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No description available.

No description available.

The investigation in this thesis is divided into two parts. In the first part, the expression and accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO), the enzyme which catalyses the final step of ethylene biosynthesis in higher plants, is examined during exposure of white clover (Trifolium repens L.) to a water deficit. The second part of this thesis is focused on the identification and characterisation of a water-deficit-associated ACC synthase (ACS), the enzyme which catalyses the production of ACC. In the first part, two white clover varieties with differing sensitivity to water deficit, a drought-tolerant Tienshan ecotype and a drought-sensitive Grasslands Challenge cv. Kopu II cultivar were exposed to two water deficit treatments: one cycle of water deficit (designated non-prestressed; NPS) and a water deficit, a rehydration period and then a second water deficit treatment (designated pre-stressed; PS) in the New Zealand Climate Environment Laboratory (NZCEL). Treatments were terminated when the petiole elongation rate (PER) in the first fully-expanded leaf reached zero. Water relations, growth responses, the expression of the white clover ACO genes, TR-ACO1 TR-ACO2 and TR-ACO3 and the accumulation of two of the corresponding proteins, TR-ACO1 and TR-ACO2, were then examined. The soil water content (SWC) and leaf water potential (LWP) measured in both varieties and in both water deficit treatments declined progressively. The rate of decline in SWC and LWP was slower in the Tienshan ecotype with no difference between the NPS and PS treatments. However, the LWP in the Tienshan ecotype at the point at which the PER ceased was less negative (ca. -1.4 MPa) compared to Kopu (ca. -1.7 MPa). In addition, the decline in the PER differed between NPS- and PS-treated Kopu. In the NPS-treated Kopu, the PER was maintained at a high rate when plants were exposed to SWC above 18%, but declined sharply as the SWC declined further. However, in the PS-treated Kopu, the PER declined more progressively in a similar pattern to that determined for NPS- and PS-treated Tienshan. Expression of TR-ACO1 and accumulation of TR-ACO1 was observed in the apical structure of the stolon. As the water deficit progressed, no significant alteration in TR-ACO1 expression and TR-ACO1 protein accumulation was observed in the apical structures of both the NPS- and PS-treated Tienshan ecotype suggesting some degree of protection of the meristem tissues in this more drought-tolerant variety. However, a discernable decline in expression of TR-ACO1 and accumulation of TR-ACO1 protein was observed in the NPS-treated Kopu suggesting some degree of tissue injury in this more drought-susceptible variety. However, after the pre-stress (PS) treatment, no real changes in TR-ACO1 expression and TR-ACO1 protein accumulation were observed, in common with the observations for the NPS- and PS-treated Tienshan ecotype suggesting that meristem protection may now be occurring. The results suggest further that the pre-stress treatment of the more drought-susceptible Kopu may result in a degree of acclimation to the water deficit. For the first-fully expanded leaves, expression of two transcripts, TR-ACO2 and TR-ACO3 and accumulation of TR-ACO2 protein was monitored as the SWC decreased. The expression of TR-ACO2 and accumulation of TR-ACO2 decreased as the water deficit progressed in both the NPS- and PS-treated Tienshan ecotype and correlated with the decrease in PER. By contrast, in the NPS-treated Kopu, TR-ACO2 expression and TR-ACO2 protein accumulation increased, but again, after a period of pre-stress, TR-ACO2 expression and TR-ACO2 accumulation decreased, in common with the Tienshan ecotype. Again, the pre-stress treatment of the drought-susceptible Kopu may result in a degree of acclimation to the water deficit such that the responses become similar to those observed in the more drought-tolerant Tienshan ecotype. However, in both NPS- and PS-treated Tienshan and Kopu there was no significant alteration in the expression of TR-ACO3 in the