Gene silencing studies in the gymnosperm species Pinus radiata

A biolistic transformation procedure was used to transform embryogenic Pinus radiata tissue with constructs containing the Zea mays UBI1 (ubiquitin)-promoter followed by the P. radiata CAD (cinnamyl alcohol dehydrogenase) cDNA in sense or anti-sense orientation or in the form of an inverted-repeat. The effect of the different constructs on silencing the endogenous CAD gene was monitored in embryogenic tissue and somatic seedlings of 28 P. radiata transclones. Quantitative CAD measurements demonstrated that the construct containing an inverted-repeat of the CAD cDNA was most efficient in triggering gene silencing in P. radiata. Northern hybridization experiments with silenced transclones revealed that reduced CAD activities were the result of reduced steady state levels of the targeted CAD mRNA. Monitoring of the activity of the UBI1-promoter in the P. radiata transclones and heat-shock experiments with transgenic somatic P. radiata seedlings indicated that gene silencing is positively correlated with the expression level of the transgene. The obtained data are also consistent with a role for the expression level of the endogenous CAD gene in gene silencing.     

Ethylene in induced conifer defense: cDNA cloning, protein expression, and cellular and subcellular localization of 1-aminocyclopropane-1-carboxylate oxidase in resin duct and phenolic parenchyma cells

Members of the Pinaceae family have complex chemical defense strategies. Conifer defenses associated with specialized cell types of the bark involve constitutive and inducible accumulation of phenolic compounds in polyphenolic phloem parenchyma cells and oleoresin terpenoids in resin ducts. These defenses can protect trees against insect herbivory and fungal colonization. The phytohormone ethylene has been shown to induce the same anatomical and cellular defense responses that occur following insect feeding, mechanical wounding, or fungal inoculation in Douglas fir (Pseudotsuga menziesii) stems (Hudgins and Franceschi in Plant Physiol 135:2134–2149, 2004). However, very little is known about the genes involved in ethylene formation in conifer defense or about the temporal and spatial patterns of their protein expression. The enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO) catalyzes the final step in ethylene biosynthesis. We cloned full-length and near full-length ACO cDNAs from three conifer species, Sitka spruce (Picea sitchensis), white spruce (P. glauca), and Douglas fir, each with high similarity to Arabidopsis thaliana ACO proteins. Using an Arabidopsis anti-ACO antibody we determined that ACO is constitutively expressed in Douglas fir stem tissues and is up-regulated by mechanical wounding, consistent with the wound-induced increase of ethylene levels. Immunolocalization showed cytosolic ACO is predominantly present in specialized cell types of the wound-induced bark, specifically in epithelial cells of terpenoid-producing cortical resin ducts, in polyphenolic phloem parenchyma cells, and in ray parenchyma cells.J.W. Hudgins and Steven G. Ralph contributed equally to this work.     

Kunitz-type trypsin inhibitor gene family in Arabidopsis and Populus trichocarpa and its expression response to wounding and herbivore in Populus nigra

Trypsin inhibitors (TIs) play important roles in plant defense against biotic stresses. In this work, we first characterized the KTI gene families in the herbaceous model system, Arabidopsis thaliana, and the woody model system, Populus trichocarpa. Genomic analysis of AtKTIs and PtKTIs including phylogenetic relationship, gene structure, and motif preservation was presented. The temporal and spatial expression pattern of AtKTI genes under biotic and abiotic stresses has been performed by mining the publicly available microarray data. Unlike Populus, the absence of AtKTI induction under herbivore attack suggested that AtKTIs may not be closely related to herbivore defense in this plant species. In order to assess the potential of PtKTI as target genes for genetic improvement of the biotic resistance in plant species of high economic impact, we isolated KTI complementary DNAs from Populus nigra on a genome-wide scale and analyzed their respective response to Apocheima cinerarius Erschoff and mechanical wounding. A basically similar subset of PnKTIs was shown to be rapidly induced by both treatments in this study, though quantitatively distinct. This study revealed a different collection of wound- and herbivore-induced P. nigra KTI genes from those reported previously for hybrid poplar infested with Malacosoma disstri. Our data demonstrated that Populus could deploy KTI genes actively and selectively in an insect-specific manner. While KTI genes constitute good candidates for genetic engineering to improve biotic resistance in transgenic plant, their selection should be herbivore-oriented to obtain sufficient effects.     

The pea plastocyanin promoter directs cell-specific but not full light-regulated expression in transgenic tobacco plants

A series of 5′ deletions of the pea plastocyanin gene (petE) promoter fused to the β-glucuronidase (GUS) reporter gene has been examined for expression in transgenic tobacco plants. Strong positive and negative cis-elements which modulate quantitative expression of the transgene in the light and the dark have been detected within the petE promoter. Disruption of a negative regulatory element at ?784 bp produced the strongest photosynthesis-gene promoter so far described. Histochemical analysis demonstrated that all petE-GUS constructs directed expression in chloroplast-containing cells, and that a region from ?176 bp to +4 bp from the translation start site was sufficient for such cell-specific expression. The petE-promoter fusions were expressed at high levels in etiolated transgenic tobacco seedlings but there was no marked induction of GUS activity in the light. The endogenous tobacco plastocyanin genes and the complete pea plastocyanin gene in transgenic tobacco plants were also expressed in the dark, but showed a three- to sevenfold increase in the light. This indicates a requirement for sequences 3′ to the promoter for the full light response of the petE gene.     

