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Item Potent inhibition of human monoamine oxidase A and B by phenolic compounds and polyunsaturated fatty acids in tobacco smoke(Elsevier B V, 2025-05-25) Hong SW; Heydari A; Watson PR; Teesdale-Spittle PH; Page R; Northcote PT; Keyzers RA; Vyssotski M; Truman PSmoking is a main cause of premature death and preventable disease in the world. Interestingly, animal studies indicate that inhibition of monoamine oxidase (MAO), key enzymes for the degradation of neurotransmitters, increased self-administration of nicotine. The purpose of this study was to identify and characterize the potential MAO inhibitors in tobacco smoke responsible for MAO inhibition in smokers. A bioassay-guided isolation from an extract of tobacco smoke showed that catechol, 4-methylcatechol, hydroquinone, α-linolenic acid, and linoleic acid all displayed potent human MAO inhibitory activity. Additionally, the tobacco catechols 4-ethylcatechol and 4-vinylcatechol were included to test their inhibitory potencies. Catechol, 4-methylcatechol, 4-ethylcatechol, and hydroquinone are potent and irreversible MAO inhibitors. Among the phenolic compounds tested, 4-methylcatechol and 4-ethylcatechol inhibited MAO A with IC50 values of 10.0 and 12.6 μM, respectively, reducing to 0.27 and 0.43 μM after 1 h preincubation. In addition, α-linolenic acid and linoleic acid competitively inhibited MAO A with Ki values of 10.50 and 6.95 μM, respectively. These results suggest that MAO inhibition by phenolics and polyunsaturated fatty acids in tobacco smoke may be important contributors to the MAO inhibition experienced by smokers and to the enhancement of nicotine dependence this MAO inhibition is believed to cause.Item Exploring the substitution of cannabis for alcohol and other drugs among a large convenience sample of people who use cannabis.(BioMed Central Ltd, 2024-11-05) Wilkins C; Romeo J; Rychert M; Graydon-Guy TBackground The substitution of cannabis for alcohol and other drugs has been conceptualised in a harm reduction framework as where cannabis is used to reduce the negative side-effects, addiction potential, and social stigma of other drugs. There is currently mixed evidence with recent reviews suggesting cannabis co-use patterns may vary by age and ethnicity. Yet few studies have had large enough samples to examine this demographic variation in detail. Aims To explore the co-use of cannabis with alcohol and other drugs within demographic subgroups of a large sample of people who use cannabis. Specifically: (1) whether cannabis is being substituted for other drugs, and (2), whether cannabis use leads to more, less or the same level of other drug use. Method Online convenience survey promoted via Facebook™ completed by 23,500 New Zealand respondents. Those who had used cannabis and any of eight other substances in the same six-month period were asked if their use of cannabis had any impact on their use of each other substance (“a lot more”, “little more”, “no impact/same”, “little less”, “a lot less”). Frequency and quantity used of each other drug was compared by co-use group. Generalised logistic regression models were developed to predict co-use categories. Results Significant proportions reported cannabis use led to “less” alcohol (60%), synthetic cannabinoid (60%), morphine (44%) and methamphetamine (40%) use. Those who reported using “less” had lower frequency and amount used of other drugs. Approximately seven-out-ten reported cannabis use had “no impact” on LSD, MDMA, and cocaine use. One-in-five reported using cannabis led to “more” tobacco use. Young adults (21–35-years) were more likely to report cannabis use led to “less” drinking and methamphetamine use. Adolescent co-users (16–20 years) reported mixed impacts. Māori were more likely to report cannabis use resulted in “less” alcohol, tobacco, methamphetamine, and LSD use. Students and those living in cities were less likely to report cannabis use lowering use of other substances. Conclusion Cannabis and other drug co-use patterns are moderated by life stages, lifestyles, cultural perspectives, and urbanicity. Harm reduction initiatives and policy reforms should take account of these moderating factors.Item Quantitative inheritance of leaf shape characters in tobacco (Nicotiana tabacum L.) