Science Collected Works - Theses
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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.
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ItemCharacterization of arabinogalactan-proteins (AGPs) from the pistil of Nicotiana alata(University of Melbourne, 1995)Arabinogalactan-proteins (AGPs) are common components of most plant tissues, plant secretions, and suspension cultured plant cells. AGPs are a family of proteoglycans that consist of mainly carbohydrate (usually >90% w/w) rich in galactose (-60%) and arabinose (-30%) residues and a protein backbone rich in hydroxyproline (Hyp), Ala, and Ser. Various physiological functions have been proposed for the AGPs, such as maintaining water balance in the plant, providing nutrients for growing pollen tubes, responding to pathogen infection, and cell-cell recognition. More recently, membrane bound AGPs have also been implicated as determinants of cellular identity. Although the structure of the carbohydrate moiety of the AGPs is relatively well studied, little is known about the protein backbone, partly because of the difficulties in the separation of individual AGPs and subsequent cloning of the genes encoding these protein backbones. This in turn has hindered our understanding of the role that AGPs play in plant growth and development. Using a combination of ion-exchange and gel filtration chromatography, a major group of buffer-soluble AGPs have been isolated from the pistils of Nicotiana alata. Carbohydrate was removed by anhydrous hydrogen fluoride and the deglycosylated AGP protein backbones were fractionated by reversed-phase HPLC into three major fractions; an unbound fraction, and two fractions which eluted from the column with retention times of 25 min (RT25) and 30-40 min (RT35). Amino acid analysis of the two bound fractions showed that the RT25 backbone was "typical" of AGPs in that it contained mainly Hyp, Ala, and Ser, while the RT35 fraction was "atypical" of AGPs and was rich in Asx, Glx, and Ala. Internal amino acid sequences were obtained from the RT25 protein backbone after protease digestion. Gene-specific oligonucleotides were designed according to the peptide sequence and polymerase chain reaction (PCR) was carried out. The PCR product was then used to screen a style cDNA library, and a cDNA clone, AGPNa1, was obtained (Du et al., 1994). The AGPNa1 cDNA (712 bp) encodes a protein of 132 amino acid residues and is consistent with our concept of a "classical" AGP dominated by Hyp/Pro, Ala, and Ser residues. The predicted AGP protein backbone contains an N-terminal secretion sequence, which agrees with the known extracellular location of the pistil AGPs. The middle part of the deduced backbone is rich in Pro, Ala, and Ser. The abundance of hydroxyl amino acid residues is consistent with the extensive O- glycosylation of the AGPs. The C-terminus of the protein is very hydrophobic. Whether this hydrophobic tail serves as a membrane anchor or is proteolytically removed in vivo is unclear. The AGPNa1 is a single-copy gene and is expressed in all plant tissues examined, which suggests that it may have a general role in plant growth and development. Using a similar strategy, internal amino acid sequences were obtained from the RT35 protein backbone and a cDNA, AGPNa3, was isolated (Du et al., 1995). The AGPNa3 cDNA (762 bp) encodes a protein of 169 amino acid residues with three domains, an N-terminal signal sequence, a central Pro-rich domain, and a C- terminal Cys-rich domain. The AGPNa3 thus encodes an AGP that is "non- classical" in that it is rich in Asx and Glx but relatively poor in Hyp. AGPNa3 is also a single-copy gene. Northern blot analysis showed that the AGPNa3 gene is only expressed in the mature pistils of N. alata but not in other tissues. Within the pistil, the AGPNa3 gene is primarily expressed in the stigma, indicating a possible role in the pollination process. To further investigate the function of AGPs, transgenic plants with sense or antisense constructs containing the AGPNa1 cDNA were generated. As expected, the sense plants produced higher amounts of the AGPNa1 transcript in the pistil than the control plants; one of the antisense plants had significantly reduced expression of AGPNa1. However, all transgenic plants grew normally and were not noticeably different from untransformed plants. Future transformation experiments to generate transgenic plants containing the antisense AGPNa3 gene and double knockout plants with reduced expression of both AGPNa1 and AGPNa3 gene will be valuable in revealing further the functions of AGPs.