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Item Low fluence UV-B as a positive regulator of photosynthesis in Arabidopsis thaliana : a thesis presented in partial fulfilment of the requirements for the degree of Doctor in Philosophy in Agriculture and Horticulture at Massey University, Palmerston North, New Zealand(Massey University, 2019) Sievers, Rixta F. T.UV-B radiation can induce a wide range of developmental responses in plants, and magnitudes of UV-B exposure can also vary greatly. Historically, research into the effects of UV-B radiation on photosynthetic processes has often utilised high fluence rates of UV-B, which have been frequently shown to impede photosynthetic performance and induce photosystem damage. More recently, a number of studies have focused on the impact of low fluence UV-B exposure, and have found that such treatments can be beneficial to photosynthesis by upregulating photosynthetic performance. The aim of this PhD was to understand the consequences of low fluence UV-B exposure on net photosynthetic rate and underlying mechanistic responses. We characterised the photosynthetic response to 0.5 μmol m⁻² s⁻¹ of UV-B and established that net photosynthetic rate increased by 12% in wild type Arabidopsis plants at 24hrs of UV-B exposure. Through analysis of knockout lines for the UV-B photoreceptor UVR8, we determined that the photosynthesis phenotype is dependent on the presence of UVR8. To determine how low fluence UV-B exposure mediates the increase in photosynthetic rate, transcriptomic analysis via RNA-seq was undertaken. Our analysis showed that UV-B exposure results in the upregulation of photosynthesis-associated genes during the initial exposure period. The most highly upregulated genes were related to chloroplast biogenesis and synthesis of photosynthetic proteins within the chloroplast, as well as chloroplastic oxidoreductase activity. We further investigated three of these candidates: RBF1, TOC33 and TFP, and found that each of those genes plays a role in the UV-B mediated increase of photosynthetic rate at 24hrs and that the upregulation of these genes in response to UV-B exposure is regulated by UVR8. Taken together, we describe here for the first time, that low fluence UV-B increases net photosynthetic rate through UVR8-mediated upregulation of key genes, resulting in increased synthesis of chloroplastic photosynthesis-associated proteins and chloroplastic oxidoreductase activity. This further extends our knowledge of UV-B plant-response and offers further potential for exploitation of UV-B photomorphogenesis in agriculture.Item Regulation of postharvest inflorescence senescence in Arabidopsis thaliana : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Palmerston North, New Zealand(Massey University, 2020) Xu, XiSenescence is critical for plant survival and fitness as it ensures the most efficient use of nutrients for development and production of offspring. Senescence is a genetically controlled and hormone-mediated programme. Besides being induced in an age-dependent manner, senescence can also be initiated precociously from harvest-induced stress such as light- and sugar-deprivation. Understanding the biological mechanisms behind dark-mediated senescence is important as it helps to provide a new strategy for extending shelf life of crop plants. This project aims to understand the regulation of dark-induced inflorescence senescence in model plant Arabidopsis thaliana by using a forward genetic approach. Arabidopsis mutants showing accelerated and delayed inflorescence senescence (named ais and dis) were identified previously. Here, I rescreened 23 mutants and confirmed the altered time to senescence phenotype of nine mutants, including two ais and seven dis mutants. Of those, the dis2, dis9, dis15 and dis51 mutants were used for further analysis. The delayed degreening phenotype was also observed in detached dark-held leaves of dis2 and dis51 mutants, indicating that the causal mutations affected genes that regulate both leaf and inflorescence senescence. Segregation analysis was used to determine the genetic nature of dis traits in the dis2 and dis51 mutants. The dis2 trait was found to be monogenic recessive while the dis51 trait is dual-genic recessive. The dis2 mutant showed an extended “stay-green” phenotype and retained higher chlorophyll (Chl) b than Chl a. These findings were consistent with a lesion in the NON-YELLOW COLORING1 (NYC1) gene. Sequencing revealed a C/T transition in exon 8 of NYC1, which caused a highly conserved proline to be substituted by serine at amino acid position 360 of the NYC1 protein. By contrast, dis51 retained a similar amount of Chl a and Chl b. One of the genetic lesions in this mutant was mapped to a ~665 kb region at the top arm of chromosome 5 by using High Resolution Melt (HRM)-based mapping technology. The ETHYLENE INSENSITIVE2 (EIN2) gene was considered as a promising candidate because similar phenotypes were observed in ein2 mutants and dis51 seedlings did not show triple response when treated with the ethylene precursor ACC in the dark. PCR-based sequencing showed