Trichome glands on the surface of many higher plants produce and secrete exudates affecting insects, microbes, and herbivores. Metabolic engineering of gland exudation has potential for improving pest/disease resistance, and for facilitating molecular farming. We identified a cytochrome P450 hydroxylase gene specific to the trichome gland and used both antisense and sense co-suppression strategies to investigate its function. P450-suppressed transgenic tobacco plants showed a > or =41% decrease in the predominant exudate component, cembratriene-diol (CBT-diol), and a > or =19-fold increase in its precursor, cembratriene-ol (CBT-ol). Thus, the level of CBT-ol was raised from 0.2 to > or =4.3% of leaf dry weight. Exudate from antisense-expressing plants had higher aphidicidal activity, and transgenic plants with exudate containing high concentrations of CBT-ol showed greatly diminished aphid colonization responses. Our results demonstrate the feasibility of significantly modifying the natural-product chemical composition and aphid-interactive properties of gland exudates using metabolic engineering. The results also have implications for molecular farming.

In this study, we have investigated the global impact of heterogeneous nuclear Ribonuclear Protein (hnRNP) H/F-mediated regulation of splicing events and gene expression in oligodendrocytes. We have performed a genome-wide transcriptomic analysis at the gene and exon levels in Oli-neu cells treated with siRNA that targets hnRNPH/F compared to untreated cells using Affymetrix Exon Array. Gene expression levels and regulated exons were identified with the GenoSplice EASANA algorithm. Bioinformatics analyses were performed to determine the structural properties of G tracts that correlate with the function of hnRNPH/F as enhancers vs. repressors of exon inclusion. Different types of alternatively spliced events are regulated by hnRNPH/F. Intronic G tracts density, length and proximity to the 5′ splice site correlate with the hnRNPH/F enhancer function. Additionally, 6% of genes are differently expressed upon knock down of hnRNPH/F. Genes that regulate the transition of oligodendrocyte progenitor cells to oligodendrocytes are differentially expressed in hnRNPH/F depleted Oli-neu cells, resulting in a decrease of negative regulators and an increase of differentiation-inducing regulators. The changes were confirmed in developing oligodendrocytes in vivo. This is the first genome wide analysis of splicing events and gene expression regulated by hnRNPH/F in oligodendrocytes and the first report that hnRNPH/F regulate genes that are involved in the transition from oligodendrocyte progenitor cells to oligodendrocytes.

In this study, we sought to investigate the mechanism by which heterogeneous nuclear ribonucleoprotein (hnRNP) H and F regulate proteolipid protein (PLP)/DM20 alternative splicing. G-rich sequences in exon 3B, G1 and M2, are required for hnRNPH-and F-mediated regulation of the PLP/DM20 ratio and, when placed between competing 5 splice sites in an ␣-globin minigene, direct hnRNPH/F-regulated alternative splicing. In contrast, the activity of the intronic splicing enhancer, which is necessary for PLP splicing, is only modestly reduced by removal of hnRNPH/F both in PLP and ␣-globin gene context. In vivo, hnRNPH reversed reduction of DM20 splicing induced by hnRNPH/F removal, whereas hnRNPF had little effect. Tethering of the MS2-hnRNPH fusion protein downstream of the DM20 5 splice site increased DM20 splicing, whereas MS2-hnRNPF did not. Binding of U1 small nuclear ribonucleoprotein (U1snRNP) to DM20 is greatly impaired by mutation of G1 and M2 and depletion of hnRNPH and F. Reconstitution of hnRNPH/F-depleted extracts with either hnRNPH or F restored U1snRNP binding. We conclude that hnRNPH and F regulate DM20 splicing by recruiting U1snRNP and that hnRNPH plays a primary role in DM20 splice site selection in vivo. Decreased expression of hnRNPH/F in differentiated oligodendrocytes may regulate the PLP/DM20 ratio by reducing DM20 5 splice site recognition by U1snRNP.Alternative splicing of a single transcript is widely utilized to generate proteomic diversity in response to developmental, cell specific, and external signals (1). Typically, alternatively spliced sites are weak and the final splicing selection depends on the interplay of enhancers and silencers and the relative abundance and/or affinity of the RNA binding factors (2, 3). Early recognition of the 5Ј splice sites is mediated by the U1snRNP 2 through direct base pairing of the single-stranded 5Ј-end of U1snRNA with six conserved nucleotides at the 5Ј splice site. Binding of U1snRNP and base pairing of the U1snRNA to the template is required for spliceosome assembly (4, 5).A number of splicing factors that bind to either enhancers or silencers have been identified and shown to influence the efficiency of splice site recognition and spliceosome assembly (6). The hnRNPs are a large family of ubiquitously expressed RNA binding factors, which, in addition to regulating constitutive splicing, play an important role in alternative splicing (7,8). hnRNPH and F are highly homologous proteins that bind to G-rich sequences present in exons, in introns, and in close proximity to the polyadenylation site (8 -12). Depending on the gene context, these splicing factors have different affinity for their cognate sequences and appear to act in concert to regulate splicing of a number of genes (13, 14). Although they are most often inhibitors of alternatively spliced exons, they can also function as enhancers (14 -18). G-rich sequences are highly conserved within introns in close proximity to splice sites (7, 10, 19 -21). In genes containing short introns, these G-rich...

Trichomes are specialized epidermal cells that produce secretions that are thought to provide a first line of defence against pests and pathogens. Many trichome-secreted compounds are used commercially as flavourings, medicines, etc. Described here is the cloning and characterization of the promoter of a tobacco trichome-specific P450 gene, CYP71D16. This promoter is shown to direct the specific expression of the reporter gene, beta-glucuronidase (GUS), in glandular trichomes of Nicotiana tabacum cv. T.I. 1068 at all developmental stages. With the full promoter, GUS activity was predominantly in the gland cell, with less in the stalk cell adjacent to the gland, and in lower stalk cells. GUS staining was also observed in the most distal trichome stalk cells of non-glandular trichomes found on variety T.I. 1112. Promoter deletion analysis revealed that the region from -223 to +111 bp is sufficient to direct trichome-specific expression, but not strong gland expression. Examination of the literature suggests that this is the first characterized trichome-specific-promoter shown to function at all stages of plant development. This promoter may provide efficient bioengineering to enhance pest and pathogen resistance, and for molecular farming based on the trichome gland system.

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