Science Collected Works - Theses
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ItemSelf-incompatibility in Raphanus raphanistrum (Wild Radish) : Insights and Applications(University of Melbourne, 2013)Wild radish (Raphanus raphanistrum) is a self-incompatible (SI) winter annual and is a major crop weed in Australia, being present in all Australian states and territories except the Northern Territory. Control of wild radish costs an estimated A$210 million per annum and the species has recently overtaken ryegrass as the worst weed to control in Western Australia (WA), with 93% of the WA populations resistant to one or more herbicides. There is thus an urgent need for herbicide-independent control measures that can target wild radish reproduction. This thesis aims to evaluate the potential of the self-incompatibility (SI) system of R. raphanistrum as a reproductive target and uses two approaches to activate the SI response in a constitutive manner: the first uses SP11 (S-locus protein 11) molecules and the second uses stigmatic ROS (reactive oxygen species) inducers. Chapter 1 is a literature review and surveys current understanding of the SI response, with a particular focus on the Brassicaceae which are the emphasis of this thesis. The SI response is controlled by a multiallellic locus, the S-locus, and pollen rejection or acceptance is determined by the interaction of the products of two tightly-linked S-locus genes: SP11, encoding a pollen-expressed protein ligand and SRK, encoding a receptor kinase expressed by the stigma. Binding of SP11 to its cognate SRK causes an allele-specific activation of the SI response resulting in rejection of self-pollen. SP11 thus has the potential to inhibit reproduction in R. raphanistrum in a specific and non-toxic manner. However, to act as a reproductive blocker, the allele-specific action of SP11 requires that the number of S-alleles present in the Australian R. raphanistrum populations to be low - information that is currently lacking. The chapter concludes with a statement of the aims of this thesis. Chapter 2 describes the identification and characterisation of S-alleles in a wild R. raphanistrum population (ML8) from WA (Western Australia). A diallel was used to demonstrate the presence of a robust SI system and compatibility relationships in the population were determined. SRK S-domain and kinase domain sequences were amplified from genomic DNA: in particular, S-domains for three S-alleles were isolated and physically linked to their respective kinase domains. SP11 sequences were amplified from floral bud RNA and their relationship to SRKs determined. Based on SRK kinase domain sequences and a theoretical estimation, a total of 30 S-alleles was predicted to be present in the entire Australian R. raphanistrum population - a number that is considered low and renders the SP11 molecule a viable reproductive blocker candidate. Chapter 3 reports further work on characterizing the gene products of SP11 and SRK, with particular emphasis on the interaction between these two SI determinants. Recombinant SP11 produced in E. coli was able to prevent the germination of otherwise compatible pollen on R. raphanistrum stigmas in an allele-specific manner. A recombinant version of SRK that was missing the kinase domain (eSRK) was transiently expressed in tobacco (Nicotiana benthamiana) leaves and the hypervariable sub-domain (HVR) of eSRK was produced in E. coli. However, none of the recombinant eSRK or HVR interacted with SP11 in pull-down and dot-blot assays. Chapter 4 describes the effect of ROS on the SI response in R. raphanistrum. Chemicals known to induce or inhibit ROS production were used to manipulate ROS levels in the stigma. Treatment with sodium pyruvate, ascorbic acid and menandione bisulphite led to >8-fold increase in ROS stigmatic levels. Stigmas with elevated ROS levels were able to reject compatible pollen in a manner that appeared identical to the rejection of incompatible pollen. The reasons why increased stigmatic ROS levels should lead to pollen rejection are discussed. This thesis concludes with a summary of key experimental findings in light of current understanding of SI and suggests potential avenues for further research. A discussion of an integrated weed management approach incorporating reproductive blockers described in this thesis with existing control strategies is also presented.