a G to A mutation in exon 6 of EIN2, resulting in a premature stop codon, which thereby resulted in a truncated EIN2 protein missing part of the C-terminal region that is required for ethylene signal transduction. In addition, the dis51 mutant emitted a pleasant aroma, which is abnormal in Arabidopsis. Four compounds (benzaldehyde, benzyl alcohol, phenylacetaldehyde and phenylethanol) were detected by using GC-MS analysis. However, it is not clear if the mutation causing the aroma phenotype also contributed to the dis51 phenotype. The mutations in the dis9 and dis15 mutants were previously mapped to chromosome 3 and chromosome 2, respectively. Here, further HRM and whole genome sequencing (WGS) data analyses were used to identify the causal mutation in the dis9 mutant. The mutation changed a highly conserved Ser-97 to Phe in the active site of strigolactone (SL) receptor gene DWARF14 (D14), likely causing loss-of-activity. Since dis15 showed similar phenotypes to dis9, I hypothesised that the genetic lesion in dis15 may also have occurred in an SL pathway gene. The MORE AXIALLY GROWTH1 (MAX1) SL biosynthesis gene was present within the previously mapped region of this mutant. By using WGS data, I found a G/A mutation in the coding region of MAX1. The mutation in MAX1 substituted a highly conserved Gly-469 (G469) with Arg (R) in the haem-iron ligand signature of the Cytochrome P450 proteins. Using a N. benthamiana transient expression system, I found that the G469R substitution caused loss-of-activity of MAX1. In addition, the delayed sepal degreening of dis9 and dis15 was also observed in planta, suggesting a role of SL in regulation of both developmental and dark-induced sepal/inflorescence senescence. nCounter transcript counting technology was used to investigate the relationship between SL biosynthesis and signalling, sugar signalling and dark-induced senescence. There was no evidence of SL biosynthesis during the normal night in the inflorescences. During the extended night, the expression patterns of the SL biosynthetic gene MAX3 and signalling gene SUPPRESSOR OF MAX2-LIKE7 (SMXL7) best correlated with the sugar-responsive senescence regulatory genes [ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN92 (ANAC092) and NAC-LIKE, ACTIVATED BY AP3/PI (NAP)] and senescence marker gene (SENESCENCE-ASSOCIATED GENE12; SAG12), suggesting an interaction between SL and sugar signalling in controlling dark-induced inflorescence senescence. ANAC092 and NAP were further induced in the max1 mutant by the SL analogue, GR24, suggesting they are SL-inducible genes. The overall findings in this project reflect a complex regulatory network, which involves multiple phytohormones and degradation pathways, during dark-induced inflorescence senescence in Arabidopsis. Here, I proposed a model in which prolonged darkness first causes sugar-starvation in the excised inflorescence; the plant hormones ethylene and SL subsequently work together to regulate inflorescence senescence, including NYC1-regulated Chl degradation.Item The molecular basis of RPS4/RRS-mediated defense activation in Arabidopsis : thesis submitted to Massey University for the degree of Doctor of Philosophy(Massey University, 2017) Newman, Toby EdwardUpon pathogen invasion, each plant cell has the ability to mount an innate immune response. Plants have evolved R genes, which typically encode nucleotide-binding domain and leucine-rich repeat-containing immune receptors (NLRs). The model plant species, Arabidopsis, harbors the paired NLRs, RPS4 and RRS1, the products of which function cooperatively to confer recognition of the Pseudomonas syringae effector, AvrRps4, and the Ralstonia solanacearum effector, PopP2. The exact mechanism underlying RPS4/RRS1-mediated effector recognition remains unclear; therefore, the function of RPS4 and RRS1 was further elucidated. Firstly, by investigating the avirulence activity of natural variants of PopP2 isolated from R. solanacearum strains from across the Republic of Korea, popP2 was demonstrated to be well-conserved and RPS4/RRS1-mediated recognition of PopP2 could tolerate multiple natural polymorphisms in the popP2 sequence. Moreover, a conserved PopP2 EAR motif was identified and characterized; the EAR motif was shown to be required for in planta PopP2 stability and recognition. Secondly, utilizing suppressor of slh1 immunity (sushi) mutants generated in a forward genetic screen on slh1 mutant seeds, insight was gained into the differential requirements for RRS1 auto-activity and effector perception. A leucine-rich repeat (LRR) mutation, L816F, was identified, which affected auto-activity but not effector recognition. Furthermore, a WRKY domain mutation, C1243Y, was identified, which conferred auto-activity with distinct features compared to other known auto-active RRS1 variants. Notably, a TIR mutant harboring a C15Y mutation was identified that impaired RPS4/RRS1 TIR/TIR heterodimer formation and full-length RRS1 function. Finally, an analagous self-association interface (DE) identified in the crystal structure of the TNL, SNC1, was investigated for its role in RPS4 