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Item Comparison of leaf senescence regulation between distantly related plant species uncovers knowledge gaps and opportunities for plant improvement strategies(Elsevier B.V., 2023-10-01) Aloryi KD; Jing H-C; Dijkwel PPLeaf senescence is a destructive process that allows the efficient recycling of nutrients from dying leaves to growing parts of the plant. It is the final stage of leaf development that can be induced in response to stress. This makes leaf senescence an adaptive process that is highly beneficial for whole plant survival under unpredictable environmental conditions. Thus, the manipulation of this process has the potential to improve crop plants to become more climate resilient. In this review we compare leaf senescence processes between distantly related species to identify knowledge gaps and opportunities for plant improvement strategies. We describe that main signalling pathways controlled by carbohydrates, reactive oxygen species and hormones are conserved. However, the role of ethylene in age-induced leaf senescence in cereals is obscure. Moreover, downstream senescence regulatory signalling events are largely unknown, and these may be considerably different between members of annual dicots and cereals. Because leaf senescence regulation is so intricately connected to basic metabolic and developmental processes, we propose to increase research efforts to discover natural variation in senescence regulation and pinpoint gene variants that are tried and tested in nature.Item Understanding kiwifruit postharvest physiology and quality changes in tropical retail market conditions : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, New Zealand(Massey University, 2022) Malavalli Veeregowda, Praveen KumarKiwifruit is New Zealand’s major horticulture crop. A challenge that exists for the New Zealand kiwifruit industry in emerging tropical markets is maintaining fruit quality during marketing. In Asia and the Middle East, high ambient temperatures (> 30 °C) are often prevalent. The domination of traditional fruit marketing practices in these regions results in fruit exposure to these conditions. Understanding of fruit postharvest responses to high ambient temperatures is limited. In this thesis, a supply chain survey was conducted in India and Singapore, where kiwifruit at edible ripeness were found to be exposed to temperatures > 30 °C and ethylene concentrations as high as 150 nL L-1 during retail. Back in the laboratory, kiwifruit were exposed to simulated tropical conditions, where 12 h of exposure to 40 °C resulted in significant changes in fruit physiology as evidenced by rapid respiratory decline. The findings from this initial study indicated that 40 °C exposure may have a lethal impact on kiwifruit whilst 33 °C may not. The role of ethylene in influencing kiwifruit physiology under tropical conditions was investigated by treating kiwifruit with 1-MCP to prevent ethylene responses. Consequently, 1-MCP treated fruit exposed to 33 °C retained firmness both in the presence or absence of exogenous ethylene whilst untreated fruit softened rapidly, indicating that the possible ethylene exposure in tropical conditions advances kiwifruit softening. A further study aimed to identify exposure temperature and time combinations that trigger detrimental outcomes for kiwifruit including after a period returned to coolstorage. Like previous, kiwifruit at 40 °C exhibited rapid respiratory decline, while at 33-38 °C, no such response was evident. This confirmed that 40 °C exposure for 12 h could have a lethal impact on kiwifruit physiology. Fruit that were exposed to high temperatures (33-40 °C) beyond 24 h and later coolstored developed internal breakdown symptoms indicating that irrecoverable degradative processes are induced. A plausible mechanism as a result of heat exposure is the occurrence of anaerobic respiratory metabolism. At 33-40 °C, Respiratory Quotient (RQ) in kiwifruit remained ≥ 1 reaching 3 at 40 °C whilst at 20 °C, RQ remained close to 1. Contrastingly, ethanol accumulation increased at high temperatures but minimal ethanol content changes occurred at 20 °C. At some high temperatures, ethanol content increased with time but no symptoms of heat injury were evident, suggesting that heat injury may not be exclusively caused by the accumulation of anaerobic metabolites. The increase in ethanol content measured in kiwifruit at high temperatures indicates the potential risk of off-flavour development at retail conditions in tropical markets. Overall, this research contributes to kiwifruit quality maintenance in tropical markets by elucidating safe exposure durations for kiwifruit and identifying a postharvest treatment of 1-MCP as a potential quality maintenance tool. Future research requirements include identifying molecular mechanisms that control physiological changes in kiwifruit at high temperatures, identifying causes for the differential responses observed between ‘Hayward’ and ‘SunGold™’ and investigating the efficacy of 1-MCP at wider ranges of high temperature conditions.Item Ethylene related ripening of 'SunGold™' kiwifruit : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand. EMBARGOED until 31 January 2026.