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    Stress adaptation and ageing is controlled by senescence-inducing age-related changes in Arabidopsis thaliana : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2018) Kanojia, Aakansha
    Senescence is the final stage of leaf development and leads to the death of a leaf. In leaves, chloroplasts are the major source of nitrogen (75%-80%), which is found mainly in proteins. The disassembly of chloroplasts during the senescence process releases a considerable amount of nitrogen, which is then remobilized to other growing parts of the plant. Thus, nutrients from dying parts of the plants are crucial for the initial development of seeds and new plant organs. Therefore, while leaf senescence is a destructive process, efficient senescence also increases viability of the whole plant and its survival to the next season or generation. However, senescence can also be induced prematurely by abiotic stress. Early senescence caused by environmental stress can be undesirable as it may affect the growth and yield of a plant. Plants grown under abiotic stress conditions such as high salinity, drought, cold or heat, display a variety of molecular, biochemical and physiological changes. Plants under environmental stress conditions activate several signalling pathways which, in coordination with hormones such as ethylene and abscisic acid, allow for an adaptive response to stress, resulting in adjustments of plant growth and development, in an attempt to maximise survival chances. Early senescence of old leaves is one of the important strategies adapted by plants for the survival of young growing tissues. The remobilisation of nutrients from old leaves to young tissues allows survival of the whole plant under stressed conditions. However, the outcome of the stress, i.e. survival or death, depends on the strength and duration of the stress in combination with the stress response. A plant’s response to stressed conditions also depends on the age of the plant. It has been reported by multiple studies that the tolerance to stress decreases with age, however, the underlying molecular mechanisms are not well understood. In chapter 1, it is reviewed and proposed that plants of different age show distinct responses to environmental stress because of senescence-inducing age-related changes (ARCs). Research work in chapter 3 sought to understand the synchrony between ageing and reduction of plant stress tolerance, using Arabidopsis thaliana as a model plant. Transcriptomic studies were carried out to examine the occurrence of senescence-inducing ARCs in Arabidopsis first rosette early expanding leaves (EEL), mid expanding leaves (MEL) and fully expanded leaves (FEL). The transcriptomic dataset showed that, as the leaf grows, genes associated to DNA repair mechanisms are downregulated and genes linked to stress hormone biosynthesis, oxidative stress, senescence and other stress responses, are upregulated. This research confirmed that Arabidopsis young, mature and adult plants, when treated with drought, salt, and dark stresses, had greater stress sensitivity with increased age, consistent with the role of senescence-inducing ARCs in stress resistance. This study suggests that young plants are more tolerant to stress because of negligible senescence-inducing ARCs in young leaves, whereas the gradual accumulation of ARCs in mature leaves, and rapid accumulation in old leaves, results in decreased resistance to stress. Next, to characterise mutants that modulate senescence-induced ARCs, I used stress-sensitive onset of leaf death (old) mutants of Arabidopsis thaliana. The mutants were characterised based on stress responses observed in old13 and old14 mutant plants compared to the wild type (WT) (Chapter 4). The old13 mutant was selected as an appropriate mutant to study the regulatory pathway of senescence-inducing ARCs as I found that the old13 mutant plants are susceptible to stress in an age-dependent manner (Chapter 5). The transcriptomes of old13 leaves compared with the WT samples illustrate that stress susceptibility in the old13 mutant is because of early acquisition of senescence-inducing ARCs. Compared to the WT leaves, old13 showed significant downregulation of genes involved in antioxidant activity, stress tolerance, and cell-wall morphology, while genes involved in oxidative stress, senescence and stress responses were upregulated. Furthermore, transcriptional and metabolomic data illustrated that an unbalanced sugar level in old13 leaves is one of the important senescence-inducing ARCs involved in ageing and stress responses. Chapter 6 includes an attempt to identify the mutated gene in old13 using high throughput next generation sequencing. Further study on old13 gene recognition will offer an exciting opportunity to gain an in-depth knowledge of the coupling between ageing and stress responses in plants. Together, this study suggests that the occurrence of senescence-inducing ARCs is an intrinsic process integrated into the stress response and ensures certain death in plants.