first fully-expanded leaf. The expression of TR-ACO2 and TR-ACO3 and accumulation of TR-ACO2 protein were also observed in the second fully-expanded leaves (an older tissue). Again similar patterns in the expression of TR-ACO2 and TR-ACO3 and accumulation of TR-ACO2 protein were observed in both NPS- and PS-treated Tienshan and Kopu. In these leaves, expression of TR-ACO2 and accumulation of TR-ACO2 protein decreased as the water deficit progressed, but expression of TR-ACO3 increased as the water deficit decreased to less than 10%. These results suggest that responses of younger tissues (apical structure; first-fully expanded leaf) maybe the critical determinant for the tolerant (or otherwise) of white clover plants to water deficit. In the second part of this thesis, four ACS genes were identified from the Tienshan ecotype exposed to water deficit and designated TR-ACS-T. Three of these were similar to previously identified TR-ACS genes from Grasslands Challenge genotype 10F while the fourth was a novel gene designated TR-ACS4-T. TR-ACS4-T is 64%, 64% and 63% homologous to TR-ACS1-T, TR-ACS2-T and TR-ACS3-T, respectively in terms of nucleotide sequence. In the GeneBank database, TR-ACS4-T shares highly homology to ACC synthase sequences from a wide range of tissues including seedlings and fruit tissues, in addition to a high homology to ACS genes induced in auxin-, wounding- and ethylene-treated tissues. The pattern of TR-ACS4-T expression observed during leaf development suggests that the gene is expressed initially in the apical structures and in the newly initiated leaves, and then again in the later mature leaves and those at the onset of senescence. Expression decreases again during senescence. TR-ACS4-T expression is not altered by water deficit, but is induced by both ethylene and NAA treatment, but the auxin-induced TR-ACS4-T is mediated by ethylene treatment.

O mamão é um fruto climatérico, cujas transformações resultantes do amadurecimento ocorrem rapidamente após a colheita. O tratamento com 1- metilciclopropeno (1-MCP) tem sido testado para ampliar sua vida útil, entretanto, com resultados até então pouco consistentes. Pesquisas com peras, bananas e ameixas foram realizadas utilizando conjuntamente 1-MCP e etileno, com resultados relevantes. Portanto, o objetivo deste trabalho foi estudar a fisiologia e a conservação pós-colheita do mamão Golden, submetido a diferentes combinações de 1-MCP e etileno, aplicados simultaneamente. Os frutos foram colhidos em pomares comerciais no estádio 1 de maturação, tratados e armazenados em câmaras frias a 11 e a 22ºC. O projeto foi dividido em três etapas. Na primeira etapa, observou-se que doses de 100 e 200 nL.L-1 de etileno combinados com iguais concentrações de 1-MCP, apresentaram resultados semelhantes aos do 1-MCP aplicado isoladamente. Na segunda etapa, o etileno nas concentrações entre 0 e 10 SL.L-1 foi combinado com 100 nL.L-1 de 1-MCP. Esse aumento na concentração de etileno diminuiu ou até mesmo anulou a ação do 1-MCP, nas concentrações mais elevadas. A mudança de cor e a perda da firmeza foram mais lentas nos tratamentos com 1-MCP na dose de 100 nL.L-1, aplicado sozinho ou associado a menor dose de etileno (2,5 SL.L-1). Estes mesmos tratamentos apresentaram atividade respiratória e produção de etileno menores que o controle. Na terceira etapa, os frutos tratados apenas com 1-MCP e 1-MCP mais etileno nas doses de 1 e 2,5 SL.L-1, mostraram maior retenção da firmeza externa, firmeza interna e cor da casca. A aparência também foi melhor nestes tratamentos, com menor porcentagem de frutos podres. O teor de sólidos solúveis foi semelhante em todos os tratamentos e o de ácido ascórbico foi mais elevado nos frutos tratados apenas com 1-MCP. A produção de etileno e a concentração de etileno endógeno foram maiores nos frutos que receberam apenas 1-MCP. A atividade respiratória e a concentração de CO2 endógeno mostraram pouca variação entre os tratamentos, durante o armazenamento. O teor de carotenóides foi menor nos tratamentos que receberam as maiores doses de etileno. O teor de clorofila foi maior no tratamento que recebeu apenas 1-MCP o qual levou mais