(Massey University, 2020) Tongonya, JeritahA key component of the success of the New Zealand kiwifruit industry is the consistent provision of high-quality produce. The projected increase in kiwifruit volumes necessitates the widening of harvest and marketing windows. Two different kiwifruit export marketing strategies are currently deployed: i) Early season fruit are delivered for immediate sale (‘KiwiStart’), ii) later season fruit have a maturity that enables extended postharvest cool storage (‘MainPack’). These marketing strategies determine harvest criteria and subsequent postharvest management practices employed in the supply cool chain. The introduction of new cultivars, e.g. ‘SunGold™’, necessitates a re-evaluation of the postharvest ripening and storage practices, tuning the requirements to the specific responses of each cultivar. Since there is minimal information on ethylene related responses for ‘SunGold™’, research to develop a fundamental understanding of these responses is crucial for optimal management and performance of the product in the market. The purpose of this PhD is to determine the effect of industry-relevant ethylene concentrations on ‘SunGold™’ quality progression (firmness and soluble solids content (SSC)). With that understanding, a semi-mechanistic model for the simultaneous description of firmness decline and soluble solids increase under dynamic temperature conditions and ethylene concentrations was developed.--Shortened abstractItem Investigation into the relationship between ethylene and sulfur assimilation in Arabidopsis thaliana and onion (Allium cepa L.) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science (with Honours) in Biochemistry at Massey University(Massey University, 2006) Sanggang, Fiona AnnThe phytohormone ethylene (C2H4) mediates the adaptive responses of plants to various nutrient deficiencies including iron (Fe)-deficiency, phosphorus (P)-deficiency and potassium (K)-deficiency. However, evidence for the involvement this hormone in the sulfur (S) deficiency response is limited to date. In this study, the effect of C2H4 treatment on the accumulation of the S-assimilation enzymes ATP sulfurylase (ATPS). adenosine-5 -phosphosulfate-reductase (APR), O-acetylserine-(thiol)-lyase (OASTL) and sulfite reductase (SiR) was examined in A. thaliana and onion (A. cepa). To complement this, the effect of short-term S-depletion on the expression of the 12-member gene family of the C2H4 biosynthetic enzyme, l-amino-cyclopropane-l-carboxylic acid (ACC) synthase (ACS) from A. thaliana, designated AtACS1-12, was also examined. Western analyses were used to show that plants of A. thaliana pre-treated with the C2H4-signalling inhibitor 1-MCP, had elevated levels of ATPS, APR and OASTL protein in leaf tissue at all time points examined, suggesting that C2H4 has an inhibitory effect on the accumulation of these enzymes. However, SiR appeared to be under dual regulation by C2H4: under S-sufficient conditions C2H4 appears to prevent the unnecessary accumulation of SiR and conversely promote the fast accumulation of SiR under S-depleted conditions. The changes in AtACS1-12 expression in the root and leaf tissues of S-sufficient and S-depleted plants of A. thaliana were examined by RT-PCR using gene-specific, exon-spanning primers. The expression patterns of AtACS2, AtACS6 and AtACS7 were comparable regardless of S availability and may therefore be housekeeping genes. In contrast, the expression of AtACS5 in leaf, and AtACS8 and AtACS9 in roots was repressed under S-depleted conditions, although the mechanism of this repression cannot be elucidated from this study. The protein products of these closely-related genes are believed to be phosphorylated and stabilised by a CDPK whose activity may be compromised by S-depletion. The inhibition of AtACS5, AtACS8 and AtACS9 expression, and the decrease in AtACS5, AtACS8 and AtACS9 accumulation, and hence less C2H4 production, may be part of the plant adaptive response to S-depletion, as the C2H4 -mediated repression of root growth is alleviated to allow the plant to better seek out the lacking nutrient. The expression of the MPK-stabilised genes AtACS2 and AtACS6 appeared to be similar regardless of S availability, although this may merely be a consequence of the scoring method used in this study, which cannot