Characterization of a F-box gene up-regulated by phytohormones and upon biotic and abiotic stresses in grapevine

F-box proteins are key components of the ubiquitin (Ub)/26S proteasome pathway that mediates selective degradation of regulatory proteins involved in a wide variety of cellular processes affecting eukaryotic cells. In plants, F-box genes form one of the largest multigene superfamilies and control many important biological functions. Among the F-box genes characterized to date only few have been involved in the regulation of plant defense responses. Moreover, no F-box genes have been studied and characterized in grapevine. Using a differential display approach we isolated a F-box gene (BIG-24.1), which is up-regulated during Botrytis cinerea infection of grapevine leaves. BIG-24.1 encodes a polypeptide of 386 amino acids with a conserved F-box domain in the N-terminus region and a kelch domain. By investigating expression profiles of BIG-24.1, we show that the gene expression is strongly stimulated in B. cinerea infected berries and in grapevine cells challenged by MAMP rhamnolipids, a non-host bacterium and an endophytic rhizobacterium. The gene is also strongly induced by abiotic stresses including UV-C and wounding or by salicylic acid, methyl-jasmonate, ethylene and abscisic acid that are known to be involved in defense signalling pathways. In addition, sequence analysis of the BIG-24.1 promoter revealed the presence of several regulatory elements involved in the activation of plant defense responses.     

Rice gene expression in response to N-acetylchitooligosaccharide elicitor: comprehensive analysis by DNA microarray with randomly selected ESTs

N-acetylchitooligosaccharides are potent elicitors to suspension-cultured rice cells, inducing a set of defense reactions. Expression of defense-related genes is considered to play an important role in defense reactions, and we employed microarray analysis of 8987 randomly selected expressed sequence tags to analyze the changes in gene expression caused by N-acetylchitooctaose. In this experiment, 166 genes were significantly induced and 93 genes were repressed. RNA gel blot analysis of 16 of these genes confirmed the microarray results. Of the 259 ESTs identified as responsive to N-acetylchytooctaose, 18 genes are related to signal transduction, including five calcium-dependent protein kinases (CDPKs). Among these, three novel CDPKs responsive to N-acetylchitooctaose were isolated.     

Expression of Lignin Biosynthetic Genes in Wheat during Development and upon Infection by Fungal Pathogens

As a major component of the cell wall, lignin plays an important role in plant development and defense response to pathogens, but negatively impacts biomass processability for biofuels. Silencing the target lignin genes for greater biomass processability should not significantly affect plant development and biomass yield but also must not compromise disease resistance. Here, we report experiments to identify a set of lignin genes that may be silenced without compromising disease resistance. We profiled the expression of 32 lignin biosynthetic candidate genes by qRT-PCR in 17 wheat tissues collected at three developmental stages. Twenty-one genes were expressed at a much higher level in stems compared to sheaths and leaf blades. Expression of seven these genes significantly correlated with lignin content. The co-expression patterns indicated that these 21 genes are under strong developmental regulation and may play a role in lignin biosynthesis. Profiling gene expression of same tissues challenged by two fungal pathogens, Fusarium graminearum and Puccina triticina indicated that expression of 17 genes was induced by F. graminearum. Only PAL1, a non-developmental-regulated gene, was induced by P. triticina. Thus, lignin biosynthetic pathway overlaps defense response to F. graminearum. Based on these criteria, 17 genes, F5H1, F5H2, 4CL2, CCR2, COMT1, and COMT2 in particular that do not overlap with disease resistance pathway, may be the targets for downregulation.     

Molecular mechanism of null expression of aldehyde dehydrogenase-1 in rat liver

In isozyme systems in general, the pattern of tissue-dependent expression of a given type of isozyme is uniform in various mammalian species. In contrast, a major cytosolic aldehyde dehydrogenase isozyme, termed ALDH1, which is strongly expressed in the livers of humans and other mammals, is hardly detectable in rat liver. Thirteen nucleotides existing in the 5′-promoter region of human, marmoset, and mouseALDH1 genes are absent in the four rat strains examined. When the 13 nucleotides were deleted from a chloramphenicol acetyltransferase expression construct, which contained the 5′-promoter region of the humanALDH1 gene and a low-background promoterless chloramphenicol acetyltransferase expression vector, the expression activity was severely diminished in human hepatic cells. Thus, deletion of the 13 nucleotides in the promoter region of the gene can account for the lack of ALDH1 expression in rat liver.