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Item Breeding and transgenic approaches to improving water use efficiency in white clover (Trifolium repens L.) : 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, 2014) Raynes, Julia Sarah MargaretThis thesis investigates the potential role of the ABA biosynthetic gene 9-cis epoxycarotenoid dioxygenase 1(NCED1) as a determinant of water-use-efficiency (WUE) in plants as part of a longer term aim to confer improved WUE to the forage legume white clover (Trifolium repens L.). Two experimental approaches have been used. The first looked at the expression of NCED1 in a range of Trifolium species that display anatomical and morphological traits that confer some adaptations to growth and survival in dryland habitats. The second approach involved over-expression of NCED1 in the model species tobacco (Nicotiana tabacum) in proof-of-concept experiments to directly determine if any changes in plant water relations can be measured. Initially the constitutive expression of TrNCED1 was assessed, using the quantitative real-time polymerase chain reaction (q-RT-PCR), in two varieties of white clover. The first is an agronomically elite cultivar with a higher water requirement for optimal growth, cv. Grasslands Kopu II, and the second a dryland-adapted ecotype, Tien Shan. It was found that for both varieties, TrNCED1 expression was highest in the younger (first-fully-expanded) leaf (FFEL) tissue, followed by root tissue, then lastly apical tissue. When comparing the two varieties under well-watered conditions, the relative expression of TrNCED1 was higher in aerial parts of the cv. Kopu, but in the roots of the Tien Shan ecotype with respect to the reference genes, TrActin and TrGAPDH. Further, in the high biomass cv. Grasslands Kopu II, expression of TrNCED1 decreased in the apical and FFEL tissues of plants experiencing a water deficit, and decreased while for the dryland ecotype Tien Shan, TrNCED1 expression did not change in the apex reguardless of plant water status. Expression in the FFEL increased in tissue experiencing water deficit, and decreased in roots, suggesting different mechanisms for drought tolerance and response in the two varieties. For the range of Trifolium species assessed, constitutive expression of the TrNCED1 homologue was measured under well watered conditions in the FFEL and apical tissues. Essentially no significant difference in expression in either tissue between species was detected, with respect to the reference genes, TrActin and TrGAPDH. model species. In other studies, over-expression of NCED1 has been found to confer some characteristics associated with increased WUE, although abnormal growth associated with high levels of ABA at key developmental stages has proved problematic. For this thesis, the two senescence associated promoters, Senescence-Activated-Gene (SAG13) and Senescence-Associated-Receptor-Kinase (SARK), were selected to drive over-expression of NCED1 from Solanum lycopersicum in tobacco. Of those plants that came through tissue culture and were successfully established in soil, a single line transformed with SAG13p::SlNCED1, and three plants transformed with SARKp::SlNCED1, were shown to be positive for transgene insertion using a polymerase chain reaction (PCR) with genomic DNA. Of these, two plants, both transformed with SARKp::SlNCED1, were found to express SlNCED1 when tested using PCR with cDNA from isolated RNA. Water relations measurements performed on all four plants that were positive for the transgene, an empty vector control, a selection of plants that were negative for transgene insertion, and some wild-type controls, found that one of the lines confirmed as expressing the transgene, line 751-1, had a very low transpiration rate and low level of stomatal conductance. To extend these measurements to determine and increase in water-use-efficiency, a comparison must be made between growth rate and water uptake, and many more transgenic plant lines must be analysed. Finally, to determine whether the eventual transformation of white clover with SAG13p::SlNCED1 and SARKp::SlNCED1 would result in co-suppression of both types of transgene and the constitutive NCED1, expression of TrNCED1 in seven tissue types, from two varieties was measured. Highest expression was determined in the root tissue and in the younger leaf tissue, but was lower in the mature tissue examined. This suggests that transformation with SlNCED1 and expression in the mature tissues is not likely to be influenced by the constitutively expressed TrNCED1.Item Characterization of an AtPAP26-like protein (TrPAP26) from white clover (Trifolium repens L.) : 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) Huang, Jennifer YPhosphate levels in soils are often in deficit in New Zealand agriculture systems, resulting in the need for phosphate supplements in the form of fertilizers. Plants are able to adapt to many environmental stresses and display a wide range of responses designed to cope with phosphate-deficiency, and the study of these may lead to the production of crop and pasture plants that can utilize added P more efficiently. One adaptive mechanism is to express purple acid phosphatase (PAP) genes, the protein products of which are able to generate, transport, and recycle inorganic phosphates from phosphate-rich compounds both intracellularly and extracellularly. Their general mechanism of action is to hydrolyze phosphate-rich esters that are found within cells, the cell wall or in the rhizosphere. One PAP, AtPAP26, has been extensively characterized in Arabidopsis thaliana and displays high levels of acid phosphatase activity during phosphate-starvation. AtPAP26 has been found to be the predominantly expressed PAP during phosphate-starvation and the enzyme plays a key role in supplying inorganic phosphate to the plant by hydrolyzing the organic phosphates present in the rhizosphere. An AtPAP26-like sequence has been identified previously in white clover and so this project firstly cloned the full-length TrPAP26 and then examined expression in response to phosphate-starvation. The protein product (TrPAP26) was also characterized and compared to AtPAP26 in terms of its putative biochemical functions. TrPAP26 was predicted to be a 55 kDa protein with three N-glycosylation sites, a signal peptide of 21 amino acid residues, and a metal-ligating motif typical of PAPs. Its observed mass was closer to 45 kDa, and preliminary experiments, using recombinant TrPAP26 partially purified from transgenic tobacco, suggested that it hydrolyzed a wide range of phosphate-rich esters including adenosine triphosphate (ATP), phosphoenolpyruvate (PEP), and pyrophosphate (PPi), but not inositol hexakisphosphate (phytate). TrPAP26 transcript levels were found to be constitutive in the roots of white clover, but correlated positively with phosphate supply in other tissues. The protein and activity levels were not directly correlated with the transcript levels suggesting other methods of regulation such as post- translational modifications, including N-glycosylation. TrPAP26 accumulated more in the mature leaves of white clover plants grown with a full supply of phosphates. Taken together, these results suggest that TrPAP26 may play a role in internal P remobilization, rather than P scavenging directly.