function. It was demonstrated that the DE interface mutations, R116A and M150R, disabled RPS4 TIR domain effector-independent cell death induction and impaired full-length RPS4 signaling.Item Identification and functional analysis of Pseudomonas syringae pv. actinidiae effector-triggered immunity in Nicotiana spp. and Arabidopsis thaliana : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Manawatu, New Zealand(Massey University, 2017) Choi, SeraPseudomonas syringae pv. actinidiae (Psa) is the causal agent of bacterial canker in commercially important cultivars of kiwifruit (Actinidia delicosa and A. chinensis) worldwide, including New Zealand. Like many gram-negative pathogens, Psa is expected to utilise type III effectors to promote virulence in host plants. In order to better understand Psa effector-triggered immunity and susceptibility, we aimed to investigate multiple molecular characteristics of Psa type III effectors and their recognition mechanisms in model plants, Nicotiana spp. and Arabidopsis thaliana. Nicotiana tabacum and N. benthamiana are widely-used model plants for Agrobacterium-mediated transient expression (agroinfiltration) of effectors for functional characterization. Firstly, we screened multiple characteristics of effectors from two Psa strains, Psa NZ V13 and Psa NZ LV5. The former is a strongly virulent and the latter is a weakly virulent strain in kiwifruit. By using agroinfiltration in Nicotiana spp. to express individual effector proteins, we observed diverse subcellular localisation for Psa effectors. Additionally, we identified multiple Psa effectors that can trigger HR-like cell death (HCD) in both N. tabacum and N. benthamiana. Using virus-induced gene silencing (VIGS), we identified that some Psa effector-triggered HCD requires the immunity regulator SGT1, suggesting that the Psa effector-triggered HCD could be a result of immunity activation. We focused on one Psa NZ V13 effector, HopZ5, which belongs to the YopJ-like acetyltransferase family. HopZ5 triggers hypersensitive response (HR) in Arabidopsis accession, Ct-1. Another Arabidopsis accession, Col-0, does not develop an HR but shows immunity in response to HopZ5. The gene that confers HopZ5-triggered HR in Ct-1 was identified as SOBER1 (SUPPRESSOR OF AVRBST-ELICITED RESISTANCE 1) by using recombinant inbred lines derived from two parental accessions, Ct-1 and Col-0. SOBER1 is a known suppressor of Xanthomonas effector AvrBsT-triggered immunity. Interestingly, AvrBsT also belongs to YopJ family. Uniquely, SOBER1 specifically suppressed HCD triggered by several YopJ-like acetyltransferase effectors in N. benthamiana, including HopZ5 and HopZ3 from Psa. This suggests a common mechanism shared between a subset of YopJ-like acetyltransferase effectors is suppressed by SOBER1. Finally, we identified one Arabidopsis accession, Ga-0, which carries a truncated SOBER1 variant but does not develop an HR upon HopZ5 delivery. Using bulked- segregant analysis of an F2 population derived from a cross between Ct-1 and Ga-0, we mapped the locus conferring HopZ5-recognition in Ct-1 to the upper arm of Chromosome 3.Item 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, RyoheiMADS-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.Item 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 A molecular genetic analysis of the requirement of TOR kinase signalling for plant growth : a thesis presented in partial fulfilment of the requirements for the degree Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand(Massey University, 2015) Rexin, DanielEukaryotes have developed a highly complex mechanism to incorporate signals from nutrient, energy, stress, developmental, and environmental cues to modulate their growth. To promote this growth, eukaryotes have to coordinate the expansion in cellular mass and size through macromolecular synthesis with the increase in cell number through division. This demands a complex orchestration of a plethora of cellular processes such as transcription, protein synthesis, metabolism and cell wall synthesis. The TARGET OF RAPAMYCIN (TOR) pathway was identified as a central integrator of this growth-regulating mechanism. Components of this pathway, including the TOR kinase and its interaction partners REGULATORY-ASSOCIATED PROTEIN OF TOR (RAPTOR) and LETHAL WITH SEC 13 PROTEIN 8 (LST8), are highly conserved among eukaryotes. This includes plants, for which the adaptation to changing environmental conditions is particularly important given their sessile lifestyle and highly plastic development. This work sought to further expand the knowledge of how TOR function was adapted to suit the requirements of plants. Therefore, I analysed genetic knock-out mutants of raptor in Arabidopsis thaliana, which resulted in a severe reduction of growth but did not cause an early developmental arrest as reported by previous studies. Detailed analysis of these mutants further revealed defects in the development of trichomes, gametophytes, and the polar extension of root hairs and pollen tubes. Potential causes for these defects were indicated by lower DNA content and limited