Submitted to: Journal of Experimental Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2004
Publication Date: 6/18/2004
Citation: Moon, H., Callahan, A.M. 2004. Developmental regulation of peach Acc oxidase promoter-Gus fusions in transgenic tomato fruits. Journal of Experimental Botany, June 18, 2004. Interpretive Summary: Peach fruit have to be harvested while still firm and thus not fully ripe, in order to arrive in the marketplace intact. This requirement results in fruit with less than optimum flavor compared to that realized in tree-ripened fruit. As part of an on-going project to enhance peach flavor by delaying fruit softening through genetic engineering, a promoter sequence has been characterized. This is a necessary step to successfully introduce a new gene that will be capable of delaying fruit softening while allowing fruit flavor development. The new gene has to be expressed at specific times in fruit development, in specific amounts, and in specific tissues in order to have a positive effect on the trait that is being modified. To delay softening in peach fruit, so they may develop more flavor on the tree, the rate of ethylene production, a plant-produced gas that accelerates ripening/softening, will be reduced by introducing an anti-ethylene gene. Ethylene is also involved in other processes such as pollination and defense mechanisms against plant diseases and environmental stresses, that we do not want to be negatively affected. To accomplish that, the decrease in ethylene should only take place in fruit tissue during the time of ripening. To this end, the promoter sequence for a gene expressed during fruit ripening was identified and isolated. Various lengths of this promoter were used to express a marker gene, whose activity is easy to measure. Based on these studies we identified three regions of that promoter that are important. One region is able to express the marker gene in fruit tissue only. Another region is able to increase expression of that marker gene. The third region is able to express the marker gene at low levels in immature fruit, high levels in ripening fruit and again at low levels in the ripest fruit. These results will allow us to design a promoter sequence that could specifically restrict the expression of an anti-ethylene gene to ripening fruit without negatively affecting plant defense mechanisms against plant diseases and environmental stresses.
Technical Abstract: A genomic DNA sequence (PpACO1) encoding 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase from peach (Prunus persica L. Batsch cv. Loring) was isolated. It has four exons interrupted by three introns and 2.9 kb of flanking region 5' of the translational start codon. Previous work with the cDNA demonstrated that accumulation of the peach ACC oxidase message occurs during the softening of ripening fruit, and that this response correlated with increasing amounts of ethylene synthesized by the fruit. To identify regulatory elements in the peach ACC oxidase gene, chimeric fusions between 403, 610, 901, 1319, 2141 and 2919 bp of the 5' flanking region of the PpACO1 sequence and the -glucuronidase (GUS) coding sequence were constructed and used to transform tomato [Lycopersica esculentum (Mill) cv. Pixie]. Fruits were harvested from individual transgenic plants and analyzed for GUS expression by histochemical GUS staining, GUS quantitative enzyme activity determination, and measuring the relative amounts of GUS RNA. Constructs with the smallest promoter of 403 bp had significant GUS expression in fruit but not in other tissues indicating the presence of a region that affects tissue specific expression. An increase in GUS expression was observed with promoters longer than 901 bp, indicating an enhancer region between -1319 and -901. The 2141 bp and the 1319 bp promoters drove GUS expression at relatively high levels in all fruit stages. The 2919 bp of PpACO1 upstream sequence directed GUS expression from the early green stage of fruit development, and increased GUS activity as fruit matured with the maximum at the final 'red' stage, indicating a regulatory region between -2919 and -2141 that controls the temporal expression of the gene in fruit. Less GUS staining was observed in the leaves and stems than in fruit. Quantitative RNA studies also indicated an enhanced amount of RNA with promoters longer than 901 bp and found that only the full-length 2919 bp promoter conferred consistent temporal expression similar to that of the native PpACO1 mRNA.