Massey Documents by Type

Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294

Browse

Has files: YesSubject: search.filters.subject.Ethylene synthesis

Search Results

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Ethylene biosynthesis during leaf maturation and senescence in white clover : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University
    (Massey University, 1997) Butcher, Stephen Mark
    Aspects of leaf senescence in relation to ethylene biosynthesis in the plagiotropic herbaceous plant white clover (Trifolium repens L.) have been studied. Two stolons growing from clonally propagated plants were trained over a dry substratum that inhibits nodal root growth, and all axillary stolons and flowers were removed. White clover plants grown using this method produce leaves at all stages of development along a single stolon from initiation at the apex, through expansion, maturity, senescence and then necrosis. The study shows that modification of the stolon growth habit of white clover by the suppression of nodal roots provides a suitable system for the study of leaf development and senescence in relation to ethylene metabolism. The pattern of leaf development (the number of leaves at each stage of development present on the stolon) and senescence (as measured by changes in leaf chlorophyll content) along the white clover stolon is consistent between plants of the same genotype growing under the same environment, but varied greatly between the different cultivars and genotypes examined. The rate of change between the different stages of leaf development and senescence within the one genotype used in this study, AgResearch Grassland genotype 10F, differed when grown under two different environments. On mature stolons (stolons with 6 or more nodes with senesced leaves) of genotype 10F grown using the modified stolon system, the number of green leaves was maintained at a constant number as the leaf appearance rate was balanced by the senescence rate. However, the number of leaves maintained on the stolons differed between the two growing environments used in this study, from 9.85 +/- 0.23 for plants grown at Levin, to 14.57 +/- 1.99 for plants grown at Palmerston North. The total chlorophyll concentration in the leaves from plants grown at Levin increased from leaf one (the youngest opened leaf; 740 μg/g.fw) to a maximum in leaf five (mature green leaf; 2240 μg/g.fw), declined rapidly from leaf five to leaf seven (senescing leaves; 1500 μg/g.fw), and then remained constant from leaf seven to leaf ten. A similar pattern of change in chlorophyll concentration was measured in leaves from plants grown at Palmerston North, but the maximum concentration was found in leaf 4 (1750 μg/g.fw), remained relatively constant to leaf 8, before decreasing in leaf 9 (750 μg/g.fw) and declining to a minimum in leaf 15 (250 μg/g.fw). The chlorophyll a:b ratio in mature green leaves from plants grown at Palmerston North (1.46:1 to 2.63:1) was lower than the ratio in leaves from plants grown at Levin (3.72:1 to 4.98:1). Leaves of white clover produce ethylene. Ethylene evolution from attached leaves varied from 1 nL/g.fw/h (mature green leaves) to 3 nL/g.fw/h (senescing leaves). Ethylene evolution from detached leaves was initially high (12.6 nL/g.fw/h at 15 min) but declined to 3.8 nL/g.fw/h by 45 min before increasing again. Detached mature green leaves (leaves four to six) of white clover are sensitive (as measured by chlorophyll loss) to low concentrations (<1 ppm) of exogenous ethylene. The chlorophyll concentration in these leaves after four days of ethylene treatment (1, 10 or 100 ppm ethylene) was significantly lower than the chlorophyll concentration in freshly harvested leaves. However, the chlorophyll concentration in leaves two and three (early mature green) treated with ethylene was not significantly different from the concentration in freshly harvested leaves. 