Expression studies of the ACC oxidase gene family of white clover (Trifolium repens L.) : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular Plant Biotechnology at Massey University, Palmerston North, New Zealand
Four ACO promoters and four ACO genomic sequences have been isolated and cloned from Trifolium repens L. The promoter sequences were cloned using Gene WalkerTM technology, and are defined as the 5' flanking sequences upstream of the ATG translation start codon, and designated pTR-ACO1 (1006 bp), pTR-ACO2 (1510 bp), pTR-ACO3 (1350 bp), and pTR-ACO4 (1250 bp). To confirm that each 5' flanking sequences represents distinct genes, Southern analysis was undertaken with each of the 5' flanking sequences used as probes. For TR-ACO1 and TR-ACO2, Southern analysis indicated that the genome of white clover contains two copies of each gene, while single copies of TR-ACO3 and TR-ACO4 are evident. However, the pattern of recognition of pTR-ACO3 differs from pTR-ACO4 confirming TR-ACO4 as a newly identified member of the ACO gene family of white clover. The four genomic sequences isolated cover sequences downstream of the ATG codon to the stop codon, and each comprises 4 exons interspersed by 3 introns. In terms of sequence identity, for exon 1, identities over the four genes ranges from 69% to 94%, with 94% identity between exon 1 of TR-ACO3 and TR-ACO4, while for exon 2, identities range from 60% to 99%, with 99% identity between TR-ACO3 and TR-ACO4. For exon 3, sequence identities ranged from 71% to 89%, with 89% identity between TR-ACO3 and TR-ACO4, while for exon 4, identities range from 62% to 100%, with 100% sequence identity between TR-ACO3 and TR-ACO4. For the intron sequences, significantly lower identities are observed, with again, highest identities were observed for TR-ACO3 and TR-ACO4. For intron 1, identities ranged from 40% to 81% with the highest identity of 81% observed between TR-ACO3 and TR-ACO4. For intron 2, an identity range of 32% to 72% was observed with 72% identity between TR-ACO3 and TR-ACO4, while identity values of 13% to 79%, with 79% between TR-ACO3 and TR-ACO4. Analysis, in silico, of the 5' flanking sequences was undertaken to identify putative transcriptional binding domains using the PLACE and Mat-Inspector programmes. The TR-ACO1 5' flanking sequence contains a higher proportion of domains that are associated with young developing tissues, while the TR-ACO2 5' flanking sequence contains domains that are associated with environmental/hormonal cues. In contrast, the TR-ACO3 and TR-ACO4 5' flanking sequences contain a higher proportion of ethylene-response and wound associated domains. The expression pattern, in vivo, directed by all four 5' flanking sequences during leaf development has been examined using GUS fusions and transformation into both tobacco and white clover. In tobacco, the pTR-ACO1 directed expression in the terminal bud and in axillary buds of younger leaves, with expression declining in the older tissues. The pTR-ACO2 directed expression in the petioles and mature-green and senescent leaves, while the TR-ACO3 and TR-ACO4 promoters directed expression in the axillary buds, petioles and leaves of mature-green tissues and those in the early stages of senescence. In white clover, the TR-ACO1 5' flanking sequence directed highest expression in the apical tissues, axillary buds, and leaf petiolules in younger tissues and then declines in the ageing tissues, while the pTR-ACO2 directed expression in the axillary buds and leaf petiolules in mature-green tissues. The TR-ACO3 and TR-ACO4 5' flanking sequences direct more expression in the ontological older tissues, including the axillary buds and leaf petiolules. However, in association with this ontological pattern, all of the 5' flanking sequences directed expression in most cell types examined during leaf ontogeny. In younger tissues, the TR-ACO1 5' flanking sequence directed expression in the ground meristem and newly emerged leaf tissue at the apical bud of the stolon, the ground meristem tissue of axillary buds, vascular tissue, pith and cortex of the internode and node, and the cortex and vascular tissue of the leaf petiolule. In ontological older tissue, the TR-ACO3 and TR-ACO4 5' flanking sequences directed expression in the ground meristem of the axillary buds, the vascular tissue of the stolon and petiolule. However, staining could be observed in the pith and cortex of the stolon, and the cortex of the leaf petiolule, but at a reduced intensity. These expression studies suggest that in leaf development of white clover, the primary cues for the transcriptional regulation of the ACO gene family are ontological in nature.