determine whether there was any difference in the level of expression of these genes. The expression of AtACS10 and AtACS12 was repressed in S-deficient plants. Although both AtACS10 and AtACS12 isozymes posses the hallmark seven conserved regions found in the ACSes of other plant species, they are also phylogenetically related to alanine and aspartate aminotransferases, and are known to encode aspartate (AtACS10) and aromatic amino acid transaminases (AtACS12). Therefore, the apparent downregulation of these genes suggests that the downregulation of amino acid metabolism may be part of the plant adaptive response to S-depletion. The downregulation of several AtACS genes, and therefore possibly also C2H4 biosynthesis, in S-deficient plants was accompanied by an accumulation of APR protein. The increase in APR protein that also occurred in 1-MCP-treated plants indicates that C2H4 may be involved in the plant response to S-depletion, because in both cases the upregulation of the S-assimilation pathway, as manifested by the accumulation of APR protein, occurred when C2H4 biosynthesis and signalling was repressed. However, the possible role of other phytohormoes in the plant response to S-depletion cannot be excluded, as there is evidence for crosstalk between the C2H4 signalling pathway and those of auxin, abscisic acid (ABA), cytokinins and jasmonic acid (JA). Furthermore, because C2H4 has been implicated in the response of various plants to Fe-deficiency, P-deficiency, and K-deficiency, in addition to S-deficiency, it may be a regulator of the plant adaptive response to nutrient stresses in general.Item Ethylene synthesis inhibitor affects postharvest kiwifruit quality : a thesis presented in partial fulfilment of the requirements for the degree of Master of Applied Science at Massey University(Massey University, 1998) Marques, Jose RobertoFirmness is a key quality criteria of kiwifruit and changes significantly during fruit ripening, with premature softening being a serious commercial problem for the industry. Ethylene is involved in regulation of fruit ripening and influences a number of processes, including ethylene production, respiration rate and changes in firmness. Kiwifruit is very sensitive to ethylene, which increases fruit softening rate and reduces storage potential. Aminoethoxyvinylglycine (AVG), an inhibitor of ACC synthase, a key enzyme in the pathway for ethylene biosynthesis, has been applied to horticultural crops, especially apples, in an attempt to regulate ethylene synthesis and its mediated processes, with a number of positive effects including reduced fruit ethylene production, reduced respiration rate, and slower softening rate. The effects of AVG (500 and 1000 mg.l a.i., or 200 and 400 g.acre a.i.) applied to 'Hayward' kiwifruit vines (6 and 4 weeks before commercial harvest) on ethylene production, respiration rate, firmness and soluble solids content of fruit at harvest and after coolstorage were investigated. Kiwifruit treated with either 500 or 1000 mg.l AVG 4 weeks before commercial harvest and maintained at 20 °C over 15 days, had a lower respiration rate, reduced ethylene production, a slower softening rate, and lower SSC than control fruit immediately after harvest and following 14 days at 0 °C, with the differences generally becoming significant after 6 days at 20 °C. These attributes are generally stimulated by ethylene, indicating that the endogenously produced ethylene was inhibited by the applied AVG, resulting in a slower fruit ripening rate at 20 °C. However, AVG effects were transitory. There were generally no differences in the above fruit variables between AVG-treated and control fruit at 20 °C up to 20 days following 30, 52, and 80 days at 0 °C. After 110 and 180 days at 0 °C, kiwifruit treated with either 500 or 1000 mg.l AVG 4 weeks before commercial harvest and maintained at 20 °C up to 10 days, had a higher respiration rate, increased ethylene production, and accelerated softening compared with control fruit. There were basically no differences in any of the above fruit variables between the treatments 500-AVG-6 and control, either immediately after harvest or following storage at 0 °C up to 180 days. The short term effect of AVG in kiwifruit during and after coolstorage and the questionable efficiency of AVG uptake in kiwifruit are issues to be further addressed before any practical application can be recommended.Item Ethephon, ethylene and abscission physiology of camellia : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Horticultural Science at Massey University, Palmerston North, New Zealand(Massey University, 1992) Woolf, Allan BrianEthylene application to leaves and floral buds of Camellia resulted in abscission with a lag period, the duration of which was dependent on ethylene