ROS accumulation in raptor mutants. High similarities between raptor and lst8 mutants indicated that the formation of TOR complexes as found in other eukaryotes might not be functionally conserved in plants. Further, I adapted a CRE/lox system for the induction of mosaic deletions of RAPTOR, which indicated no tissue-specific requirement for RAPTOR functions within the root of A. thaliana, but demonstrated a role in the regulation of meristem size. To conclude, this data presents further evidence for an altered requirement of RAPTOR and LST8 function for TOR signalling in plants compared to fungi and animals. This thesis revealed novel functions of TOR in plant development, ROS homeostasis and endoreduplication. It further draws attention to the connection with other signalling pathways to regulate growth and development in plants.Item Modulations of visible light irradiance effects the photosynthetic phenotype in UV-B exposed Arabidopsis thaliana :|ba 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, 2015) Nelson, Briana C WPhotosynthesis is dependent upon energy provided by visible light from the electromagnetic spectrum. While such wavelengths of light are vital for resource assimilation to take place, we now also understand that other wavelengths of light may likely alter a plant’s photosynthetic capability, including the ultraviolet (UV) radiation spectrum. The ultraviolet spectrum includes UV-A (315nm-400nm) and UV-B radiation (280nm-315nm). UV-B light has been of particular interest in recent years as changes in the ozone has resulted in increased UV-B radiation levels reaching the Earth’s surface. Such scientific interest has resulted in many subsequent studies trying to understand how plants protect themselves against this powerful waveband. UV-B response in plants has been linked to both physiological and molecular changes in plants. That could be manipulated to protect plants against pathogens and increase crop yields. The quite recent discovery of the UV-B specific photoreceptor UVR8 showed how plants to respond to UV-B. A molecular pathway has begun to take shape for UVR8, with interactions with the transcription factors COP1 and HY5 necessary for activation. What is less understood are the subsequent interactions genes have with UVR8, to cause responses such as flavonoid accumulation and photosynthetic competency. After previous research showed an increase in photosynthetic rate in lettuce in response to UV-B radiation this study aimed to find the photosynthetic response of Arabidopsis thaliana and possibly re-create the increase. To do this the photosynthetic rate was studied under various PAR levels alongside UV-B exposure to characterise the photosynthetic response. The accumulation of photo-protective compounds was also studied to see if their accumulation affected photosynthetic responses. Three different lines were studied; Columbia-0, Landsberg erecta and uvr8-1. The uvr8-1 plants provided information on whether UVR8 is necessary for photosynthetic competency in Arabidopsis. qPCR studies of genes linked to the UVR8 pathway were also considered for their role in photosynthetic competency. The results in this thesis will show that manipulations of PAR, changes the UV-B photosynthetic response and that UVR8 is necessary for photosynthetic competency. ELIP1 and SIG5 are not mediated by UVR8 for photosynthetic competency. ELIP1 and SIG5 are possibly involved in UVR8 mediated accumulation of photo-protective compounds.Item Identification of genetic regulators of longevity in dark-held detached Arabidopsis inflorescences : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology, Massey University, Palmerston North, New Zealand.(Massey University, 2014) Jibran, RubinaHarvested green plant tissues experience a number of stresses including energy deprivation, water disruption, and changes in hormone levels. These stresses accelerate the senescence of the tissues, which causes their deterioration. A comprehensive understanding of how these stresses cause senescence is essential if this unwanted deterioration is to be minimised. In this thesis, I used detached dark-held immature inflorescences of Arabidopsis thaliana (Arabidopsis) to investigate the regulatory programme responsible for the senescence of harvested energy-deprived tissue. Detached dark-held Arabidopsis inflorescences completely degreened at day 5 when held in the dark at 21°C. The degreening was accelerated by exogenously applying ACC, ethrel, MeJA, and ABA that have previously been shown to accelerate senescence in detached dark-held leaves. Higher MeJA concentrations unexpectedly delayed rather than accelerated degreening of the detached dark-held inflorescences and this was associated with reductions in transcripts for the senescence-associated genes SEN4, ANAC029, NAC3, and SAG12. To identify key genetic regulators of inflorescence senescence an untargeted forward genetics approach was utilized. This involved detaching the immature inflorescences grown from ~20,000 ethyl methane-sulfonate-treated (EMS-treated) Arabidopsis (Landsberg erecta) seeds, holding them in the dark