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ItemMutations affecting self-incompatibility in the genus Nicotiana(University of Melbourne, 1997)Fertilisation in flowering plants begins with a pollen grain bearing the male gametes landing on the female stigma. Several mechanisms enable the stigma and style to discriminate between the different types of pollen that it may receive, the best studied being self-incompatibility. If a pollen grain from a self-incompatible plant lands on its own stigma or on the stigma of a genetically related plant, the pollen will either fail to germinate or will germinate to produce a pollen tube that grows poorly in the style and does not reach the ovary. In many cases, this process is controlled by a single, multi-allelic locus called the S-locus. In Nicotiana alata (ornamental tobacco), the product of the S-Iocus in the pistil is an extracellular ribonuclease (S-RNase) but the product of the S-locus in pollen has not been identified. Mutations affecting the pollen component of the N. alata S-locus were generated using gamma (?) rays. Pollen mother cells in developing flower buds were irradiated. Pollen was collected from the irradiated flowers at anthesis and used to pollinate styles of the same S-genotype. Only those pollen grains with mutations that affected their S-phenotype would be expected to grow through such a pistil. Several seeds formed following these pollinations and the plants that grew from these seeds were self-compatible. Breeding experiments indicated that the pollen component of the S-locus, or the pollen and style components, had been affected in each plant. Plants in which only the pollen component had been affected were called pollen-part mutants (PPMs). Cytological analysis of some PPM plants found the genome contained an extra chromosome. The genomes of the other PPM plants had no extra chromosomes. The extra chromosome was smaller in size than other N. alata chromosomes and appeared to have a central constriction, indicative of a centromere. These chromosomes have previously been called centric fragments. The inheritance of the pollen-part mutation in five plants was followed in families obtained by either self-pollinating the mutated plant, backcrossing it to an unmutated plant of the same S-genotype or outcrossing it to an unmutated plant of a different S-genotype. Characterisation of the progeny in the selfed, backcrossed and outcrossed families showed that all PPM plants contained a duplicated S-allele. The duplicated S-allele was present on a centric fragment in three plants, but had been integrated into the genome of the remaining PPM plants. DNA blot analysis showed that only some of the duplications had an S-RNase gene, a second genetic marker of the N. alata S-locus, 48A, was present on all the duplications. Each duplication, therefore, included either part of the S-locus (only the 48A gene present) or an entire S-locus (both the 48A and S-RNase genes present). It is assumed the pollen component of the S-locus was also present on the duplicated fragments, and that this gene(s) is adjacent to the 48A. A breakdown of a pollen's self-incompatibility phenotype occurred when the duplicated S-allele, or the duplicated part of an S-allele, was different from the allele present at the S-locus. Observations made in this study support an "inhibitor" model of self-incompatibility. In this model, the pollen component of the S-locus encodes an inhibitor that inactivates S-RNases entering the pollen tube. The inhibitor does not inactivate S-RNases encoded by a matching S-allele and, consequently, these are able to degrade the pollen's tube rRNA. The reduced capacity to synthesise proteins will inhibit pollen tube growth. The breakdown of self-incompatibility in the pollen-part mutants occurs because some pollen tubes contain inhibitors encoded by two, non-identical S-alleles. The two types of inhibitors can inactivate all S-RNases and will allow the pollen tube to grow through an otherwise incompatible style. A second part of the thesis looks at the role mutations at the S-locus may have played in the evolution of self-compatibility in the genus Nicotiana. A survey of stylar ribonuclease activity found uniformly high levels in all self-incompatible species and low levels in most self-compatible species. One of the self-compatible species surveyed, N. sylvestris, had a level of stylar ribonuclease activity comparable to that in the styles of self-incompatible species, however. A ribonuclease was purified from the N. sylvestris styles and sequenced, and a cDNA corresponding to this protein was cloned from a stylar library. The sequence of the N. sylvestris ribonuclease could be aligned with those of the solanaceous S-RNases, suggesting an evolutionary relationship. Several features of the N. sylvestris RNase gene, including its copy number and the site at which carbohydrate is attached to the encoded protein, are discussed in the context of the origin of self-compatibility in this species.