concentration and cultivar. During this period, cellulase activity doubled in leaf abscission zones, and when abscission commenced, activity increased more rapidly. However, no increase in cellulase activity was observed in floral bud abscission zones. Propylene application revealed that autocatalytic ethylene production increased in leaf abscission zones prior to and decreased after abscission. However, in the leaf blade, no change in endogenous ethylene production was measured, nor were any signs of leaf senescence observed. Application of(STS) completely inhibited leaf abscission and delayed and reduced floral bud abscission in response to applied ethylene. This pointed to a similar role for ethylene in both organs, but that the abscission process of floral buds occurred at a faster rate than that of leaves. Application of ethylene for differing durations to floral buds and leaves demonstrated that regardless of ethylene treatment duration, abscission ceased less than 24 hr after ethylene removal indicating that continuous ethylene exposure is required to promote abscission of Camellia organs. Measurement of abscission rate (time to 50% abscission) in response to a range of ethylene concentrations determined that floral buds were more sensitive (that is; responded more rapidly to lower ethylene concentrations) than leaves. Ethylene-sensitivity was i nfluenced by organ maturity. As floral buds mature d from initiation t o flower opening, the rate of ethylene-promoted abscission i ncreased, i ndicati ng g reater sensitivity. Leaves were most sensitive to ethylene directly after bud break and sensitivity decreased untiiU weeks after cessation of stem extension ; after this time, sensitivity did not change significantly over the next 3 years. Low temperatures reduced the ethylene-promoted abscission rate of both leaves and floral buds with an exponential relationship. Low temperatures increased the ethylene concentration required to saturate the abscission response. Endogenous ethylene production of Camellia leaves increased with higher temperatures and peaked at 20 to 25c. Measurement of abscission rate (time to 50% abscission) in response to a range of ethylene concentrations determined that floral buds were more sensitive (that is; responded more rapidly to lower ethylene concentrations) than leaves. Ethylene-sensitivity was influenced by organ maturity. As floral buds matured from initiation to flower opening, the rate of ethylene-promoted abscission increased, indicating greater sensitivity. Leaves were most sensitive to ethylene directly after bud break and sensitivity decreased until 12 weeks after cessation of stem extension; after this time, sensitivity did not change significantly over the next 3 years. Low temperatures reduced the ethylene-promoted abscission rate of both leaves and floral buds with an exponential relationship. Low temperatures increased the ethylene concentration required to saturate the abscission response. Endogenous ethylene production of Camellia leaves increased with higher temperatures and peaked at 20 to 25c. Since ethylene release from ethephon may be described in terms of concentration and duration of ethylene exposure, the effect of time, temperature , cultivar, organ type and organ maturity on organ abscission response to ethephon application could be explained in terms of the ethylene-promoted response. The level of ethylene- and ethephon-promoted abscission were explained in terms of the interaction of ethylene concentration and duration of exposure with organ type, organ maturity and temperature which determined the level of abscission response. Three mechanisms were important in determining the response to ethylene; ethylene-sensitivity, and rate of reaction and reversibility of the abscission process. The rate of the abscission process was determined by ethylene concentration, temperature, organ type and maturity. Since abscission was reversible in Camellia, the duration of exposure interacted with the abscission rate to determine the extent of abscission in response to ethylene or ethephon application. In conclusion , the greatly expanded understanding of the ethylene-promoted abscission process carried out in this study facilitates control (promotion or inhibition) of abscission in Camellia. This enhances the possibility for culture and transportation of high quality Camellia plants from New Zealand.Item Effects of carbon dioxide on the ethylene-forming enzyme in Japanese pear and apple fruits : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Horticultural Science at Massey University(Massey University, 1991) Tian, Mei-shengThe aim of this thesis was to investigate the effects of carbon dioxide on ethylene-forming enzyme (EFE) and its regulation in both Hosui and Granny Smith fruit discs. 