tempo para tornar-se amarelo. A atividade da enzima pectinametilesterase (PME) foi menor nos tratamentos com 1-MCP e 1-MCP mais etileno na dose de 1 SL.L-1. A atividade das enzimas 1-aminociclopropano-1- carboxílico sintase (ACS) e oxidase (ACO) alternaram períodos de elevada e de baixa atividade em todos os tratamentos, durante o armazenamento. A aplicação simultânea de 1-MCP e do etileno mostrou resultados promissores no controle do amadurecimento do mamão Golden. / Papaya is a climacteric fruit which ripening resulting transformations occur rapidly after harvest. Treatment with 1-methylcyclopropene (1-MCP) has been tested to extend its shelf life, however, with inconsistent results so far. Researches with pears, bananas and plums were carried out using 1-MCP and ethylene with relevant results. Therefore, the aim was to study the physiology and postharvest storage of \'Golden\' papaya subjected to different combinations of 1-MCP and ethylene applied in the same time. The fruits were harvested from commercial orchards in stage 1 of ripening, they were treated and stored in cold chambers. The study was divided into three stages. In the first step it was observed that doses of 100 and 200 nL.L-1 of ethylene combined with equal concentrations of 1-MCP showed similar results to the 1-MCP applied alone. In the second step, ethylene concentrations between 0 and 10 L.L-1 were combined with 100 nL.L-1 of 1-MCP. This increase in ethylene concentrations, decreased or even nullified the action of 1-MCP. The change in color and the firmness loss were slower in the treatments using 100 nL.L-1 of 1-MCP applied alone or associated with a lower dose (2.5 L.L-1) of ethylene. These same treatments showed less respiratory activity and ethylene production than the control. In the third step, the fruits treated only with 1-MCP and 1-MCP more ethylene at the doses of 1 and 2.5 L.L-1, showed higher retention of external and internal firmness and the green color of peel. The appearance was also better in these treatments, with a lower percentage of rotten fruit. The soluble solids content was similar for all treatments and the ascorbic acid was higher in the fruits treated only with 1-MCP. The ethylene production and endogenous ethylene concentration was higher in fruits treated only with 1-MCP. Respiratory activity and endogenous CO2 concentration showed few variation among the treatments during the storage. The carotenoid content was lower in the treatments that received the highest doses of ethylene. The chlorophyll content was higher in treatments with only 1-MCP, which took longer to become yellow. The pectinmethylesterase (PME) enzyme activity was lower in treatments only with 1-MCP and 1-MCP more ethylene at a dose of 1 L.L-1. The 1- aminocyclopropane-1-carboxylic acid synthase (ACS) and oxidase (ACO) enzyme activity, alternate periods of high and low activity in all treatments during storage. Simultaneous application of 1-MCP and ethylene showed promising results in the ripening of papaya \'Golden\'.

O mamão é um fruto climatérico, cujas transformações resultantes do amadurecimento ocorrem rapidamente após a colheita. O tratamento com 1- metilciclopropeno (1-MCP) tem sido testado para ampliar sua vida útil, entretanto, com resultados até então pouco consistentes. Pesquisas com peras, bananas e ameixas foram realizadas utilizando conjuntamente 1-MCP e etileno, com resultados relevantes. Portanto, o objetivo deste trabalho foi estudar a fisiologia e a conservação pós-colheita do mamão Golden, submetido a diferentes combinações de 1-MCP e etileno, aplicados simultaneamente. Os frutos foram colhidos em pomares comerciais no estádio 1 de maturação, tratados e armazenados em câmaras frias a 11 e a 22ºC. O projeto foi dividido em três etapas. Na primeira etapa, observou-se que doses de 100 e 200 nL.L-1 de etileno combinados com iguais concentrações de 1-MCP, apresentaram resultados semelhantes aos do 1-MCP aplicado isoladamente. Na segunda etapa, o etileno nas concentrações entre 0 e 10 SL.L-1 foi combinado com 100 nL.L-1 de 1-MCP. Esse aumento na concentração de etileno diminuiu ou até mesmo anulou a ação do 1-MCP, nas