Theses

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Included in the Research Theses collection are Masters, PHD and Professional Doctorate theses.
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Open Access
Evaluating populations derived from complex crosses involving both bread wheat and durum wheat parentage for partial resistance to crown rot
(2014) Deserio, Domenico; Able, Jason Alan; Mather, Diane Elizabeth; School of Agriculture, Food and Wine
Crown rot in durum, caused by Fusarium pseudograminearum and Fusarium culmorum, can reduce yields up to 90% in seasons characterised by limited spring rainfall. To decrease this potential loss, breeding of partially resistant cultivars could complement agronomic approaches. However, the limited variation in durum has meant that development of partially resistant lines is still a major objective to overcome. The aim of this study was to evaluate, through genotypic and phenotypic-based approaches, durum lines with partial resistance to crown rot. The germplasm under study consisted of 252 durum lines obtained by crossing durum parents with partially resistant bread wheat varieties. Phenotypic assessment of the symptoms, accomplished by visual assessment of the fungal necrosis of the stems, led to the identification of 120 partially resistant lines. Genotypic assessment, performed through a SNP array, identified associations between marker genotype and crown rot severity for the family originating from the parents EGA Bellaroi 38a and Sumai 3. Moreover, the frequency of QTL for crown rot partial resistance already published was investigated in the populations under study through the multiplex ready PCR technique. These findings confirm that bread wheat varieties can be exploited to reduce crown rot severity in durum.
Open Access
The genetic basis of barley black point formation.
(2008) March, Timothy; Able, Amanda Jane; Able, Jason Alan; Schultz, Carolyn Jane; School of Agriculture, Food and Wine : Plant and Food Science
Black point of barley grain refers to a discolouration of the embryo end of the grain. Historically black point has been proposed to be due to fungal colonisation of the grain. However, Koch’s postulates have yet to be satisfied. The discolouration occurs during grain fill in response to high humidity or rainfall during the grain filling period. In wheat, which is also affected by black point, the discolouration has been proposed to be due to the oxidation of phenolic acids within the grain to form discoloured end products. Within this study, two approaches were investigated in order to understand the proteins and genes associated with this disorder. Firstly, a proteomics approach enabled the identification of individual proteins associated with black point. Two-dimensional gel electrophoresis was used to compare the proteome of the husk and whole grain tissue of mature black pointed and healthy grain. Very little watersoluble protein was extracted from the husk tissue. However, a significantly larger amount of protein was extracted using a salt extraction buffer, indicating the husk proteins were mostly cell wall bound. Due to the effect of residual salt and low protein concentrations these proteins were not conducive to analysis using two-dimensional gel electrophoresis. Further experiments using acid hydrolysis of the husk tissue and subsequent amino acid analysis revealed that the proteins were bound to the husk cell walls via covalent bonds. In contrast, large quantities of protein were obtained from the whole grain samples. This allowed statistically significant comparisons to be made between gels from healthy and black pointed grains. Two proteins were identified as being more abundant in black pointed grains. Mass spectrometry identified these as isoforms of barley grain peroxidase 1 (BP1). In addition, three proteins were identified as being more abundant in healthy grain. Mass spectrometry revealed these to be isoforms of the same protein with sequence similarity to a partial EST sequence from barley. Using 3' RACE the entire coding sequence of the gene was isolated which revealed that it encoded a novel putative late embryogenesis abundant (LEA) protein. Northern blot analysis was performed for BP1 and LEA and showed that gene expression differences could not account for the differences seen in protein quantities. Western blot analysis revealed that the LEA protein was biotinylated in vivo which is consistent with similar LEA proteins from other plant species. To further understand the role of these proteins in black point, antibodies were raised against the two proteins. Subsequent immunolocalisation studies indicated BP1 was present throughout all tissues of the grain whilst LEA was most abundant in the embryo and aleurone tissue. The second major area of investigation within this thesis was to further delineate the previously identified quantitative trait loci (QTL) associated with black point in barley. Previous studies have reported QTL for black point and kernel discolouration in both barley and wheat. Comparison of the published QTL revealed a locus on the short arm of chromosome 2H to be of particular interest. To identify genes underlying this QTL the genomes of barley, wheat and rice were compared. An in silico approach showed that the QTL shared macro-synteny with rice chromosomes 4 and 7. From the rice genome sequence, barley ESTs with sequence similarity were selected. In total, 20 ESTs were selected based on two main criteria: their putative role in black point and also being evenly spread across the region of the QTL length. These QTL were mapped within the Alexis x Sloop double haploid population. This approach revealed that there was some conservation of synteny but also identified clear boundaries where synteny between barley and rice had been lost since divergence. Significantly, the additional markers mapped to this region have enabled the initial black point QTL to be reduced from approximately 30cM to 20cM. In conclusion, this study has added significant knowledge our understanding of the genetics of black point in barley through the use of two approaches. The proteomics approach has aided in understanding the biochemical processes occurring within the grain in response to black point. The comparative genetics approach has aided in understanding the genetic control of an important region of the genome influencing black point susceptibility. Combined, these findings will direct future research endeavours aimed at producing black point resistant barley cultivars.