1. From measurement of respiration rate and ethylene production after harvest and response of them to propylene treatment, it has been shown that Japanese pear Hosui grown in New Zealand is a nonclimacteric fruit. Granny Smith apple showed different respiration and ethylene production patterns in different seasons. Because fruit produced typical climacteric respiration and ethylene peaks, Granny Smith apple is a climacteric fruit. 2. The rapid rise of ethylene production and respiration measured at 21°C showed no time lag in Granny Smith fruit after storage for 20 days at low temperature (1°±1°C) compared to those immediately measured after harvest at 21°C. Ethylene production and respiration rate also increased faster and to a greater extent in fruit exposed to low temperature than in fruit measured immediately after harvest. This ripening behavior is similar to that of European pears and Golden Delicious apple. In contrast to Granny Smith apple, ethylene production and respiration patterns of Hosui were not changed by low temperature storage. 3. A reliable method for testing ethylene forming enzyme (EFE) activity in fruit discs of Hosui and Granny Smith was developed. 4. At harvest time EFE activity was present in Hosui fruit, but not in Granny Smith apple. EFE development showed a similar pattern in both fruits, increasing steadily in fruit stored at low temperature. 5. Carbon dioxide stimulated EFE synthesis in fruit discs of preclimacteric Granny Smith, but not in those of Hosui. Carbon dioxide stimulated EFE activity in Hosui fruit discs during the short term storage at 1°±1°C, after CO2 lost its stimulatory effect. In contrast to Hosui, CO2 stimulated EFE activity in Granny Smith fruit discs through the measuring period tested. 6. Carbon dioxide was not able to reverse Co++ (an EFE activity inhibitor) inhibitory effect on EFE activity in types of fruit discs. This result showed that CO2 could directly stimulate EFE activity. 7. The stimulatory effect of CO2 on EFE activity was dependent on exogenous ACC. EFE activity in both types of fruit discs was lower in 0.4 M mannitol solution than in 0.8 M solution, but EFE lost the capability to respond to CO2 in 0.8 M mannitol solution. This suggests that the EFE located in the plasma membrane is the main form to respond to CO2 in both types of fruit discs. 8. Results from kinetic studies indicated that EFE in discs of both Hosui and Granny Smith fruits was not allosteric. The apparent Km values of EFE for ACC were 0.166 mM for Hosui, and 0.193 mM for Granny Smith apple. Carbon dioxide increased the maximum reaction rate of conversion of ACC to ethylene without changing apparent Km values of EFE for ACC in discs of both Hosui and Granny Smith fruits. It suggests that the mechanism of the direct stimulatory effect of CO2 on EFE activity was due to the formation of a CO2-EFE-ACC complex and/or EFE-ACC-CO2 complex which increased the maximum rate of the reaction which resulted in the conversion of ACC to ethylene. 9. Silver ions inhibited ethylene production in Granny Smith fruit discs, but not in Hosui fruit discs which did not produce detectable ethylene. Low concentrations (<0.25 mM) Ag+ stimulated, but high concentrations (>0.5 mM) inhibited, EFE activity in both types of fruit discs. Carbon dioxide did not reverse the inhibitory effect of Ag+. Because only System I ethylene receptor is thought to occur in Hosui, a nonclimacteric fruit, and the inhibitory effect of Ag+ on EFE activity in discs of both types of fruit were similar, results suggest that EFE activity was regulated by the System I ethylene receptor. 10. Norbornadiene (NDE) is a competitive ethylene action inhibitor, which inhibits ethylene synthesis by binding to the ethylene binding site of ethylene receptors. At 0.5% (v/v), NDE inhibited EFE activity in both types of fruit discs, and this inhibition was partially reversed by CO2 in discs from unripe Hosui and preclimacteric Granny Smith fruit. These results suggest that CO2 might indirectly stimulate EFE activity by binding competitively to the System I ethylene receptor at the ethylene binding site. When Granny Smith was at the climacteric stage CO2 did not reverse the NDE inhibitory effect on EFE activity. 