An effective plant alkaloid chemical defense requires a variety of transport processes, but few alkaloid transporters have been characterized at the molecular level. Previously, a gene fragment encoding a putative plasma membrane proton symporter was isolated, because it was coordinately regulated with several nicotine biosynthetic genes. Here, we show that this gene fragment corresponds to a Nicotiana tabacum gene encoding a nicotine uptake permease (NUP1). NUP1 belongs to a plant-specific class of purine uptake permease-like transporters that originated after the bryophytes but before or within the lycophytes. NUP1 expressed in yeast cells preferentially transported nicotine relative to other pyridine alkaloids, tropane alkaloids, kinetin, and adenine. NUP1-GFP primarily localized to the plasma membrane of tobacco Bright Yellow-2 protoplasts. WT NUP1 transcripts accumulated to high levels in the roots, particularly in root tips. NUP1-RNAi hairy roots had reduced NUP1 mRNA accumulation levels, reduced total nicotine levels, and increased nicotine accumulation in the hairy root culture media. Regenerated NUP1-RNAi plants showed reduced foliar and root nicotine levels as well as increased seedling root elongation rates. Thus, NUP1 affected nicotine metabolism, localization, and root growth.

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Plants disease resistance (R) genes encode specialized receptors that are quantitative, rate-limiting defense regulators. R genes must be expressed at optimum levels to function properly. If expression is too low, downstream defense responses are not activated efficiently. Conversely, overexpression of R genes can trigger autoactivation of defenses with deleterious consequences for the plant. Little is known about R gene regulation, particularly under defense-inducing conditions. We examined regulation of the Arabidopsis thaliana gene RPP8 (resistance to Hyaloperonospora arabidopsidis, isolate Emco5). RPP8 was induced in response to challenge with H. arabidopsidis or application of salicylic acid, as shown with RPP8-Luciferase transgenic plants and quantitative reverse-transcription polymerase chain reaction of endogenous alleles. The RPP1 and RPP4 genes were also induced by H. arabidopsidis and salicylic acid, suggesting that some RPP genes are subject to feedback amplification. The RPP8 promoter contains three W box cis elements. Site-directed mutagenesis of all three W boxes greatly diminished RPP8 basal expression, inducibility, and resistance in transgenic plants. Motif searches indicated that the W box is the only known cis element that is statistically overrepresented in Arabidopsis nucleotide-binding leucine-rich repeat promoters. These results indicate that WRKY transcription factors can regulate expression of surveillance genes at the top of the defense-signaling cascade.

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Small, multigene families organized in a tandem array can facilitate the rapid evolution of the gene cluster by a process of meiotic unequal crossing-over. To study this process in a multicellular organism, we created a synthetic RBCSB gene cluster in Arabidopsis thaliana and used this to measure directly the frequency of meiotic, intergenic unequal crossing-over between sister chromatids. The synthetic RBCSB gene cluster was composed of a silent ⌬RBCS1B::LUC chimeric gene fusion, lacking all 5 transcription and translation signals, followed by RBCS2B and RBC3B genomic DNA. Expression of luciferase activity (luc ؉ ) required a homologous recombination event between the ⌬RBCS1B::LUC and the RBCS3B genes, yielding a novel recombinant RBCS3B͞ 1B::LUC chimeric gene whose expression was driven by RBCS3B 5 transcription and translation signals. Using sensitive, single-photon-imaging equipment, three luc ؉ seedlings were identified in more than 1 million F2 seedlings derived from self-fertilized F1 plants hemizygous for the synthetic RBCSB gene cluster. The F2 luc ؉ seedlings were isolated, and molecular and genetic analysis indicated that the luc ؉ trait was caused by the formation of a recombinant chimeric RBCS3B͞1B::LUC gene. A predicted duplication of the RBCS2B gene also was present. The recombination resolution break points mapped adjacent to a region of intron I at which a disjunction in sequence similarity between RBCS1B and RBCS3B occurs; this provided evidence supporting models of gene cluster evolution by exon-shuff ling processes. In contrast to most measures of meiotic unequal crossing-over that require the deletion of a gene in a gene cluster, these results directly measured the frequency of meiotic unequal crossingover (Ϸ3 ؋ 10 ؊6 ), leading to the expansion of the gene cluster and the formation of a novel recombinant gene.Genome organization can directly affect the evolution of a gene. Single-copy genes or dispersed members of a multigene family evolve independently. In contrast, members of a multigene family organized as a gene cluster can exploit this organization to generate further gene duplications and novel recombinant genes by a process of unequal crossing-over. For example, a single intergenic unequal crossover event in a gene cluster results in four simultaneous alterations: a deletion, a duplication, and two reciprocal, recombinant genes. The impact of such unequal crossover events evidently have been important in the evolution of complex loci such as HOX (1), amylase (2), globin (3), MHC (4), Ig (5), the maize R-r complex (6), RBCS (7), and plant disease-resistance loci (8-10). Although DNA sequencing of gene clusters provides information about past changes in a particular gene cluster, it can only estimate the rate of unequal crossing-over in terms of geological time scales.In multicellular organisms, unequal crossing-over is implicated in several genetic disorders. This was demonstrated first with the Drosophila bar locus (11). Subsequent research with the bobbed locus demonstrated ...