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in Plant Science at Massey University, Palmerston North, New Zealand(Massey University, 1982) Yaacob, Musa binAn F₁ half diallel cross experiment with 8 parents (i.e. ½ p (p = 1) combinations) was used to study the quantitative inheritance of leaf shape characters in tobacco (Nicotiana tabacum L.). The effect of stalk positions on the inheritance of these characters was also included. The study was carried out under a glasshouse conditions. The parental lines used in the crosses represent a random sample of leaf shape characters available in New Zealand germplasm collection. Except for wing area (2nd leaf), phenotypic analysis showed that there was a high genetic variability for other characters. The genetic analysis of the diallel indicated that inter-locus interaction (epistasis) was of little importance for most of the characters studied. Additive genetic variance was the main component of the total genetic variance. Heritability estimates ranged from moderate (approximately 40 %) to moderately high (approximately 70 %) for most characters. Near similar values were obtained from both the narrow and broadsense heritability estimates. Very little hybrid vigour was observed for both leaf area and leaf dry weight. Both the phenotypic and genotypic correlation coefficients between selected pairs of characters were in good agreement with each other in terms of direction and levels of significance. The estimates were generally high and highly significant. The components of genetic variance (i.e. additive and dominance genetic variance), heritability and correlation coefficient estimates were generally larger in the middle as compared to the top or bottom leaves.Item Diallel analysis of varying late season night temperatures on the development of a range of fluecured tobacco (Nicotiana tabacum l.) genotypes : a thesis presented in partial fulfillment of the requirements for the degree of Masterate of Agricultural Science in Plant Science at Massey University(Massey University, 1977) Beatson, Ronald AlanA study was conducted in the climate room facilities, at D.S.I.R. Plant Physiology Division, Palmerston North, on the effect of varying late season night temperatures on the development of a range of flue-cured tobacco genotypes. The study involved imposing three night temperatures, 10°C, 15°C and 20°C, when the plants came into flower. Ten F1 genotypes of a five parent diallel cross (no parents, no reciprocals) were grown at each night temperature with three replications per temperature. Fourteen morphological, physical and chemical characters were measured. The effect of late season night temperature was negligible but there was some evidence of genotype environment interaction for some of the characters. The experiment was conducted using single plants as plots and the statistical analysis showed acceptable coefficients of variation for biological studies. The genetic analysis of the diallel showed that general combining ability variance is the most important type of genetic variance in the characters examined. This agrees with the majority of other tobacco diallel studies. As general combining ability variance is largely a measure of additive genetic variance, breeding homozygous lines from a heterozygous base population should be the best approach to follow. Heritability values were of sufficient size for several of the commercially important characters to indicate that improvement through selection was possible. General combining ability and phenotypic simple correlations between pairs of characters were generally in good agreement, demonstrating that phenotypic selection will result in altering the genotypes in the desired direction for the characters in question. The experiment showed a large negative correlation between the two economically important characters, yield and total nicotine alkaloids. This result is in agreement with similar studies carried out by other workers in this field. The experiment revealed a number of improvements which could be useful in the conduct of future tobacco climate room studies.Item Evaluation of the efficacy of the inducible over-expression of 9-cis-epoxycarotenoid dioxygenase1 (NCED1) to confer improved water use efficiency in transgenic plants : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Palmerston North, New Zealand(Massey University, 2013) Sixtus, Caleb D.In order to trial the concept of inducible overproduction of the plant hormone abscisic acid (ABA) to confer