Open Access
Investigating chromosome pairing in bread wheat using ASYNAPSIS I.
(2008) Boden, Scott Andrew; Able, Jason Alan; School of Agriculture, Food and Wine : Plant and Food Science
Pairing and synapsis of homologous chromosomes are required for normal chromosome segregation and the exchange of genetic material during meiosis. Pairing is defined as the recognition and alignment of chromosomes that occurs either pre-meiotically or during early prophase I to ensure that associations via synapsis and recombination occur only between homologues. Synapsis is the intimate juxtaposition of homologous chromosomes that is complete at pachytene following formation of a tri-partite proteinaceous structure known as the synaptonemal complex (SC). In yeast, HOP1 is an essential component of the SC that localises along chromosome axes during prophase I and promotes homologous chromosome interactions. Homologues in Arabidopsis (AtASY1), Brassica (BoASY1) and rice (OsPAIR2) have been isolated through analysis of mutants that display decreased fertility due to severely reduced synapsis of homologous chromosomes. Analysis of these genes has indicated that they play a similar role to HOP1 in pairing and formation of the SC through localisation to axial/lateral elements of the SC. In this study, we have characterised the bread wheat homologue of HOP1, TaASY1, and its encoded protein. The full length cDNA and genomic DNA clones of TaASY1 have been isolated, sequenced and characterised. TaASY1 is located on chromosome group 5 and the open reading frame displays significant similarity to OsPAIR2 (84%) and AtASY1 (63%). In addition to OsPAIR2 and AtASY1, the deduced amino acid sequence also displays sequence similarity to ScHOP1, with all four proteins containing a HORMA domain. Transcript and protein analysis showed that expression is largely restricted to meiotic tissue, with elevated levels during the stages of prophase I when pairing and synapsis of homologous chromosomes occurs. Antibodies specific to TaASY1 were used in immuno-fluorescence microscopy and immuno-gold transmission electron microscopy to investigate the localisation of TaASY1 in meiotic cells. Immuno-fluorescence analysis initially detected ASY1 in pollen mother cells (PMCs) during meiotic interphase as foci randomly distributed over the chromatin. The ASY1 signal became increasingly continuous during leptotene, reflecting the changes occurring in chromosome morphology. Throughout zygotene, the signal became progressively more continuous, localising along the entire length of the axial elements as chromosomes synapsed. This signal appeared to persist until pachytene, before disappearing from the chromatin as the SC disassociated through late pachytene and early diplotene. The immuno-gold based electron microscopy displayed that TaASY1 localises to chromatin that is associated with both axial elements before SC formation as well as chromatin of lateral elements within formed SCs. Analysis of RNAi Taasy1 mutants was performed to further define the role of ASY1 in bread wheat meiosis. ASY1 localisation was disrupted in these mutants, with a diffuse and non-continuous signal observed through leptotene and zygotene. Feulgen staining of meiotic chromosomes displayed reduced synapsis during prophase I, as well as multivalents at metaphase I and abnormal chromosome segregation during anaphase I. These observations are consistent with the presence of homoeologous chromosome interactions. TaASY1 expression and localisation was also investigated in the bread wheat pairing mutant, ph1b. Quantitative real-time PCR (Q-PCR) revealed that TaASY1 is significantly up-regulated in ph1b, with greater then 20-fold expression compared to wild-type Chinese Spring, while maintaining the same pattern of expression as wild-type through progressive stages of meiosis. ASY1 localisation was significantly disrupted in ph1b, with irregular loading on axial elements during mid to late zygotene, indicative of abnormal chromatin remodelling and multiple axial element associations that have previously been reported in ph1b. Taken together, these results indicate that TaASY1 is essential for promoting homologous chromosome interactions during meiosis, and that impairment of ASY1 function in bread wheat meiosis results in reduced restriction of chromosome associations to homologues.
