Promoters and regulation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase genes from loblolly pine (Pinus taeda L.)
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This dissertation describes investigations into the gene structure and regulation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase in loblolly pine (Pinus taeda L.). A cDNA clone, PtACO1, and its genomic sequence were isolated and characterized. The gene contained four exons separated by three introns. Quantitative PCR analyses showed that the PtACO1 transcript was expressed in a regulated fashion in various tissues of loblolly pine. Transcription of the gene responded to bending stress and IAA treatments. To study regulation of the ACC oxidase gene in more detail, the promoter region, upstream of the gene was isolated from loblolly pine genomic DNA. The promoter, designated pACO1, was recovered by PCR amplification along with a second promoter, designated pACO2, from another ACC oxidase gene. Using promoter-reporter gene constructs in transgenic Arabidopsis plants, these two promoters were shown to be differentially expressed in various Arabidopsis tissues. Both promoters were strongly expressed in rapidly dividing and expanding cells in several tissues, and particularly in roots. Subjecting the transgenic plants to a variety stresses and perturbations, it was found that the pACO1 promoter responded to fungal infection, acute bending stress and IAA treatment. The pACO2 promoter was upregulated by IAA treatment, and responded in an asymmetric fashion during gravitropic response of stems. It was concluded that the ACC oxidases from loblolly pine from which these promoters were cloned were differentially expressed and could be up-regulated in response to bending stresses and hormone treatments. The potential relationships between bending stress, gravitropism, ethylene biosynthesis and auxin with respect to compression wood formation in conifers were discussed in light of these results. In addition to experimental work with the loblolly pine ACC oxidase, an extensive computational analysis was completed to place the pine gene within a detailed phylogenetic tree generated for the 2-oxoglutarate-dependent dioxygenase (2-ODD) genes identified in the completed Arabidopsis, rice and poplar genomes.