increased water use efficiency, the forage legume Trifolium repens (L.) (white clover) and the model plant species Nicotiana tabaccum (L.) (tobacco) were transformed with the construct 9-cis-epoxycarotenoid dioxgenase 1 (NCED1) gene from Solanum lycopersicon (SlNCED1) driven by the RUBISCO small subunit promoter (SSUp). For white clover, a total of 18 putatively genetically-independent transgenic lines were obtained through selection in tissue culture, and these were further cloned by vegetative propagation to give 56 plants in total. Ten of these tested positive for NCED1 insertion using polymerase chain reaction (PCR) with genomic DNA. Establishment of transgenic clover on soil was problematic, but seven putatively transformed lines were established. Of these, only one line potentially expressed SlNCED1, but the transcriptional levels were too low, as determined by semi-quantitative reverse-transcriptase dependent PCR (sqRT-PCR), for any further analysis. This low expression, and the fact that only one line was identified, led to the decision to discontinue investigations with white clover. In parallel, the more amenable tobacco transformation system was also used with the SlSSUp::NCED1 construct to act as proof-of-concept. Fourteen putatively genetically independent transgenic lines of tobacco were obtained through tissue culture, six of which were successfully established onto soil. A range of integrated gene copy numbers and NCED1 expression levels were identified in the To lines using genomic PCR and sqRT-PCR. Self-fertilised seed was collected from each transgenic line, but the germination rate from all of the transgenic lines was significantly lower than wild-type. Those lines that did germinate often displayed a range of aberrant growth phenotypes. After trialing methods to evaluate water use efficiency, a total of 47 T1 seedlings displaying a normal seedling phenotype were established on soil. A range of water use efficiencies were observed as determined by analysis of plant growth rates against water use, followed by a transpiration assay of plants deemed „efficient‟ and „poor‟ users of available water. No correlation of SlNCED1 expression with the more efficient users of available water was observed, and so ultimately it was concluded that the transgene was ineffective at raising the water use efficiency of tobacco as determined by the parameters measured in thesis.Item Digestion and metabolism of sulphur containing amino acids in sheep fed fresh forage diets : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Palmerston North, New Zealand(Massey University, 1990) McNabb, Warren CharlesSulphur amino acids (SAA) are important in the sheep industry because they appear in many instances, to limit wool protein synthesis. Experiments were carried out to study two methods with the potential to increase the absorption of SAA from the small intestine of sheep fed fresh forage diets. The first experiment evaluated the effects of condensed tannins (CT) in Lotus pedunculatus upon the following; (1) Rumen-S metabolism, and (2) the digestion of methionine and cystine. (3) The metabolism of plasma SAA and inorganic sulphate, and (4) The solubility and (5) degradation of protein in the rumen. The second experiment identified proteins that contained a high proportion of SAA, and assessed their potential for expression in the leaves of forage legumes using genetic engineering techniques. Two aspects were measured; (6) Rates of rumen degradation of ovalbumin and sunflower albumin 8 (SF8) proteins in vitro, and (7) The expression of SF8 protein from sunflower seeds in the leaves of transgenic tobacco. The nutritional consequences of CT in Lotus pedunculatus were assessed by infusing polyethylene glycol (PEG), into the rumen of one group of sheep (PEG sheep; CT not operating), whilst a separate group of sheep received an infusion of water (CONTROL sheep; CT operating). PEG selectively binds CT, preventing CT from binding plant proteins in the rumen, so that CT effects on digestion could be evaluated. Polyethylene glycol was also added to in vitro incubations as required. The principal results were; (1) Condensed tannins had a major effect on rumen sulphur (S) metabolism. The irreversible loss rate (IRL) of reducible-S (total non-protein S in rumen fluid, measured as sulphate after performic acid oxidation of rumen fluid) from the rumen was lower (P<0.001) in control (0.84gS/d) than PEG (2.49gS/d) sheep. This was due in part to a higher (P<0.001) flux of methionine (2.75 and 2.09g/d) and cystine (3.33 and 2.52g/d) through the abomasum in control than PEG sheep. There was no net loss of dietary methionine or cystine from the rumen in control sheep, whilst 29% of methionine (P<0.001) and 28% of cystine (P<0.001) intake disappeared from the rumen in PEG sheep. The proportion of microbial-non-ammonia-nitrogen (NAN) in whole rumen digesta-NAN was lower (P<0.001) in control (0.44) than PEG (0.71) sheep, although it was calculated that the rumen microbial-NAN pool size was similar in control (2.9g) and PEG (3.1g) sheep. These observations suggest CT reduced the degradation of forage protein and SAA in the rumen. (2) The apparent absorption of methionine from the small intestine was higher (27%; P<0.001) in control (2.11g/d) than PEG (1.66g/d) sheep, but the apparent absorption of cystine from the small intestine was similar (4%; P>0.05) for control (1.40g/d) and PEG (l.34g/d) sheep. The apparent digestibility of methionine in the small intestine was similar (0.78) for both groups, whilst for cystine, it was lower (P<0.01) in control (0.42) than PEG (0.53) sheep. The increased absorption of methionine from the small intestine with CT was due to an increased flux from the rumen, whereas the digestibility of cystine in the small intestine may have been lower as a consequence of binding to CT complexes. (3) Condensed tannin had a major effect on plasma SAA fluxes, especially cystine. Plasma cystine concentration and IRL were higher (P<0.001) in control (41.7μmol/l and 39.8:μmol/min) than PEG (27.5μmol/l and 22.4μ.mol/min) sheep. Condensed tannins resulted in a 79% increase in the transulphuration of methionine to cystine (11.7 and 6.5μ:mol/min; P<0.05) and a decrease (P<0.05) in the oxidation of cystine (3.33 and 5.2μ.mol/min) and methionine (0.2μ.mol/min and 1.2μmol/min) to sulphate in control compared to PEG sheep. The net effect of CT was to increase (P<0.05) the flux of plasma cystine to productive processes and maintenance by 110% in control (36.5μmol/min) compared to PEG (17.4μmol/min) sheep. This represented 91% of total plasma cystine flux in control sheep, compared to only 74% of total cystine flux in PEG sheep (P<0.05). Since wool growth is generally accepted as the major productive process utilising plasma cystine, these results indicate that a major effect of CT in the diet would probably be to increase wool growth. The IRL of plasma methionine was similar in control (20.5μmol/min) and PEG (19.9μmol/min) sheep, whilst the IRL of plasma sulphate was lower (P<0.01) in control (35.9μmol/min) than PEG (50.lμmol/min) sheep. (4) The rate of protein solubilization in the rumen was studied by measuring the loss of N, corrected for microbial-NAN contamination (true), from fresh minced (FM) and freeze-dried and ground (FD) Lotus pedunculatus, incubated in polyester-fibre bags suspended in the rumen of control and PEG sheep fed Lotus pedunculatus. Freshly minced Lotus was chosen as one treatment because it more closely represented the effects of chewing on cell rupture and CT release than was likely with freeze drying. Mincing resulted in a much greater initial N loss (47%) than freeze drying (14%). The true loss of N from FD Lotus was higher in PEG than control sheep at 2, 4, 6.5, 11 and 24 hours of incubation, whilst with FM Lotus, N losses were similar. Microbial-NAN adhering to FD residues was higher in PEG than control sheep at 2, 4, 6.5 and 11 hours, but was similar at 24 hours of incubation. However, microbial-NAN adhering to FM Lotus was higher in PEG compared to control sheep, only at 6.5 and 24 hours. These observations suggest that CT reduced protein solubility and microbial colonization of FD Lotus to a much greater extent than FM Lotus. (5) The rate of protein degradation in the rumen was studied in vitro by incubating FM and FD Lotus pedunculatus in rumen fluid, with and without PEG, and using sodium-dodecyl-sulphate gel electrophoresis (SDS-PAGE). After 4 hours of incubation protein from FD and FM Lotus was clearly degraded when PEG was present, whilst after 8 hours of incubation it was essentially undetectable in both incubations. In contrast, after 8 hours, leaf protein from FM and FD Lotus was still readily detectable in incubations without PEG. Therefore, CT substantially reduced the rate at which soluble protein was