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    Functional characterization of two plant type I MADS-box genes in Arabidopsis thaliana : AGL40 and AGL62 : 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, 2008) Kaji, Ryohei
    MADS-box transcription factors (TF) are a family of evolutionary conserved genes found across various eukaryotic species. Characterized by the conserved DNA binding MADS-box domain. MADS-box TF has been shown to play various roles in developmental processes. MADS-box genes can be based on MADS-box structural motifs divided into type I and type II lineages. In plants very limited functional characterization have been achieved with type I genes MADS-box genes. In this project we attempted to functionally characterize 2 closely related members of the type I lineage MADS-box genes AGL40 and AGL62 and give further support to the hypothesis that plant type I MADS-box genes are also crucial to normal plant development. Based on our expression domain characterization assay using AGL62: GUS fusion construct, we have shown expression of AGL62 in various tissues but especially strong in developing seeds, pollen and seedling roots and shoots. The web based microarray data suggesting that AGL62 may have a function in seed, pollen and seedling development backed up this result. Interestingly when we carried out PCR based genotyping with segregating population of heterozygous AGL62 T-DNA insertion lines (agl62/+) to identify the homozygous T-DNA insertion lines we detected no homozygous T-DNA insertion line indicating loss-of-function of AGL62 may be lethal to plant. With reference to the AGL62 expression in pollen, seed and seedling root and shoot, we carried out phenotypic assay on each of these tissues in agl62/+ background to investigate whether there was any phenotypic defect observed. Significant reduction in number of seeds was observed in agl62/+ indicating possible role of AGL62 in seed development. Our microscopic observation of seeds from agl62/+ plants showed defective embryos and confirmed that AGL62 plays a role in seed development. Our data on AGL62 is the first report that confirms AGL62's involvement in plant development and can be a ground work for further works on functional characterization of other members of plant type I MADS-box genes.
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    Characterization of the Arabidopsis MADS-box transcription factor, AGL104 : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2007) Reddy, Arti S
    AGL104 is an Arabidopsis MADS-box transcription factor belonging to the MIKC* clade. The exclusive expression of MIKC* genes in the gametophyte generation of both mosses and angiosperms has fueled questions regarding the function of these genes in both these taxa and the notion that the developmental program of the gametophyte generation in both these taxa may be fundamentally similar even though the structures themselves differ greatly in their phenotype. Since transcription factors control development and changes in the developmental control genes is thought to be a major source of evolutionary changes in morphology, characterization of MIKC* genes is expected to provide clues to the evolutionary changes in land plant body form. In angiosperms. AGL104 is reported to be expressed late and exclusively in the male (pollen) and female (embryo sac) gametophyte. Since late pollen development, such as pollen germination and pollen tube elongation, is thought to occur independently of transcription, the exclusive and high level of expression of a transcription factor is thus intriguing. We report the expression of AGL104 in developing anthers, mature pollen, pollen tubes and the egg apparatus of the embryo sac. Our study is the first report of AGL104 expression in the pollen tubes. Our data showing spatial expression of AGL104 in the different developmental stages of pollen, with weak expression in the uninucleate microspore that increases and culminates in the mature pollen, is also novel since spatial expression of this gene during pollen development had not been previously reported. Functional characterization through gain-of-function and loss-of-function analyses shows that AGL104 promotes pollen germination and an increased pollen tube length when measured 4 hours after pollination. The implication of this data is that, despite popular notions, active gene regulation is taking place during pollen germination and tube elongation. Further functional analysis in the pollen and the embryo sac is required to establish the precise role of AGL104 in the angiosperms. This information will then lay the groundwork for future comparisons of MIKC* activity in the basal and higher plants and determine if changes in MIKC* gene function were responsible for evolutionary changes in land plant body form.
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    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 Ann
    The 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.