11. According to the response of EFE to CO2 and the mechanisms of the response, cells in Hosui and Granny Smith fruits were distinguished into four different types. During ripening, cells in fruits changed their type, thereafter the responsiveness of EFE to CO2 changed. A model is presented to explain the mechanism of CO2 on EFE synthesis and its activity through interactions with EFE directly and the ethylene receptors indirectly.Item The role of ethylene in kiwifruit softening : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University(Massey University, 1999) Kim, Hyun OkPremature fruit softening during storage at 0°C is a serious and costly problem for the New Zealand kiwifruit industry. Ethylene gas (C2H4) is a potent promoter of fruit softening; it is involved in regulation of fruit ripening and influences a number of processes, including ethylene production, respiration rate and fruit softening. Exogenous ethylene increases softening rate in kiwifruit at 20°C and at 0°C; a concentration of 0.01µl/1 will enhance softening at 0°C. The precise relationship between kiwifruit softening and endogenous ethylene concentration is not known. The influences of low temperature, an ethylene synthesis inhibitor (aminovinylglycine (AVG)), an inhibitor of ethylene action (1-methylcyclopropene (1-MCP)) and application of ethylene at different maturities were investigated in an attempt to elucidate ethylene's role in initiating kiwifruit softening. Exposing fruit to 0°C for more than 52 days hastened ethylene production upon removal to 20°C compared with fruit maintained at 20°C continuously after harvest; this enhanced ethylene was associated with increased 1-aminocyclopropane-1-carboxylic acid (ACC) concentration and ACC oxidase (ACO) activity. Kiwifruit softening at 0°C occurred even though ethylene production was low and constant between 0.02 to 0.06µl/kg/h (corresponding to internal ethylene concentrations (IEC) 0.2 to 0.6µl/1). This softening was associated with low ACC concentrations varying between 0.2 to 0.5nmole/g and ACO activity varying between 0.01 to 0.66nl/g/h These results indicate that very low concentrations of ethylene may play an essential role in kiwifruit softening. In kiwifruit treated with AVG (500ppm) 4 weeks before harvest, ethylene biosynthesis, manifested as reduced ACC concentration, ACO activity and ethylene production, was significantly inhibited both at 20°C and after storage at 0°C. AVG application resulted in a slower softening rate and firmer fruit than in untreated controls. Rates of softening were 0.4N/day and 1.9N/day in the AVG treated fruit and control fruit respectively during 14 days at 20°C. However, the AVG effect was reduced after storage at 0°C for 14 days, indicating that the AVG effect was only temporary and may not be sufficient to warrant possible commercial use for longer storage. Application of 250ppm AVG 2 or 4 weeks before harvest, and 500ppm AVG 2 weeks before harvest had no effect on ethylene production or fruit firmness. Fruit infected with B. cinerea produced more ethylene than non-infected fruit at 0°, 4°, 10° and 20°C. An increase in ethylene production, induced by B. cinerea infection, occurred in tissue slices from the invasion zone (the infection front containing both infected tissue and sound tissue immediately ahead of the infection front) and adjacent zone (sound tissue ahead of the invasion zone) of kiwifruit. The increased ethylene in infected fruit was associated with increased ACO activity in tissue from adjacent and distal (sound tissue at the distal end of the fruit) zones. Application of 500ppm AVG to kiwifruit vines before harvest reduced ACC concentration and ACO activity and ethylene production induced by B. cinerea at 20°C. However this AVG reduced ethylene production from B. cinerea infected fruit after 4 weeks at 0°C was insufficient to prevent rapid growth of B. cinerea. Therefore, such an AVG treatment can not be used to reduce B. cinerea infection during storage at 0°C. As kiwifruit matured, softening was enhanced increasingly by exogenous ethylene, indicating that tissue sensitivity to this growth regulator increased with time. The sensitivity of kiwifruit was reduced by treatment with 1-MCP, an inhibitor of ethylene action. When applied to kiwifruit at harvest, 1-MCP reduced ethylene production and respiration rate, resulting in a slower fruit softening and firmer fruit during storage at both 0°C and 20°C. Kiwifruit treated with 1-MCP plus ethylene at harvest, remained firmer than fruit exposed to ethylene alone for 4 days at 20°C and 8 days at 0°C, after which this 1-MCP effect disappeared with firmness being the same for both ethylene plus 1-MCP and ethylene treated fruit at the two temperatures. When both 1-MCP plus ethylene were applied after storage at 0°C, 1-MCP negated the ethylene-induced softening with treated fruit having a softening rate 4 times less than for fruit treated with ethylene alone. Softening rate of control and 1-MCP treated fruit was the same as those treated with 1-MCP plus ethylene (approximately 0.06 ('Kiwifirm'unit)/day) compared with 0.2 ('Kiwifirm'unit)/day for fruit treated with ethylene alone. Since 1-MCP binds to the ethylene receptor sites irreversibly, it is suggested that kiwifruit can synthesis new ethylene receptors with time during storage at 0°C and 20°C, making kiwifruit increasingly