1-aminocyclopropane-1-carboxylic acid (ACC) concentration was low in leaves one to four (<1 nmoles/g.dw), increased to reach a maximum concentration of 11.4 nmoles/g.dw in leaf seven and declined to 2 nmoles/g.dw in leaf ten. 1-aminocyclopropane-1-carboxylic acid (MACC) concentration was highest in leaf one (11.3 nmoles/g.dw), declined to 6 nmoles/g.dw in leaf two, and remained constant for all other leaves. ACC synthase activity could not be determined in protein extracts from white clover leaf tissue. ACC oxidase activity in protein extracts varied in the different leaves examined. The activity versus substrate concentration curve for leaves one, three, five and six displayed saturation kinetics with respect to the substrate, ACC, whereas the data for leaves eight and ten did not show saturation kinetics over the range of ACC concentrations used. The ACC oxidase activity varied from 0.81 nL/mg.protein/h in extracts from leaf six, to 1.64 nL/mg.protein/h for leaf five. The apparent Km varied from 61 μM for leaf six to 138 μM for leaf five, while the Vmax varied from 0.92 for leaf six to 2.06 for leaf five. Degenerate oligonucleotide primers corresponding to conserved regions found among diverse ACC synthases were used for reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify DNA fragments from RNA extracted from white clover leaf tissue. A DNA clone, ACS7, showed 88% homology at the nucleotide level to ACC synthase from Glycine max. The ACS7 sequence contained the three conserved domains (including the reaction centre, and the three residues known to bind the pyridoxal phosphate coenzyme) identified in published ACC synthase sequences. The derived amino acid sequence for the conserved domains are identical with other published sequences. Southern analysis indicates ACC synthase is represented by a multigene family in white clover. Northern analysis of the expression of ACC synthase using ACS7 as a hybridisation probe was unsucessful. Preliminary screening of a white clover leaf cDNA library produced a clone with 72.5% homology to a putative cysteine proteinase from Pisum sativum, and 63.4% homology to a cysteine proteinase from Vicia sativa.
  • Thumbnail Image
    Item
    Molecular characterisation of ethylene biosynthesis during leaf ontogeny in white clover (Trifolium repens L.) : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Massey University
    (Massey University, 1999) Yoo, Sang Dong
    Ethylene (C2H4) biosynthesis has been investigated during leaf ontogeny in white clover (Triforium repens L. cv. Glassland challenge, genotype 10F) with a particular emphasis on the production of the hormone in the apex and newly initiated leaves (designated as leaves 1 and 2). In these developing tissues, a relatively higher rate (5 to 6-fold) of ethylene production (0.5 to 0.9 nL C2H4 gFwt-1 hr-1) was associated with a higher accumulation (1.5-fold) of 1-aminocyclopropane-1-carboxylate (ACC), when compared with mature green leaves. Genes encoding the ethylene biosynthetic enzymes, ACC synthase (ACS) and ACC oxidase (ACO) have been cloned to characterise ethylene biosynthesis at the molecular level. The partial protein-coding regions of ACS genes have been cloned using reverse transcriptase-dependent polymerase chain reaction (RT-PCR) with degenerate nested primers using cDNA templates from RNA isolated either the apex or leaf 2. These ACS genes were identified and designated as TRACS1, TRACS2 and TRACS3. TRACS1 (680 bp) is 72% and 64% homologous to TRACS2 (674 bp) and TRACS3 (704 bp), respectively, and TRACS2 and TRACS3 are 63% homologous, in terms of nucleotide sequence. TRACS1 shows highest homology with PS-ACS2, an ACS cloned from etiolated pea (Pisum sativum) seedlings which is induced by IAA and wounding. TRACS2 shows highest homology with MI-ACS1, an ACS cloned from mature mango (Mangifera indica) fruit. TRACO3 shows highest homology with VR-ACS7, an ACS cloned from etiolated hypocotyls of mung bean (Vigna radiata) and also PS-ACS1, an ACS cloned from etiolated pea seedlings which is induced by IAA, but not by wounding. The TRACS3 gene was expressed as a 1.95 kb transcript mainly in the apex and newly initiated leaves as determined by northern analysis. TRACS1 has not been detected in the tissues examined. The expression of TRACS2 has not been determined. Three ACO genes, comprising ca. 1100 bp of the protein-coding region and 3'-untranslated region (3'-UTR) have been cloned using a combination of RT-PCR and 3'-rapid amplification of cDNA ends (3'-RACE), and designated as TRACO1, TRACO2 and TRACO3. Comparison of a 813 bp protein-coding region