Browsing by Author "Yang, Zhenbiao"

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    Farnesyltransferase: Gene Expression in Plants and Role in Plant Development
    Zhou, Dafeng (Virginia Tech, 1997-03-14)
    Protein farnesyltransferase (FTase, E. C. 2.5.1.21) post-translationally modifies regulatory proteins involved in controlling cell growth, division, and differentiation. Recently, a cDNA clone (PsFTb) encoding a pea (Pisum sativum) FTase b subunit was isolated. Initial studies led to the hypothesis that FTase plays a role in the regulation of plant cell division. To gain insight into FTase function in plants, a detailed study of the expression pattern of FTase genes was carried out. A cDNA (NgFTb) encoding the b subunit of tobacco FTase was cloned from a Nicotiana glutinosa cDNA library to initiate studies in tobacco. In tobacco BY-2 suspension culture, levels of NgFTb mRNA and FTase activity transiently increased at the early log phase of cell growth and rapidly declined before cells entered stationary phase. These data, along with inhibitor studies in the BY-2 system, support our hypothesis. To understand the expression and regulation of pea FTase subunit genes, 5'-upstream sequences of both pea FTase subunit genes (PsFTb and PsFTa) were cloned from a pea genomic library. The 5'-upstream sequence (~2 kb) of PsFTa was fused to GUS (b-glucuronidase) and GFP (green fluorescent protein) reporter genes and introduced into tobacco plants. This 2 kb upstream region appears insufficient to provide PsFTa promoter function. On the other hand, 3.2 kb of PsFTb 5'-upstream sequence expressed as a PsFTb:GUS construct is fully functional in transgenic tobacco plants. GUS expression was most prominent in actively growing cells supporting FTase involvement in plant cell cycle control. GUS activity was also found in mature and imbibed embryos but not premature embryos, consistent with the role of FTase in abscisic acid (ABA) signaling. An unexpected pattern of GUS activity, not correlated with dividing cells or ABA signaling, was also observed in the transgenic plants. GUS activity was detected in vascular bundles adjacent to actively-growing tissues and in regions that connect two organs, e.g., junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls. Auxin promotes PsFTb expression while light and sucrose inhibit expression. These spatial and temporal expression patterns strongly suggest that FTase has a broader role associated with regulation of nutrient transportation or allocation in plants.
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    Gene regulation in a pathogen-plant interaction: soft rot erwinias versus potato tubers
    Yang, Zhenbiao (Virginia Tech, 1990)
    Erwinia soft rot is a widespread disease destructive to numerous important crop plants. Damage to plants is primarily due to celldegrading enzymes (CDEs) secreted by the bacteria. I am interested in potato (Solanum tuberosum) soft rot because it is of agricultural importance and it represents an ideal model system for understanding molecular events in plant-pathogen interactions. Much has been learned in vitro about the molecular genetics of CDEs in the past decade; however, little is known about their expression in plantae To study expression of genes for these enzymes during pathogenesis and plant responses to erwinias or their enzymes, I developed a membrane-separated system for simultaneous studies of potato and bacterial gene expression. This system facilitates the isolation of plant tissue-free bacterial cells and bacteria-free plant tissue for subsequent analysis of gene expression by RNA blot hybridization. Using this system, I demonstrated that in compatible interactions, rnRNAs for three Erwinia carotovora subsp. carotovora (Ecc) CDE genes were induced to high levels and were induced sequentially: exo-pectate lyase (PL), endo-PL, and then endopolygalacturonase (PG) with maximal mRNA accumulations at 6, 9, and 12 hr, respectively. Induction of these mRNAs was well correlated with tissue maceration. In the incompatible interaction, however, induction of all three Ecc genes was reduced several-fold compared to the compatible interaction. The kinetics of mRNA accumulation during pathogenesis were distinct from those of in vitro accumulation induced by polygalacturonic acid. My results confirm that in planta expression of these genes was induced by exo-PL reaction products as suggested by other researchers. In studies of plant genes correlated with plant responses to pathogens and environmental stresses [plant defenseresponse (PDR) genes], I also showed Ecc triggered active responses distinct from wound responses. I used gene probes for phenylalanine ammonia- lyase (PAL) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), key genes in the biosynthesis of phenylpropanoid- and terpenoidderived compounds believed to be important in plant defenses. Ecc inoculation caused much more rapid and greater increases in PAL mRNA and enzyme activity levels in potato tuber than wounding alone. Escherichia coli, a non-plant pathogen, carrying a plasmid which encodes Ecc endo-PL, also induced PAL mRNA accumulation. Ecc induced a specific HMGR isogene (HMGR1) not activated by wounding. My results support the existence of an HMGR mul-ci-gene family. Wounding resulted in a rapid and transient accumulation of HMGR2 mRNA followed by a slower accumulation of HMGR3 mRNA. These isogenes are distinct from the Ecc-induced HMGRI gene.