Open Access
An investigation of bread wheat meiosis via proteomics and gene-targeted approaches: the isolation and characterisation of four meiotic proteins.
(2011) Khoo, Kelvin Han Ping; Able, Jason Alan; Able, Amanda Jane; School of Agriculture, Food and Wine
During the early stages of meiosis, three key processes occur: chromosome pairing, synapsis and DNA recombination. Chromosomes are first replicated during interphase, after which they are aligned together in a non-random fashion to enable the installation of the synaptonemal complex (SC) along the chromosome axes leading to synapsis. Recombination machinery then enables strand invasion to occur, which then leads to the formation of chiasmata and ultimately, genetic recombination. Meiosis is further complicated in organisms with multiple genomes such as allohexaploid bread wheat (Triticum aestivum L.) which has three genomes (inherited from similar yet distinct progenitors), each with seven chromosomes. Thus a large number of proteins are likely to be required for the successful execution of this biological process. The first approach in this study used proteomics to identify proteins that have possible roles during the early stages of wheat meiosis. Total protein samples isolated from staged meiocytes (specifically from pooled stages of pre-meiotic interphase to pachytene and from telophase I to telophase II) of wild-type Chinese Spring and the Pairing homoeologous deletion mutants, ph1b and ph2a, were analysed by 2-dimensional gel electrophoresis (2DGE). This resulted in identifying six differentially expressed protein spots (designated KK01 to KK06); from which three full-length coding sequences and one partial coding sequence of the candidate genes encoding these proteins were isolated (a putative speckle-type POZ protein, a pollen-specific SF21-like protein, a putative HSP70-like protein, as well as a partial hexose transporter peptide). Southern blot analysis revealed that these genes were spread across four different chromosome groups (2, 7, 5 and 1 respectively) with a copy on each of the three genomes (A, B and D). Q-PCR analysis of these four genes across the two pooled meiotic stages and various genotypes suggests that both KK01 and KK06 have roles during the early stages of meiosis and that they may be directly/indirectly regulated by a combination of elements within the Ph1 and Ph2 loci. The high level of KK03 mRNA transcript detected in the later stages of meiosis is consistent with its role as a pollen-specific protein-encoding gene. In contrast, KK04 expression suggests that it is post-transcriptionally regulated resulting in KK04 being translated in the ph2a mutant. Both the speckle-type POZ protein and putative dnaK/HSP70 protein were also shown to interact with DNA in vitro. The second approach of this study focused on isolating and characterising wheat homologues of two known meiotic proteins, namely PHS1 and ZYP1. In the maize PHS1 mutant Zmphs1-0, homologous chromosome pairing and synapsis are significantly affected, with homoeologous chromosome interactions occurring between multiple partners. More recently, co-immunolocalisation assays using anti-PHS1 and anti-RAD50 antibodies showed that both proteins had similar localisation patterns in the wild-type maize plants and that RAD50 localisation into the nucleus was affected by the absence of PHS1 thus implicating PHS1 as a regulator of RAD50 nuclear transport. In this study, the full-length coding transcript of wheat PHS1 (TaPHS1) was isolated, sequenced and characterised. TaPHS1 is located on chromosome group 7 with copies on the A, B and D genomes. Expression profiling of TaPHS1 in both wild-type and the ph1b mutant during and post-meiosis show elevated levels of TaPHS1 expression in the ph1b background. The TaPHS1 protein has sequence similarity to other plant PHS1/PHS1-like proteins but also possesses a unique region of oligopeptide repeat units. DNA-binding assays using both full-length and partial peptides of TaPHS1 show conclusively that TaPHS1 is able to interact with both single- and double-stranded DNA in vitro, even though no known conserved DNA-binding domain was identified within the TaPHS1 sequence, indicating TaPHS1 possesses a novel uncharacterised DNA-binding domain. Immunolocalisation data from assays conducted using an antibody raised against TaPHS1 demonstrates that TaPHS1 associates with chromatin during early meiosis, with the signal persisting beyond chromosome synapsis. Furthermore, TaPHS1 does not appear to co-localise with the asynapsis protein – TaASY1 – possibly suggesting that these proteins are independently coordinated. Combined, these results provide new insight into the potential functions of PHS1 during early meiosis in bread wheat. Similar to PHS1, Arabidopsis knock-down mutants of ZYP1 also display non-homologous chromosome interactions. ZYP1 has previously been characterised as a SC protein required for holding homologous chromosomes together in other species. In this study, the full-length coding sequence of the wheat ZYP1 (TaZYP1) homologue was isolated, sequenced and characterised. Expression of TaZYP1 analysed by Q-PCR across wild-type, ph1b and multiple Taasy1 mutants during meiosis showed an approximate 1.3-fold increase in the ph1b mutant. In addition, DNA-binding assays demonstrate