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    Molecular genetic analysis of plant Mei2-like genes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2002) Alvarez, Nena de Guzman
    Molecular and genetic methods were used to analyse how a novel class of genes, plant Mei2-like genes may be involved in the regulation of morphogenesis in plants. The study specifically aimed to 1) further characterise maize te1 (the first plant Mei2-like gene to be genetically analysed) and understand the morphological basis of the te1 mutant phenotype and 2) analyse the function of Arabidopsis Terminal Ear Like (TEL) genes using expression analyses and reverse genetics strategy. te1 maize mutants are initially characterised by abnormal phytomer formation and development. A more detailed morphological analysis shows that mutant plants 1) have smaller vegetative shoot apices than the wild type, 2) initiate leaves at a higher, more distal position on the apical dome and 3) have higher plastochron ratio. Molecular analyses of kn1 expression pattern, a marker of leaf founder identity, show that dowregulation of kn1 transcripts occur higher up the dome. Clonal analyses show that fewer number of leaf founder cells are recruited to form the leaf. TEL1 and TEL2 are expressed in distinct overlapping domains in the undifferentiated region of the shoot apical meristems during the embryo, vegetative and reproductive stages of Arabidopsis development suggesting involvement of these genes in regulating meristem development and subsequent maintenance. The distinct expression of TEL1 in both the embryonic SAM and RAM raises the possibility of a unifying regulatory mechanism in the formation of the root and the shoot. The absence of TEL single knockout phenotypes supports the idea of functional redundancy. When the TEL genes were both knocked out, double mutant phenotypes show apical-basal pattern defects, ectopic production of numerous secondary shoots, production of numerous leaves and basic embryonic pattern defects such as deletions of apical and/or basal region of the seedling. Results of this study support the hypothesis that plant Mei2-like genes are important in regulating morphogenesis in plants and that they are required in the early patterning of the basic plant body.
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    Characterisation of tomato MADS-box genes involved in flower and fruit development : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2001) Ampomah-Dwamena, Charles
    MADS-box genes encode transcription factors that are involved in various aspects of plant development, by regulating target genes that control morphogenesis. Over the last decade, plant MADS-box genes have been studied extensively to reveal their control of floral development, especially in the model plants Arabidopsis and Antirrhinum. Their functions are however, not restricted to the flower but are involved in various aspects of plant development (Rounsley et al., 1995; Jack, 2001). By virtue of their extensive roles in the flower, these genes are expected to function in fruit development, which is a progression from flower morphogenesis. The aim of this study was to examine the role of MADS-box genes during flower and fruit development. Two new members of the tomato MADS-box gene family, TM10 and TM29 were identified. TM29 was isolated from a young fruit cDNA library by screening with homologous MADS-box fragments and TM10 was amplified by polymerase chain reaction from fruit cDNA templates. These genes were characterised by sequence and RNA expression patterns and their functions examined using molecular genetic techniques. Sequence analyses confirmed that both genes belong to the MADS-box family. TM29 shows 68% amino acid sequence identity to Arabidopsis SEP1 MADS-box protein. TM29 expression pattern showed similarities as well as differences to SEP1 (Flanagan and Ma. 1994). TM29 is expressed in shoot, inflorescence and floral meristems unlike SEP1, which is expressed exclusively in floral meristems (Flanagan and Ma. 1994). TM29 is expressed in all the four whorls of the flower. During floral organ development, it is highly expressed at early stages of the organ primordium but decreases as the organ differentiates and matures. In the mature flower bud, TM29 is expressed in the anther and ovary pericarp. During fruit development, TM29 is expressed from anthesis ovary to fruit of 14 days post-anthesis with its transcript localised to the pericarp and placenta. TM10 showed 64% amino acid identity to Arabidopsis AGL12. across the entire sequence. This notwithstanding, TM10 expression differed from AGL12. TM10 was expressed in shoot tissues of tomato and was not detected in roots. In contrast, the AGL12 gene transcript was only present in the roots of Arabidopsis (Rounsley et al., 1995). Expression was detected in leaves, shoot growing tips, floral buds and fruit. During fruit development, TM10 is expressed in anthesis ovary and in fruits at different growth stages. The functions of TM29 and TM10 were examined by transgenic techniques and phenotypes generated were consistent with their spatial and temporal gene expression patterns. TM29 transgenic phenotypes suggested it might be involved in the control of sympodial growth, transition to flowering, proper development of floral organs. parthenocarpic fruit development and maintenance of floral meristem identity. TM10 affected apical dominance and flowering time, development of floral organs and parthenocarpic fruit development.