Developmental regulation of 1‐aminocyclopropane‐1‐carboxylate synthase gene expression during leaf ontogeny in white clover

Abstract

Reverse‐transcriptase‐dependent polymerase chain reaction (RT‐PCR) was used to identify three distinct DNA sequences (designated TR‐ACS1, TR‐ACS2 and TR‐ACS3), each with identity to 1‐aminocyclopropane‐1‐carboxylate synthase (ACS). Southern analysis confirmed that these sequences represented three distinct genes, and phylogenetic analysis showed that each gene was more closely related to homologues from other plant species than to each other. The expression of these three ACS genes during leaf ontogeny in white clover was examined. Northern analysis revealed that TR‐ACS1 was expressed in the apical structure of the stolon in mature‐green leaf tissue and in leaf tissue at the onset of senescence; RT‐PCR analysis showed that TR‐ACS2 was expressed in the apical structure of the stolon and in newly initiated leaf tissue. Furthermore, the expression of TR‐ACS1 was regulated by the exogenous application of auxin. The third gene, TR‐ACS3, was expressed in senescent leaf tissue, but translation of the nucleotide sequence provided an enzyme with a deleted binding site for pyridoxal‐phosphate and S‐adenosyl methionine (AdoMet), suggesting that this was a pseudogene. Antibodies, raised to the protein product of TR‐ACS1 expressed in Escherichia coli, recognized a protein of 55 kDa in leaf extracts of white clover using Western analysis, with the pattern of protein accumulation corresponding to the pattern of gene expression determined using Northern analysis. The significance of these results is discussed with respect to the developmental expression of ACS genes during leaf ontogeny.