that TaZYP1 interacts with dsDNA under in vitro conditions while immunolocalisation (using an anti-TaZYP1 antibody) across wild-type, ph1b and Taasy1 revealed the spatial and temporal localisation pattern of TaZYP1. Taken together, these results show that TaZYP1 plays an identical role to its homologues in other species as a SC protein and is affected by reduced levels of TaASY1 in wheat. This body of work utilised a two-pronged approach to investigate meiosis in wheat with the overall outcome of identifying new meiotic proteins as well as characterising the wheat equivalents of two known meiotic proteins previously reported in other organisms. To this end, two previously uncharacterised wheat proteins with possible roles (involving interactions with chromatin) during meiosis have been successfully identified using the proteomics approach while both TaPHS1 and TaZYP1 have been characterised with antibodies raised against both these proteins. The characterisation of TaPHS1 and its DNA-binding capabilities, both in vitro and in planta, has shed light on a previously unknown function of the PHS1 protein while the localisation profile of TaZYP1 in Taasy1 mutant lines has contributed to our understanding of how ASY1 levels can affect chromosome pairing in wheat.
Open Access
Isolation and characterisation of wheat genes with early meiotic expression.
(2010) Jolly, Hayley Rebecca; Able, Jason Alan; Milligan, Andrew Simon; School of Agriculture, Food and Wine
For all sexually reproducing organisms meiosis is an essential process. Cells undergoing meiosis go through one round of DNA replication followed by two successive rounds of chromosome segregation and cellular division, meiosis I and meiosis II. During chromosome alignment and subsequent segregation, recombination or the exchange of genetic information between homologous chromosomes takes place, which effectively maintains genetic diversity through new allelic combinations. The majority of meiotic research to date has been limited to diploids, such as yeast, mice and Arabidopsis. However, as more than 70% of all flowering plants are polyploid, exclusively using diploids could result in over simplifying the process in more complex species. A recent transcriptomics study in bread wheat conducted by Crismani and colleagues (2006) identified 1,350 transcripts which were temporally regulated during the early stages of meiosis. A number of the meiotically-regulated transcripts have annotated functions for chromatin condensation, synaptonemal complex formation, recombination and fertility; however 1,094 transcripts were deemed to have either no annotations or purely predicted annotations. The meiotic gene DMC1, previously described in several diploid species, was one of the 1,350 transcripts having meiotic regulation in wheat. Isolation of the bread wheat DMC1 homologue, and the genes encoding TaHOP2 and TaMND1 which are known to interact closely with DMC1, was successful. TaDMC1 and TaMND1 were located on chromosome group 5, while TaHOP2 was located on chromosome group 4. All three genes have significant similarity to the rice (98%, 97%, 95%, respectively) and Arabidopsis (91%, 85%, 87%, respectively) homologues. TaHOP2 and TaMND1 transcript expression was found to be highly correlated (r = 0.98) across the germline and somatic tissues examined. Using an anti-AtMND1 antibody, the TaMND1 protein localised to meiotic chromosomes during pre-meiotic (PM) interphase through to pachytene, while localisation attempts for the TaDMC1 protein with an anti-AtDMC1 antibody proved unsuccessful. The functions of the TaDMC1 and TaHOP2 proteins were found to be conserved, with these proteins having the ability to bind preferentially to single-stranded DNA. Another gene identified from the transcriptomics study, and within the 1,350 transcripts, was TaASY1 (bread wheat ASYnapsis1). While TaASY1 is known to be involved in chromosome pairing and synapsis, the mechanics of how this gene is regulated still remains somewhat unknown. To elucidate potential protein partners of the TaASY1 homologue, a yeast two-hybrid approach was conducted. Three proteins were found to interact with the full length TaASY1 protein and the HORMA domain of the ASY1 protein, along with one protein which interacted only with the isolated HORMA domain. Characterisation of two of the proteins which interact with TaASY1 revealed a Replication Protein A (RPA14) homologue and a novel plant protein. A further objective of the research conducted within this study was to isolate and characterise novel meiotic candidates to further the understanding of the process in bread wheat. From the previously identified 1,094 novel transcripts, those with either no or putative annotations underwent selective filtering. This resulted in 40 candidates being targeted for further analysis, after which eight candidates were selected for in-depth analysis. Arabidopsis homologue mutants were obtained for these candidates to determine whether a meiotic role could be assigned. Mutant plants from one candidate, AtMN29, showed altered phenotypic features that suggest this gene has a role during meiosis in Arabidopsis. Abnormalities included decreased silique length, decreased seeds per silique, decreased pollen viability and altered expression of the meiotic genes ASY1 and DMC1.