at 21°C and visually identifying those that showed a different timing of degreening to wild type. This approach successfully identified inflorescences that were completely degreened at day 3 of dark incubation (two days earlier than wild type) that were designated accelerated inflorescence senescence (ais) and inflorescences that were more green than wild type at day 5 that were designated delayed inflorescences senescence (dis). A total of 10 ais and 20 dis mutants were identified. Interestingly, most of the dis mutants were specific for inflorescence senescence as they did not show delayed senescence in detached dark-held leaves. By utilizing a traditional map-based cloning approach, five dis mutants were mapped to particular chromosomal regions. dis9 was mapped to the top arm of chromosome 3, dis15 was to the bottom of chromosome 2, and dis1, dis34, and dis58 were mapped to chromosome 4. Whole genome sequencing of dis15 and 58 identified the EMS-induced lesions as G to A transitions in the eukaryotic ASPARTYL PROTEASE (AT2G28030) and NON-CODING RNA (AT4G13495), respectively. Transformation of the AT4G13495 DNA fragment into dis58 reverted the dis58 phenotype to wild-type confirming that the non-coding RNA is involved in regulating inflorescence senescence. In addition to these fertile mutants, a sterile agamouslike mutant that had a sepal-petal-petal phenotype was identified. The mutant showed delayed degreening of detached dark-held inflorescences. This prompted me to investigate the mechanism behind the delayed senescence of the sterile homeotic ag-1 mutant. The sepals of the ag-1 inflorescences were found to have both delayed in planta and detached dark-induced senescence. They were also found to be devoid of JA and like wild-type senesced when treated with MeJA. The delayed in planta sepal senescence appeared to be due to the lack of produced JA as the dde2 mutant (defective in JA biosynthesis and devoid of JA) also showed delayed in planta sepal senescence. However, the dde2 mutant did not show delayed darkinduced senescence suggesting that the delayed dark-induced senescence of ag-1 may be through a mechanism that is unrelated to the JA hormone. Taken together, in addition to identifying common regulators of inflorescence and leaf senescence, this screen has also identified novel regulators specific to inflorescence senescence that traditional screens based on leaf senescence would have missed. This suggests that there are both similarities and differences in the genetic pathways regulating leaf and inflorescence senescence. The identification of a range of mutants, some of which appear to be novel, also indicates that the immature detached Arabidopsis inflorescences are a useful system for studying energydeprivation driven senescence. Understanding the role of the dis58 non coding RNA and the other regulators in the mutant collection offers a new and exciting opportunity for ascertaining the regulatory genetic network initiated in energy-deprived tissues that control the deterioration of harvested produce.Item Natural variation in the serially duplicated Production of Anthocyanin Pigment loci and anthocyanin accumulation in Arabidopsis thaliana (Brassicaceae) : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Plant Biology at Massey University, Palmerston North, New Zealand(Massey University, 2013) Butcher, MatthewThe TTG1-regulatory gene network regulates the development of all epidermal cell fates in Arabidopsis thaliana. Four members of the TTG1 complex, the serially duplicated R2R3-MYB PRODUCTION OF ANTHOCYANIN PIGMENT (PAP) genes, have previously been implicated in regulating the late stages of anthocyanin bionsynthesis in Arabidopsis thaliana. To study the effects of gene duplication, we sought to determine the extent of variation in each PAP gene compared to a single copy gene of the TTG1 network, WEREWOLF, using 48 naturally occurring A. thaliana accessions. It appears that the predominantly expressed PAP1 gene demonstrates a biallelic pattern, consistent with other A. thaliana genes. All four genes fall below the average nucleotide diversity levels observed across A. thaliana; however, WEREWOLF demonstrates almost complete sequence conservation across the 48 accessions used in this study. We attempted to determine the relative ages of the four PAP genes, though this does not appear to correlate with accumulation of genetic variation. To investigate the genetic architecture of anthocyanin accumulation in A. thaliana, we performed an heritability and quantitative trait loci mapping analysis using a recombinant inbred line population derived from 19 natural A. thaliana accessions. While QTL were mapped for anthocyanin accumulation near several of the PAP genes, we observed a number of loci with no obvious candidate genes, providing novel insights into the genetic architecture of anthocyanin accumulation in A. thaliana. This work contributes to a greater understanding of the roles of regulatory genes in biosynthesis and the molecular basis of regulation as well as the effects of gene duplication on nucleotide variation in the resulting genes.