concentrações mais elevadas. A mudança de cor e a perda da firmeza foram mais lentas nos tratamentos com 1-MCP na dose de 100 nL.L-1, aplicado sozinho ou associado a menor dose de etileno (2,5 SL.L-1). Estes mesmos tratamentos apresentaram atividade respiratória e produção de etileno menores que o controle. Na terceira etapa, os frutos tratados apenas com 1-MCP e 1-MCP mais etileno nas doses de 1 e 2,5 SL.L-1, mostraram maior retenção da firmeza externa, firmeza interna e cor da casca. A aparência também foi melhor nestes tratamentos, com menor porcentagem de frutos podres. O teor de sólidos solúveis foi semelhante em todos os tratamentos e o de ácido ascórbico foi mais elevado nos frutos tratados apenas com 1-MCP. A produção de etileno e a concentração de etileno endógeno foram maiores nos frutos que receberam apenas 1-MCP. A atividade respiratória e a concentração de CO2 endógeno mostraram pouca variação entre os tratamentos, durante o armazenamento. O teor de carotenóides foi menor nos tratamentos que receberam as maiores doses de etileno. O teor de clorofila foi maior no tratamento que recebeu apenas 1-MCP o qual levou mais tempo para tornar-se amarelo. A atividade da enzima pectinametilesterase (PME) foi menor nos tratamentos com 1-MCP e 1-MCP mais etileno na dose de 1 SL.L-1. A atividade das enzimas 1-aminociclopropano-1- carboxílico sintase (ACS) e oxidase (ACO) alternaram períodos de elevada e de baixa atividade em todos os tratamentos, durante o armazenamento. A aplicação simultânea de 1-MCP e do etileno mostrou resultados promissores no controle do amadurecimento do mamão Golden. / Papaya is a climacteric fruit which ripening resulting transformations occur rapidly after harvest. Treatment with 1-methylcyclopropene (1-MCP) has been tested to extend its shelf life, however, with inconsistent results so far. Researches with pears, bananas and plums were carried out using 1-MCP and ethylene with relevant results. Therefore, the aim was to study the physiology and postharvest storage of \'Golden\' papaya subjected to different combinations of 1-MCP and ethylene applied in the same time. The fruits were harvested from commercial orchards in stage 1 of ripening, they were treated and stored in cold chambers. The study was divided into three stages. In the first step it was observed that doses of 100 and 200 nL.L-1 of ethylene combined with equal concentrations of 1-MCP showed similar results to the 1-MCP applied alone. In the second step, ethylene concentrations between 0 and 10 L.L-1 were combined with 100 nL.L-1 of 1-MCP. This increase in ethylene concentrations, decreased or even nullified the action of 1-MCP. The change in color and the firmness loss were slower in the treatments using 100 nL.L-1 of 1-MCP applied alone or associated with a lower dose (2.5 L.L-1) of ethylene. These same treatments showed less respiratory activity and ethylene production than the control. In the third step, the fruits treated only with 1-MCP and 1-MCP more ethylene at the doses of 1 and 2.5 L.L-1, showed higher retention of external and internal firmness and the green color of peel. The appearance was also better in these treatments, with a lower percentage of rotten fruit. The soluble solids content was similar for all treatments and the ascorbic acid was higher in the fruits treated only with 1-MCP. The ethylene production and endogenous ethylene concentration was higher in fruits treated only with 1-MCP. Respiratory activity and endogenous CO2 concentration showed few variation among the treatments during the storage. The carotenoid content was lower in the treatments that received the highest doses of ethylene. The chlorophyll content was higher in treatments with only 1-MCP, which took longer to become yellow. The pectinmethylesterase (PME) enzyme activity was lower in treatments only with 1-MCP and 1-MCP more ethylene at a dose of 1 L.L-1. The 1- aminocyclopropane-1-carboxylic acid synthase (ACS) and oxidase (ACO) enzyme activity, alternate periods of high and low activity in all treatments during storage. Simultaneous application of 1-MCP and ethylene showed promising results in the ripening of papaya \'Golden\'.