sensitive to endogenous ethylene. Over several different experiments in 3 seasons, kiwifruit softened from ≈ 90N to 10~19N while endogenous ethylene production remained low (below 0.1~0.2 µl/kg/h) and constant at 20°C. Increased ethylene production only occurred as fruit softened from 10N~19N to eating ripe (6~8N). These results have led to a model being proposed for ethylene action during ripening of kiwifruit. Kiwifruit softening (phases 1 and 2) occurs even though ethylene production is low as is ACS and ACO activity. It is possible that this basal level of ethylene corresponds to System 1 ethylene production which is thought be associated with basal metabolic maintenance; this ethylene probably binds to System 1 ethylene receptors. The changing ability of fruit to soften with increased maturity is due to increasing sensitivity to such low ethylene concentration resulting from the progressive formation of new ethylene receptors in the fruit. Application of AVG and 1-MCP reduced ethylene production, leading to a delay in ethylene-induced softening. It is possible that low endogenous ethylene (System 1 ethylene) is sufficient to induce starch degradation and solubilization of pectins in cell walls caused by hydrolase enzymes such as amylase, β-galactosidase and xyloglucan endotransglycosylase early in ripening. Alternatively oligomers derived from cell wall breakdown may induce ethylene production even though such oligomer elicitors have not been reported to exist in kiwifruit. Maximal cell wall swelling, depolymerization of the solubilized pectin by polygalacturonase and breakdown of the middle lamella only occur when kiwifruit already have softened to <20N (phase 3 of softening), and these events are associated with or co-ordinated by System 2 autocatalytic ethylene. This autocatalytic ethylene is associated with high ACS and ACO activity and may bind to System 2 ethylene receptors, leading to ethylene dependent responses such as PG activation which results in ready-to eat fruit with a firmness of 6~8N. In conclusion, kiwifruit softening from 90N to 10~19N occurs with low and constant ethylene production. Because new ethylene receptors of kiwifruit can be formed with time at both 0°C and 20°C, it appears that kiwifruit sensitivity to low ethylene concentration also increases with time in storage. It is possible that different cultivars of kiwifruit with different softening rates, may have different amounts or rates of formation of new ethylene receptors. By comparing physiological, biochemical and molecular attributes of Hayward and other kiwifruit cultivars and selections, it should be possible to provide information on their responsiveness and sensitivity to ethylene. This will allow plant breeders to create new cultivars that have both low ethylene production and low sensitivity to ethylene that would provide a range of commercial cultivars with prolonged and different storage lives for the international kiwifruit market. Application of inhibitors of ethylene biosynthesis (AVG) and ethylene action (1-MCP) reduced ethylene production and softening rate of kiwifruit with 1-MCP being more effective for longer than AVG. Although 1-MCP showed promise as a tool to delay softening during storage at 0°C and shelf life at 20°C, further research is required to determine optimum concentration, time and frequency of application, and efficiency when applied at 0°C in order to derive treatments that may have significant commercial applications.Item Role of cytokinin and ethylene during senescence in broccoli (Brassica oleracea var. Italica) : a thesis submitted for the degree of Doctor of Philosophy at Massey University(Massey University, 2003) Gapper, Nigel EstevenBroccoli (Brassica oleracea var. italica) deteriorates rapidly following harvest. The two plant hormones ethylene and cytokinin are known to act antagonistically on harvest-induced senescence in broccoli: ethylene acts by accelerating the process, whereas additional cytokinin delays it. The overall aim of this thesis was to gain a better understanding of how these two hormones control postharvest senescence. The effects of exogenous cytokinin (6-benzyl aminopurine, 6-BAP), 1-aminocyclopropane-1-carboxylic acid (ACC) and sucrose on senescence-associated gene expression were measured in both wild-type plants and transgenic plants harbouring an antisense tomato ACC oxidase gene (pTOM13). Exogenous cytokinin caused both a reduction (BoACO) and an increase (BoACS) in ethylene biosynthetic gene expression as well as reduced expression of genes encoding sucrose transporters and carbohydrate metabolising enzymes, indicating a significant role for cytokinin in the delay of senescence. Transgenic broccoli was produced using Agrobacterium tumefaciens-mediated transformation. Ethylene biosynthesis