ABSTRACT: The expression of two 1-aminocyclopropane-1-carboxylate (ACC) oxidase (E.C. 1.4.3)(ACO) genes, TR-ACO2 and TR-ACO3 from white clover has been examined in leaf tissue in response to physiological stimuli. In detached mature-green leaves,
the expression of TR-ACO3 (a leaf-senescence-associated ACC oxidase) is induced over a 6h time-course, while expression of TR-ACO2 (expressed constitutively in mature-green leaf tissue) decreased over the same time-course. An almost identical pattern of
expression was observed with detached mature-green leaves treated with ethylene, while wounding also did not influence the
timing of TR-ACO3 induction, significantly. No increase in overall ACO enzyme activity was detectable in crude extracts from wounded and detached
leaf tissue, but after separation of these extracts using hydrophobic interaction column chromatography, two ACO isoforms
were identified in non-wounded mature-green leaves (designated MGI and MGII), and the specific activity of one of these (MGII)
increased in wounded tissues. In attached leaf tissue, TR-ACO2, but not TR-ACO3 expression was induced by treatment with ethylene; an induction that was not observed with prior treatment of plants with
1-MCP. This induction in response to ethylene was also observed at the translational level using antibodies to TR-ACO2. Analysis,
in silico, of the 5′ flanking sequences to identify putative transcriptional binding domains reveals that both the TR-ACO2 and TR-ACO3 5′ flanking sequence contain domains that are associated with an ethylene-response. The significance of these observations
in terms of how the expression of these ACO genes is regulated during leaf ontogeny in vivo is discussed.
Trifolium repens L.-ACC oxidase-Ageing-Ethylene-Leaf development
Plant Growth Regulation 04/2012; 62(1):31-41. · 1.60 Impact Factor
ABSTRACT: Lines of transgenic tobacco have been generated that are transformed with either the wild-type peanut peroxidase prxPNC2 cDNA, driven by the CaMV35S promoter (designated 35S::prxPNC2-WT) or a mutated PNC2 cDNA in which the asparagine residue (Asn189) associated with the point of glycan attachment (Asn189) has been replaced with alanine (designated 35S::prxPNC2-M). PCR, using genomic DNA as template, has confirmed the integration of the 35S::prxPNC2-WT and 35S:prxPNC2-M constructs into the tobacco genome, and western analysis using anti-PNC2 antibodies has revealed that the prxPNC2-WT protein product (PNC2-WT) accumulates with a molecular mass of 34,670 Da, while the prxPNC2-M protein product (PNC2-M) accumulates with a molecular mass of 32,600 Da. Activity assays have shown that both PNC2-WT and PNC2-M proteins accumulate preferentially in the ionically-bound cell wall fraction, with a significantly higher relative accumulation of the PNC2-WT isoenzyme in the ionically-bound fraction when compared with the PNC2-M isoform. Kinetic analysis of the partially purified PNC2-WT isozyme revealed an affinity constant (apparent Km) of 11.2 mM for the reductor substrate guaiacol and 1.29 mM for H2O2, while values of 11.9 mM and 1.12 mM were determined for the PNC2-M isozyme. A higher Arrenhius activation energy (Ea) was determined for the PNC2-M isozyme (22.9 kJ mol(-1)), when compared with the PNC2-WT isozyme (17.6 kJ mol(-1)), and enzyme assays have determined that the absence of the glycan influences the thermostability of the PNC2-M isozyme. These results are discussed with respect to the proposed roles of N-linked glycans attached to plant peroxidases.
Phytochemistry 09/2005; 66(16):1869-79. · 3.35 Impact Factor
ABSTRACT: We have previously cloned a cDNA, designated SAT1, corresponding to a gene coding for a serine acetyltransferase (SAT) from onion (Allium cepa L.). The SAT1 locus was mapped to chromosome 7 of onion using a single-stranded conformation polymorphism (SSCP) in the 3' UTR of the gene. Northern analysis has demonstrated that expression of the SAT1 gene is induced in leaf tissue in response to low S-supply. Phylogenetic analysis has placed SAT1 in a strongly supported group (100% bootstrap) that comprises sequences that have been characterised biochemically, including Allium tuberosum, Spinacea oleracea, Glycine max, Citrullus vulgaris, and SAT5 (AT5g56760) of Arabidopsis thaliana. This group can be divided further with the SAT1 of A. cepa sequence grouping strongly with the A. tuberosum sequence. Translation of SAT1 from onion generates a protein of 289 amino acids with a calculated molecular mass of 30,573 Da and pI of 6.52. The conserved G277 and H282 residues that have been identified as critical for L-cysteine inhibition are observed at G272 and H277. SAT1 has been cloned into the pGEX plasmid, expressed in E. coli and SAT activity of the recombinant enzyme has been measured as acetyl-CoA hydrolysis detected at 232 nm. A Km of 0.72 mM was determined for l-serine as substrate, a Km of 92 microM was calculated with acetyl-CoA as substrate, and an inhibition curve for L-cysteine generated an IC50 value of 3.1 microM. Antibodies raised against the recombinant SAT1 protein recognised a protein of ca. 33 kDa in whole leaf onion extracts. These properties of the SAT1 enzyme from onion are compared with other SAT enzymes characterised from closely related species.
Phytochemistry 07/2005; 66(12):1407-16. · 3.35 Impact Factor