Open Access
Linkage disequilibrium analysis of hexaploid wheat (Triticum aestivum L.)
(2007) Kruger, Sherri Anne; Langridge, Peter; Able, Jason Alan; School of Agriculture, Food and Wine
There has recently been a renewed interest in using a whole-genome approach for identifying regions with relatively small effect on a particular trait of interest. One method that has proven effective in human populations is association mapping or linkage disequilibrium (LD) mapping. With focus on identifying the statistical correlations between marker allele frequency and phenotypes, association mapping, as a result, typically requires a high density marker map and a firm understanding of the extent and patterns of LD in the population. This study assesses the feasibility of applying LD mapping in hexaploid wheat research for the fine mapping of traits. Adequate marker coverage of the large wheat genome was attained providing a framework enabling the examination of the extent of LD in this species. Results presented in this thesis illustrate how extensive LD is in locally adapted populations of hexaploid wheat, extending up to I00cM in some cases. It is also apparent that statistical associations are not limited only to markers on the same chromosome but include those on different genomes and chromosome groups. One of the main focuses of this study was to evaluate the effect of genetic and evolutionary factors on the levels of statistically significant LD. Type- I error rate was successfully reduced by accounting for population structure and the presence of rare alleles in the data sets. This research has provided a base from which patterns of LD can begin to be understood in other populations and subsequently assess the applications of association mapping in inbreeding crop species, specifically Triticum aestivum L.
Open Access
Regulation of candidate genes in black point formation in barley.
(2012) Walker, K. Ryan; Able, Amanda Jane; Able, Jason Alan; Mather, Diane Elizabeth; School of Agriculture, Food and Wine
Black point of barley refers to discolouration of the embryo end of the grain. Downgrading of malting barley to feed grade due to black point results in significant economic loss to the Australian barley industry. Given that black point normally occurs in regions of Australia that experience high humidity during grain fill, humidity most probably contributes to the severity of black point in susceptible varieties. Previous studies have excluded fungal infection as a cause but enzymatic browning reaction has been recently hypothesised as responsible for black point. More specifically, a role for peroxidases has been proposed. The first major focus of this study was to confirm under what environmental conditions black point formation was likely to occur and whether there was genetic variation contributing to the phenotype. The occurrence of high humidity and low temperatures was associated with the formation of black point in susceptible varieties, with early maturing varieties being more susceptible to black point. These environmental conditions probably create a moist environment during grain development in which the developing grain cannot dry out, enabling stress or wounding to the embryo that subsequently results in black point formation. Analysis combining two South Australian sites (Hatherleigh and Port Wakefield, SA) identified QTL for black point formation on chromosomes 2H (QBpt.AlSl-2H) and 3H QBpt.AlSl-3H) at positions 83.4 cM and 102.6 cM respectively. Additive by environment effects were substantial at both QTL. Linkage of the QTL on chromosome 2H with the earliness per se (eps2) locus and the observation that early maturing varieties were usually more susceptible to black point established a probable association between earliness and black point susceptibility. When an early maturing(susceptible) variety was planted later so that it matured at the same time as a later maturing (tolerant) variety there was no significant difference in black point scores. The second focus of this study was to characterise a number of candidate genes more than likely linked to black point by investigating expression levels during grain fill and subsequently mapping the genomic regions responsible for those changes in expression. Candidate genes chosen were Quinone Reductase (HvQR), Phenylalanine Ammonia Lyase (HvPAL), Barley Peroxidase 1 (HvBP1), stress-related Peroxidase (HvPrx7) and Lipoxygenase A (HvLoxA). Differential expression as detected using northern analysis, between susceptible and tolerant varieties, was only observed for HvBP1, HvPrx7 and HvQR. Quantitative PCR (qPCR)confirmed that HvBP1 and HvPrx7 expression was up to two times higher in black point susceptible varieties during all stages of grain development, while HvQR expression was significantly higher in the hard dough and mature stages of grain fill in susceptible varieties. Increased expression for HvBP1 and HvPrx7 (approximately two-fold) was also apparent in the tolerant variety Alexis between symptomatic and asymptomatic grains. The qPCR data was then used as a quantitative trait, to score the expression of these candidate genes in an Alexis/Sloop double haploid (DH) mapping population. Areas of the genome potentially