distal ends by retrotransposon blocks. Thus, the maize genome appears to have scattered regions of high gene density similar to those found in other plants. The unusually high rate of intragenic recombination seen in bz may be related to the very high gene density of the region.

Plants can defend themselves from herbivorous insects by emitting volatile chemical signals that attract natural enemies of the herbivore. For example, maize seedlings attacked by beet armyworm larvae (Spodoptera exigua) produce a mixture of terpenoid and indole volatiles that serve to attract parasitic wasps. A key step in terpenoid biosynthesis is the conversion of acyclic prenyl diphosphates to terpenoid compounds by specific terpenoid synthases (cyclases). We have cloned a maize sesquiterpene cyclase gene, stc1, by transposon tagging and have identified two deletion mutations of the gene. The stc1 gene is located on chromosome 9S and does not seem to have a closely related ortholog in the maize genome. It is induced 15-to 30-fold in maize leaves by beet armyworm larvae feeding or by application of purified volicitin, the insect-derived elicitor, at a mechanically wounded site. stc1 induction is systemic, because undamaged leaves of the same plant show a similar increase in stc1 transcription. Analysis of volatiles from volicitin-treated seedlings revealed that a major naphthalene-based sesquiterpene was present in wild-type seedlings but absent in the Ac-insertion and x-ray-deletion mutants. Therefore, we have identified a maize gene that responds to caterpillar herbivory by producing a chemical defense signal that most likely serves to attract natural enemies of the herbivore.

Using a homologous transgenic rice system it is demonstrated that 5' distal and proximal cis-acting transcriptional regulatory elements are required for developmental control of a rice seed storage protein glutelin gene. Analyses of gene expression of nine progressively truncated 5' promoter sequences in developing endosperm indicated the existence of at least one major positive element located from the -5.1 to -1.8 kb region. The functional importance of proximal elements in the context of 1.8 kb promoter was demonstrated by single substitution mutations in the TATA box (-28/-23), AACA motif (-73/-61), and protein-binding boxes I (-103/-86), II (-124/-110), III (-175/-158) and IV (-200/-217). A simultaneous mutation of five protein-binding sites (-410/-86) essentially eliminated the activity of the 1.8 kb promoter. Although temporal control of the Gt1 gene during endosperm development was retained in plants of constructs from -5.1 kb to -155 bp, spatial control of the glutelin gene was altered when the 5.1 kb promoter was deleted to -507 bp or -154 bp as the reporter gene activities of these constructs were detected in phloem of leaves, and in stems, sheaths and roots of plants.

The seed storage protein P-phaseolin of the common bean (Pbaseolus vulgaris L.) was expressed i n the endosperm of transgenic rice (Oryza sativa L.) plants. The 5.1 -or 1.8-kb promoter fragment of the rice seed storage protein glutelin C t l gene was fused transcriptionally to either the genomic or cDNA coding sequence of the P-phaseolin gene. The highest quantity of phaseolin estimated by enzyme-linked immunosorbent assay was 4.0% of the total endosperm protein in the transgenic rice seeds. The phaseolin trait was segregated as a single dominant trait with a positive gene dosage effect and was stably inherited through three successive generations. 60th phaseolin genomic and cDNA coding sequences were used to synthesize four isoforms of mature phaseolin protein with apparent molecular masses of 51, 48, 47, and 45 kD. Enzyme deglycosylation experiments indicated that the 51 -kD form contains high-mannose N-glycans; the 48-and 47-kD forms have further modified N-glycans; and the 45-kD form is a nonglycosylated protein. lmmunolabeling studies using light and electron microscopy demonstrated that phaseolin accumulates primarily in the vacuolar type-I1 protein bodies located at the periphery of the endosperm near the aleurone layer. We discuss the implications of these results on nutritional improvement of rice grains.