of TRACO1, generated by RT-PCR using degenerate primers on cDNA templates from RNA isolated from the apex shows 77% and 75% homology to the protein-coding region sequences of TRACO2 (804 bp) and TRACO3 (816 bp), respectively, generated by RT-PCR using degenerate primers on cDNA templates from RNA isolated from leaf 2. The TRACO2 and TRACO3 protein-coding region sequences show 84% homology. The technique of 3'-RACE generated 3'-UTR sequences of 301 bp (TRACO1), 250 bp (TRACO2), and 92 bp (TRACO3) each amplified using mRNA extracted from the apex. The 92 bp 3'-UTR of TRACO3 has been shown to be a truncated version of a 324 bp sequence amplified from TRACO3 expressed in senescent leaf tissue of white clover (Dr. D. Hunter, IMBS, Massey University, personal communication), and it is this full-length version that was used in further experiments. The 3'-UTR sequences of the three ACO genes are more divergent, when compared with the protein-coding regions, showing 61%, 55%, and 59% homology between TRACO1 and TRACO2, TRACO1 and full-length TRACO3, and TRACO2 and full-length TRACO3, respectively. Using these 3'-UTR sequences as gene-specific probes, Southern analysis revealed that the three ACO genes are encoded by distinct genes in the white clover genome. Northern analysis, using either protein-coding regions or 3'-UTRs as probes, determined that the TRACO1 gene was expressed as a 1.35 kb transcript almost exclusively in the apex and the TRACO2 gene was expressed as a 1.35 kb transcript mainly in newly initiated leaves and mature green leaves, with maximum expression in newly initiated leaves. This pattern of gene expression coincides with the high rate of ethylene production from the apex and newly initiated leaves in white clover. The apex tissue-specific TRACO1 gene was also detected in axillary buds, and the mature leaf-associated TRACO2 gene was expressed in other mature vegetative tissues, including internodes, nodes and petioles. Both TRACO1 and TRACO2 genes are highly expressed in roots. TRACO3 gene expression was not detected in apex and mature green leaf tissues examined using the 3'-UTR gene-specific probe, but two transcripts (1.17 kb and 1.35 kb) were visualised using the protein-coding region probe and with an extended exposure time. ACO enzyme activity, in vitro, was highest in just fully expanded mature green leaves (leaf 3 and leaf 4) during leaf ontogeny in white clover. Apex, axillary bud and floral bud tissues show a relatively higher enzyme activity, when compared with mature nodes and internode tissues, but lower than that measured in petiole tissue. Highest activity of ACO, in vitro, has been detected in root extracts. Polyclonal antibodies, raised against the TRACO1 gene product expressed in E. coli, recognised a high molecular weight (ca. 205 kD) protein complex with highest accumulation in the apex. This complex was also detected in axillary bud, floral bud, and leaf 1 tissue. Immunoaffinity-based purification of the ca. 205 kD protein was carried out to obtain sufficient protein for amino acid sequencing. However, no sequence was obtained. Polyclonal antibodies, raised against the TRACO2 gene product in E. coil, recognised ACO protein (ca. 36 kD) in newly initiated leaves and mature green leaves as well as petioles and roots. This recognition pattern coincides with ACO enzyme activity, in vitro, as well as TRACO2 gene expression in the tissue. Expression of the TRACO2 and TRACO3 genes has been characterised in response to a combination of wounding, ethylene, indole-3-acetic acid (IAA), aminoethoxyvinylglycine (AVG), and 1 -methylcyclopropene (1-MCP) treatments using mature green leaves. Expression of TRACO2 gene is enhanced in response to ethylene and IAA. Ethylene-induced TRACO2 gene expression was not blocked by 1-MCP. Expression of TRACO3 was induced in response to wounding in mature green leaves. Wound-induced TRACO3 gene expression was not induced by the ethylene produced in the leaf tissue, indicating ethylene-independent ACO expression in the wounded leaves. Induction of either TRACO2 by IAA treatment or TRACO3 by wounding was delayed with AVG treatment of mature green tissue, suggesting that changes in ACS activity in the tissue is associated with induction of ACO gene expression.