involved in the regulation of these candidates (expression QTL or eQTL) were mapped on chromosomes 2H (for HvPrx7 and HvBP1) and 5H (for HvQR and HvBP1). The eQTL for HvPrx7 and HvQR were located in the same regions as the corresponding genes, suggesting their expression is regulated via cis-acting factors. In contrast, while HvBP1 is located on 3H, eQTL were located on 2H and 5H suggesting trans-acting factors were involved. The use of comparative mapping studies between barley and rice identified a number of transcription factor genes within these eQTL. The final component of this study was to investigate how HvBP1 and HvPrx7 expression might be affected by examining their promoters and potential interactors with those promoters. Promoter regions for the susceptible variety Sloop and tolerant variety Alexis were isolated, compared and analysed for known motifs. Particular emphasis was placed on those elements that were associated with embryo and endosperm specific expression or responses to environmental stresses. Several regions containing single nucleotide polymorphisms (SNPs) between the promoters from the tolerant and susceptible varieties were identified. A 160 bp region for HvBP1 and 380 bp region for HvPrx7 were used in Yeast One Hybrid (Y1H) screening to identify potential regulatory proteins. In particular, a potential bZIP-containing factor which interacted with the promoter of HvPrx7 was further characterised. Interaction was confirmed by a gel shift assay and gene expression by northern analysis showed expression at the milk, soft dough and hard dough stages of grain development. Increased expression was apparent in the susceptible variety Sloop. The eQTL, Y1H and environmental studies have furthered our understanding of genes that could be involved in the regulation of black point formation under conditions of low temperature and high humidity. This information will contribute to assessing the roles these genes play in black point formation under certain environmental conditions, and more broadly, will assist in improving breeding for resistant barley varieties.
Open Access
Understanding the physiological mechanisms of ripening in capsicum.
(2014) Wan Kamaruddin, Wan Mohd Aizat; Able, Amanda Jane; Able, Jason Alan; Stangoulis, James Constantine Roy; School of Agriculture, Food and Wine
Capsicum (Capsicum annuum L.) is considered a non-climacteric fruit, exhibiting limited respiration and ethylene levels. The physiological mechanisms of ripening in capsicum have not been fully understood to date, especially the probable reason behind the non-climacteric behaviour. In this thesis, the protein (Chapter 3) and metabolite (Chapter 4) profiles of capsicum at different ripening stages have been reported. Several proteomic and metabolic candidates, for example ACC oxidase (ACO) enzyme, sugars (glucose, fructose, sucrose) and malate were chosen and analysed in different tissue types (peel, pulp and seeds/placenta) and cultivars with different ripening times (Chapter 5). The results suggested that some of these candidates were differentially present in different tissues and cultivars which implied that ripening could be regulated spatially and temporally. Furthermore, proteomic analysis also identified an ACO isoform 4 (CaACO4) which was found during capsicum ripening onset and corresponded to the increase in the overall ACO activity (Chapter 3). The expression of several ACO isoforms including CaACO4, and other identified ACC synthase (ACS) and Ethylene receptor (ETR) isoforms were therefore characterised to shed some light on their roles in this non-climacteric fruit (Chapter 6). CaACO4 was the only ACO isoform expressed significantly higher during capsicum ripening onset, confirming the earlier proteomic results. The expression of several ACS and ETR isoforms, normally associated with the climacteric increase of ethylene in tomato (a close relative of capsicum), was also limited as was ACS activity and ACC content. The production of ACC, as an ethylene precursor, may therefore be the rate-limiting step for ethylene production in non-climacteric capsicum. The postharvest application of ethylene did not promote capsicum ripening or induce the expression of most ACO, ACS and ETR isoforms, suggesting they are not regulated by ethylene as usually observed in climacteric fruit such as tomato. However, 1-methylcyclopropene treatment significantly delayed capsicum ripening postharvest, particularly when applied at Breaker stage (the onset of ripening), suggesting that blocking ethylene perception could affect ripening and that the basal level of ethylene normally produced in non-climacteric fruit may be (partially) required for ripening (Chapter 6). Other proteomic candidates such as Copper chaperone, TCP chaperone, Cysteine synthase and Spermidine synthase were also isolated and investigated due to their possible roles in capsicum ripening. However, unlike CaACO4, the RNA expression of these candidates did not follow their respective proteomic trends, suggesting a regulation at the post-translational level (Chapter 7). The identified candidates including CaACO4 are now a resource for further investigation to identify factors that may be involved in capsicum ripening.