degraded by rumen microorganisms but had little effect on the rate at which it was solubilized, particularly when minced. (6) The rate of degradation of SF8 protein was compared to the degradation of the LSU and small subunit (SSU) of Fraction 1 leaf, vicilin and ovalbumin proteins using in vitro incubations and SDS-PAGE. The SF8 protein had a rate of proteolysis of 0.23h-1 and a half-life of 3.0 hours, but the principal degradation product of SF8, which had a half-life of 69 hours, was extremely resistant to rumen degradation. Proteolysis of the LSU of Fraction 1 leaf protein was resolved into two components. The first product had a degradation rate of 0.06h-1 and a half-life of 11.6 hours, whilst the second component of proteolysis, which occurred from 12 hours onwards, had a degradation rate of 0.45h-1 and a half-life of 4.6 hours. The proteolysis of the SSU of Fraction 1 leaf protein had a degradation rate of 0.04h-1 and a half-life of 17.3 hours. Ovalbumin was not degraded during the initial 16 hours of incubation, but was then degraded at a rate of 0.08h-1, with a half-life of 8.7 hours. Vicilin had a rate of proteolysis of 4.3h-1 and a half-life of about 10 min. Both SF8 protein and ovalbumin were found to be more resistant to rumen proteolysis than the LSU of Fraction 1 leaf protein, but different mechanisms were involved in conveying resistance. Therefore it is worthwhile to introduce expression of genes coding for these proteins into the leaves of important agricultural legumes, using genetic engineering techniques, with a view to increasing the availability of SAA for sheep. (7) The gene for SF8 is normally expressed only in seeds. Therefore a SF8 cDNA clone was genetically engineered for expression in the leaves of tobacco plants and inserted into a gene delivery system in Agrobacterium tumefaciens and transferred to tobacco. Transcription of the SF8 synthetic (chimeric) gene occurred in the leaves of transformed tobacco, with the level of SF8 mRNA varing over a 100 fold range, but in the highest expressor, it represented 14% of the SF8 mRNA level found in sunflower seeds. However, SF8 protein was not detected in the leaves of transformed tobacco. Consequently, the level of SF8 in the leaves of transgenic tobacco must have been less than 0.03% of total leaf protein. The highest SF8 mRNA expressor contained nine times more SF8 mRNA than an ovalbumin-transformed tobacco contained ovalbumin mRNA. As the ovalbumin transformed tobacco produced 0.01% of its leaf protein as ovalbumin, there is at least sufficient SF8 mRNA to support up to nine times that level of protein expression. As this level of SF8 protein expression would be detectable using sensitive immunological procedures, it seems that either SF8 mRNA is not translatable in tobacco plants or SF8 protein is very unstable in tobacco leaves. Translatability of SF8 mRNA was tested in E. coli using an expression vector, pJLA602 without success. If SF8 protein was unstable in tobacco leaves, then SF8, which is a seed storage protein, should be stably accumulated in tobacco seeds. The expression of the SF8 chimeric gene was monitored in tobacco seeds, and again the results were negative. It would appear that the SF8 cDNA coding region was untranslatable so that DNA sequencing of the SF8 chimeric gene will be necessary to correct the DNA sequence by oligonucleotide-directed, site specific mutagenesis. (8) Both CT and proteins, resistant to degradation in the rumen and with a high proportion of SAA, have the potential to increase the absorption of SAA from the small intestine in sheep grazing fresh forages. However, further research is required to examine; (a) the effectiveness of lower dietary CT concentrations than were examined in the present studies, on increasing SAA absorption and metabolism, and (b) what level of foreign gene expression is required in transgenic legumes to stimulate wool growth.Item A nopaline-type overdrive element, and its influence upon Agrobacterium-mediated transformation frequency and T-DNA copy number in Nicotiana tabacum : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics at Massey University, Palmerston North, New Zealand/Aotearoa(Massey University, 1996) Griffiths, Andrew GilbertOverdrive is an enhancer element located outside and adjacent to the right border of the T-DNA in Agrobacterium tumefaciens octopine-type tumour-inducing (Ti) plasmids. This element is