  • Thumbnail Image
    Item
    Characterisation of ACC synthase during leaf development in white clover (Trifolium repens L.) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University
    (Massey University, 2001) Murray, Patricia Alison
    ACC synthase catalyses the rate limiting step in the ethylene biosynthetic pathway, and in all plants studied has been shown to be encoded by a highly divergent gene family. These different ACC synthase genes are differentially regulated in response to a variety of developmental and environmental stimuli. In this thesis, ACC synthase gene expression during leaf ontogeny in white clover (Trifolium repens L.,) has been studied. This study utilises the stoloniferous growth pattern of white clover, which provides leaf tissue at different developmental stages, ranging from initiation at the apex, through mature green to senescent, and then finally necrotic. RT-PCR, using degenerate primers to conserved regions of ACC synthase genes in the database, was used to amplify putative ACC synthase sequences from mRNA isolated from white clover leaf tissue. Sequencing and GenBank database alignment of the PCR products revealed that ACC synthase sequences comprising approximately 670 bp of the reading frame were amplified. Sequence alignments indicate that the sequences from white clover represent three distinct ACC synthases, and these were designated TR-ACS1 (Trifolium repens ACC synthase 1), TR-ACS2 and TR-ACS3. TR-ACS1 is 62% and 71% homologous to TR-ACS2 and TR-ACS3 respectively, and TR-ACS2 and TR-ACS3 are 63 % homologous, in terms of nucleotide sequence. Genomic Southern analysis, using the amplified reading frame of each gene as a probe, confirmed that the sequences are encoded for by distinct genes. In a GenBank database search, TR-ACS1 shows highest homology to an ACC synthase sequence from IAA-treated apical hooks of pea, and TR-ACS2 shows highest homology to an ACC synthase isolated from etiolated hypocotyls of mungbean. An ACC synthase isolated from white lupin, which was found to have increased expression during germination and in response to IAA and wounding, was most similar to TR-ACS3. Phylogenetic analysis determined that the three white clover ACC synthase genes are highly divergent. Phylogenetic analysis also determined that TR-ACS1 groups with ACC synthase sequences isolated from IAA-treated apical hooks of etiolated pea seedlings and IAA-treated mung bean hypocotyls. TR-ACS2 was found to group with ACC synthases isolated from etiolated hypocotyls of mung bean and TR-ACS3 was closest to a cDNA clone isolated from Citrus parapdisi. Northern analysis has shown that two of these genes are expressed differentially during leaf ontogeny. TR-ACS1 is expressed in mature green leaves and TR-ACS3 is expressed in senescent leaf tissue. The expression of TR-ACS2 was unable to be determined by northern analysis, and so the more sensitive method of RT-PCR was used. This procedure determined that TR-ACS2 is expressed predominantly in the apex, newly initiated and mature green leaves, and again at the onset of senescence. Sequence analysis of TR-ACS3 revealed that the coded protein is missing the active site of the enzyme. Using a primer specific for the conserved active site of ACC synthase, a sequence which was similar to TR-ACS3, but not completely homologous, was amplified by RT-PCR and designated TR-ACS3A. This sequence included the region encoding the active site of the enzyme, but contained an additional four nucleotides in the sequence. Thus the sequence may also encode a non-functional protein, and the possible roles of such non-functional proteins are discussed. The pattern of TR-ACS gene expression observed during leaf ontogeny suggests that these genes are under precise developmental control. Further, an indication as to the nature of the stimuli that may regulate the expression of the ACC synthase genes was provided by the phylogenetic analysis. To learn more of these physiological stimuli, white clover leaves were treated with two of the primary stimuli of ACC synthase gene expression, IAA and wounding; factors that were also shown to regulate the expression of sequences phylogenetically related to each TR-ACS gene. While the results of this experiment do not appear to be definitive, TR-ACS1 was the only gene that hybridised to timepoints in the IAA-treated tissue, and TR-ACS3 was the only gene to hybridise to the wound-induced tissues. The significance of ACC synthase gene expression during leaf ontogeny in white clover and its regulation is discussed. Antibodies were raised to the gene product of TR-ACSI expressed in E. coli. Using western analysis, the antibodies were shown to recognise the gene products of all three TR-ACS genes and a protein of 55 KD with highest intensity in mature green leaf extracts. Minor recognition of proteins of 29, 34, 37, 69 and 82 KD was also observed.