Open Access
Whole genome approaches to identify genes involved in early meiosis.
(2009) Crismani, Wayne Matthew; Able, Jason Alan; School of Agriculture, Food and Wine : Plant and Food Science
Meiosis is a process which occurs in sexually reproducing organisms to halve the genetic complement prior to fertilisation. During meiosis a single round of DNA replication is followed by two successive rounds of chromosome segregation and cell division. The meiotic pathway in plants is complex from multiple perspectives. From a mechanical view; prior to the first meiotic division the chromosomes must replicate during meiotic interphase, then while retaining sister chromatid cohesion the homologous chromosomes must align, physically synapse and also concomitantly recombine (with the majority of sites being non-randomly positioned). Further complexities arise in allopolyploids such as bread wheat, which contains three very similar genomes from slightly diverged progenitors. Despite having homoeologous chromosomes present in the same nucleus, bread wheat displays diploid-like behaviour during meiosis I. Such an involved physical process as meiosis also has complexity reflected in the transcriptome and proteome, whether the organism be a simple eukaryote such as yeast, or a more complex eukaryote such as bread wheat. Initially, this study utilised whole genome approaches to identify novel genes that could be involved in early meiosis, focusing on bread wheat in particular. Analysis of the wheat meiotic transcriptome over seven stages of anther development identified at least 1,350 transcripts which displayed meiotic regulation. The expression profiles of a subset of selected transcripts were analysed with Q-PCR and found to correlate strongly to those obtained in the microarray. Available meiotic transcriptome data from rice was compared to the wheat data, which enabled the identification of similar sequences, many previously unidentified, which also displayed meiotic regulation. Selected candidate genes from the microarray study were also mapped in bread wheat. This data was combined with available literature and approximately 70% of candidate meiotic loci were located on chromosome group 3 or 5, which historically has been shown to contain multiple loci involved in chromosome pairing control. One of the candidates located on chromosome group 3, a plant-specific mismatch repair gene, Triticum aestivum MSH7 (TaMSH7), has previously been speculated to suppress homoeologous chromosome associations. Independent transgenic wheat plants produced using RNA interference (RNAi) were functionally characterised to ascertain a greater understanding of the role TaMSH7 has during early meiosis in bread wheat. Localisation of a synaptonemal complex-associated protein (TaASY1) displayed subtle abnormalities in these mutants when compared to wild-type. Feulgen staining of meiotic chromosomes at metaphase I in these mutants revealed some interlocking and multivalent associations. These results suggest that TaMSH7 may be linked to the mechanism underlying the phenotype that is observed in the ph2a/ph2b mutant, however further research still needs to be conducted to conclusively demonstrate that this is the case. A component of the research presented in this study was performed in the model plant Arabidopsis thaliana due to the limitations of bread wheat. Extensive mutant banks and a sequenced genome have aided a decade of meiotic research in Arabidopsis and the identification of close to 50 meiotic genes. One of these, AtMER3, has been shown to control the non-random location of well above half of the recombination events that occur in many species. AtMER3 was localised in meiotic nuclei in wild-type Arabidopsis and found to form foci on freshly synapsed regions of chromosomes in quantities far in excess of the average number of crossovers, indicating that AtMER3 does not localise exclusively to sites of crossovers. AtMER3 localisation was also analysed in several mutant backgrounds and found to act in an AtSPO11-dependent manner. However, AtMER3 loading onto meiotic chromosomes was not affected in Atrad51, Atdmc1 or Atmsh5 mutant backgrounds.

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