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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.