necessary for maximal enhancement of T-strand production and subsequent A. tumefaciens-mediated plant transformation frequency, and only the octopine-type overdrive had been characterised in any detail. A putative overdrive has been identified in the nopaline-type Ti-plasmid pTiT37 on the basis of its homology with known octopine-type overdrive sequences, particularly the eight base-pair so-called overdrive consensus core. The putative nopaline-type overdrive core, however, is only 75% homologous to that of all known overdrive core regions. Furthermore, as there are other sequences throughout the nopaline-type T-region that share 75% homology with the overdrive consensus core, the precise location of the nopaline-type overdrive is undetermined, although all nopaline-type T-region fragments exhibiting overdrive-like activity contained the putative overdrive core adjacent to the right border. The role of this particular putative core in T-DNA transfer has never been established. Deletions were made in the putative nopaline-type overdrive consensus core adjacent to the right border of a binary plant transformation vector derived from pTiT37. This was to establish whether this putative overdrive core does have a role as a transmission enhancer as proposed (Peralta et al., 1986; Van Haaren et al., 1988; Culianez-Macia and Hepburn, 1988). Two deletions were selected for the full study. The first encompassed the putative nopaline-type overdrive core flanked by 3 bp (5') upstream, and 4 bp (3') downstream, and was located in pANDY9. The other, located in pANDY10, encompassed the putative consensus core plus the entire region sharing homology with the octopine-type overdrive. This second deletion was to determine whether the core alone could account for overdrive-like activity, or whether further sequences are necessary to produce the effect. The vector with no deletions in the putative nopaline-type overdrive region was pANDY8. As determined by quantitative Nicotiana tabacum transformation assays, both deletions of the putative nopaline-type overdrive core (pANDY9, pANDY10) equally decreased the rate at which calli appeared, and equally decreased transformation frequency by 47% compared with that of pANDY8. That deletion of the putative core influenced plant transformation frequency provided strong evidence that it was indeed an overdrive-like core. Furthermore, in a virC2 mutant environment, the plant transformation frequency was reduced markedly for all three plasmids (approximately 90% reduction compared to when in the wild-type vir environment). However, there was no difference in the plant transformation frequencies of the pANDY8-10 series in a virC2 environment. This indicated that the mechanism by which the deletions influenced plant transformation frequency did not act independently of the virC operon, which is further evidence of overdrive-like activity. The type of vir regulon influenced the effect of the deletions in the putative overdrive. The transformation frequency of the plasmid with the intact putative overdrive region (pANDY8) was very similar in both an octopine-type vir environment (21.7 organogenic calli per 10 leaf discs in LBA4404) and a nopaline-type vir environment (18.7 organogenic calli per 10 leaf discs). However, in an octopine-type vir environment, deletions in the putative core resulted in a 47% decrease in transformation frequency, whereas in a nopaline-type vir environment the deletions had no effect upon transformation frequency. This may be due to a higher level of vir gene products (a feature associated with nopaline-type vir regulons), particularly VirDl and VirD2 compensating for the lack of a fully active putative overdrive. Southern analysis of plants arising from the transformation experiments (in an octopine-type vir environment) revealed that removal of the putative nopaline-type overdrive core halved the incidence of multiple T-DNA insertion events from 34.7% (pANDY8, intact nopaline-type overdrive) to 12.2% (pANDY9) and 14.3% (pANDY10). Deletion of the nopaline-type overdrive core also restricted the insert number to a maximum of two, rather than four or more. This is the first time that deletions in the regions outside the T-DNA have been shown to influence T-DNA copy number.Item 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(Massey University, 2005) Chen, Chih-MingFour 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.