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ItemRNases expressed in pollen and pistils of Nicotiana alata and Lycopersicon peruvianum : analysis of their role in self-incompatibility(University of Melbourne, 1996)Self-incompatibility in the Solanaceae is controlled by a single multi- allelic locus, the S-locus. Pollen expressing the same S-allele as the pistil on which it germinates is unable to grow through the pistil and fertilise the ovules. The products of the S-locus in the pistil are extracellular glycoproteins with ribonuclease activity, known as S-RNases. However, the product of the S-locus in pollen, and the nature of the recognition process between the pollen product and the S-RNase is not understood. In this thesis I show that S-RNases are expressed after meiosis by the developing pollen of at least two self-incompatible species from the Solanaceae; Nicotiana alata, an ornamental tobacco, and Lycopersicon peruvianum, a wild tomato. Initially I used RNA gel blots to show that transcripts hybridising to S-RNase probes were present in immature anthers. I then used in situ hybridisation to show these transcripts accumulated in the developing pollen grains but not the surrounding sporophytic tissues. In N. alata, I used a polyclonal antibody that specifically recognises the S2-RNase to show that a protein detected by this antibody was in S2 pollen within the inner layer of the in tine and close to the plasma membrane. Together, the timing of expression and cellular location of S-RNases suggested a role for these proteins in determining the self-incompatibility phenotype of pollen. I tested this hypothesis by transforming L. peruvianum plants with constructs that contained either a sense or an antisense version of the S3- RNase gene from L. peruvianum. Expression of these constructs was controlled by the promoter of the pollen-specific gene, LAT52. As a control, I used a construct in which the GUS reporter gene was fused to the LAT52 promoter. In plants transformed with the GUS reporter gene, GUS activity was detected only in developing anthers and mature pollen. Thus, this promoter should cause the sense or antisense versions of the S3-RNase to accumulate in post-meiotic pollen. Plants transformed with the sense construct produced the S3-RNase protein in pollen, and this protein was localised to the plasma membrane. These pollen were viable, but were not rejected by an S3 pistil. Twenty-eight plants transformed with the antisense construct expressed the S3-RNase antisense transcript. Only one plant was self-compatible, but analysis of the inheritance of the antisense construct in progeny of this plant showed that the construct was not the cause of self-compatible phenotype. Together, these results show that S-RNase expression in pollen is neither sufficient nor necessary for self-incompatibility. I therefore concluded that the solanaceous S-locus includes at least one other gene that is expressed in pollen and determines the self-incompatibility phenotype of the pollen. I characterised a cDNA encoding an S-like RNase (RNase NE) from N. alata that is 86% identical in sequence to an extracellular RNase from tomato cell cultures, RNase LE. RNase NE is expressed in styles, petals and immature anthers of N. alata under normal growth conditions, but is not expressed in the vegetative tissues. Under phosphate-limited conditions, RNase NE expression is induced in roots but not leaves. A transcript hybridising to RNase NE is also induced in N. plumbaginifolia cell cultures in response to phosphate starvation. RNase NE is likely to play a role in the response of N. alata to phosphate limitation, possibly by scavenging phosphate from sources of RNA in the root environment. DNA hybridisation experiments indicate that there are approximately 5-6 sequences related to RNase NE in the N. alata genome and that RNase NE is not linked to the S-locus. Cross-hybridising genes were found in the genomes of other species in the Solanaceae and the evolutionary relationships between the S- and S-like RNase genes are discussed. In addition, while characterising the intron of RNase NE, I discovered a novel short interspersed repetitive element (SINE) that was also present in four other Nicotiana genes from the DNA database. This SINE, which I designated TS2, is highly repeated in the genomes of several Nicotiana species and at one Solanum species, but not was not detected in DNA from any other solanaceous genera examined. Many SINEs are related in sequence to tRNAs and, like these SINEs, TS2 contains sequence motifs characteristic of RNA polymerase III promoters. The sequence of TS2 however does not have sequence similarity to any known tRNA.