was targeted via an antisense BoACO2 gene fused to the harvest-induced asparagine synthetase (AS) promoter from asparagus. In addition, broccoli was transformed with constructs harbouring the Agrobacterium tumefaciens isopentenyl transferase (ipt) gene using the senescence-associated SAG12 and floral-associated MYB305 gene promoters to enhance the levels of cytokinin either during senescence or in floral tissue, respectively. The presence of the antisense AS-ACO construct was associated with an increased rate of transformation when compared to control constructs. Physiological analyses of mature plants showed that the antisense AS-ACO gene construct caused delayed senescence in both detached leaves and detached heads. Gene expression analyses of harvested floret tissue from AS-ACO lines showed decreases in transcript levels of senescence marker genes compared to wild-type and transgenic control lines, as well as a reduction in expression of sucrose transporter and carbohydrate metabolising genes, confirming the key role of ethylene in the promotion of senescence. In addition, genes involved with cytokinin biosynthesis and metabolism were isolated by PCR using primers based on Arabidopsis clones. The four broccoli ipt sequences aligned closely to four of the Arabidopsis sequences and were subsequently named BoIPT4, BoIPT5, BoIPT6 and BoIPT7. A cytokinin oxidase clone (BoCKX) was also isolated from broccoli. The four BoIPT genes were expressed in a number of different tissues, suggesting that the different genes may be involved in different biological processes in the plant. BoIPT4 was expressed early and BoCKX expressed late in florets during senescence. A model depicting the regulation of senescence in broccoli through the expression of cytokinin biosynthesis and metabolism genes, and their interaction with ethylene and carbohydrate metabolism is presented and discussed.Item The role of ethylene and auxin in responses of roots to phosphate supply in white clover (Trifolium repens L.) : a thesis presentation in partial fulfilment of the requirements for the degree of Master of Science in Plant Molecular Biology at Massey University, Palmerston North, New Zealand(Massey University, 2009) Dinh, Phuong Thi YenPhosphate (P) supply is one of the major determining factors to plant productivity, since the element affects the growth and the development of plants. In response to Pdeficiency treatment, plants display alterations in root system architecture caused by changes in primary root (PR) and lateral root (LR) length and LR density. In this thesis, the root growth of the agronomically important legume, white clover (Trifolium repens L.) was found to be slightly stimulated in terms of PR length, LR number and total LR length when plants were grown in a P-deficient media (0.01 mM orthophosphate; Pi) when compared with plants grown in a P-sufficient media (1.00 mM Pi) when using a hydroponic growth system. When plants are grown in a P-sufficient media, treatment with 100 nM exogenous 1- aminocyclopropane-1-carboxylic acid (ACC) and exogenous auxin (5 nM 1- naphthylacetic acid, NAA) resulted in significant increases in white clover PR length, LR number and LR length. However, when ethylene action or auxin transport were inhibited using 300 ppm 1-methylcyclopropene (1-MCP) and 100 nM 1-Nnaphthylphthalamic acid (NPA), respectively, root growth was significantly reduced which suggests roles for ethylene and auxin in mediating white clover root growth. To examine the effects of these hormones on plants grown in P-deficient media, 100 nM ACC treatment significantly enhanced the stimulatory effects of growth on Pdeficient media only, while exposure of plants to P-deficiency alone was sufficient to significantly neutralise the inhibitory effects of 1-MCP on root growth. Hence, exposure to P-deficiency is proposed to increase either ethylene biosynthesis or ethylene sensitivity in white clover roots. In contrast, for plants grown in P-deficient media, treatment with 5 nM NAA significantly abolished the stimulation of white clover root growth observed with P-deficiency so it is proposed that exposure to Pdeficiency increases either auxin biosynthesis or auxin sensitivity, but the 5nM NAA concentration used was too high to stimulate root growth. Using DR5p::GUS transgenic white clover, auxin activity was found in the root tips and root primordia. Using these plants, it is suggested that P-deficient treatment and ACC treatment influenced white clover root growth through an increase in auxin sensitivity. Overall, ethylene and auxin are found to be essential in mediating white clover root growth in P-sufficiency, and also in mediating root responses to P-deficiency through changes in terms of the biosynthesis and the sensitivity of these two hormones.
