SummaryThe NaGSL1 gene has been proposed to encode the callose synthase (CalS) enzyme from Nicotiana alata pollen tubes based on its similarity to fungal 1,3-b-glucan synthases and its high expression in pollen and pollen tubes. We have used a biochemical approach to link the NaGSL1 protein with CalS enzymic activity. The CalS enzyme from N. alata pollen tubes was enriched over 100-fold using membrane fractionation and product entrapment. A 220 kDa polypeptide, the correct molecular weight to be NaGSL1, was specifically detected by anti-GSL antibodies, was specifically enriched with CalS activity, and was the most abundant polypeptide in the CalS-enriched fraction. This polypeptide was positively identified as NaGSL1 using both MALDI-TOF MS and LC-ESI-MS/MS analysis of tryptic peptides. Other low-abundance polypeptides in the CalS-enriched fractions were identified by MALDI-TOF MS as deriving from a 103 kDa plasma membrane H + -ATPase and a 60 kDa b-subunit of mitochondrial ATPase, both of which were deduced to be contaminants in the productentrapped material. These analyses thus suggest that NaGSL1 is required for CalS activity, although other smaller (<30 kDa) or low-abundance proteins could also be involved.Keywords: GSL, callose synthase, product entrapment, MALDI-TOF MS, LC-ESI-MS/MS, Nicotiana alata. IntroductionPlant cells are surrounded by a wall that defines their shape and size, provides mechanical strength and support, plays a role in communication, and is the cell's primary defence against pathogens (Bacic et al., 1988). Walls and their components are also of great value to human society as sources of fuel, textiles, timber and paper. The wall is composed mostly of polysaccharide, generally with cellulosic microfibrils embedded in a matrix of non-cellulosic polysaccharides and pectic polysaccharides (Bacic et al., 1988;Carpita and Gibeaut, 1993). Some cells have more specialized walls characterized by the presence of particular polysaccharides. For example, callose, a 1,3-b-D-glucan, is abundant in the walls of pollen tubes (constituting 86% by weight of the carbohydrate in walls of Nicotiana alata pollen tubes; Li et al., 1999). Callose is also found at the cell plate, around the plasmodesmata and in sieve plates, and is deposited in response to abiotic stress, wounding or pathogen attack (Stone and Clarke, 1992).In the last decade, a number of genes putatively encoding processive polysaccharide synthase enzymes have been identified based on their similarity to microbial genes. These include the CesA genes that are now known to encode cellulose synthases (Doblin et al., 2002;Pear et al., 1996;Saxena and Brown, 2000), the cellulose synthase-like (CSL) genes, which have been suggested to encode both cellulose synthases (Doblin et al., 2001;Favery et al., 2001) and the synthases that make the backbone of various non-cellulosic matrix polysaccharides of the wall (Burton et al., 2006;Dhugga et al., 2004;Liepman et al., 2005;Richmond and Somerville, 2000;Saxena and Brown, 2000), and the glucan synthase-li...
Boron (B) phytotoxicity affects cereal-growing regions worldwide. Although B-tolerant barley (Hordeum vulgare) germplasm is available, molecules responsible for this tolerance mechanism have not been defined. We describe and use a new comparative proteomic technique, iTRAQ peptide tagging (iTRAQ), to compare the abundances of proteins from B-tolerant and -intolerant barley plants from a ‘Clipper’ × ‘Sahara’ doubled-haploid population selected on the basis of a presence or absence of two B-tolerance quantitative trait loci. iTRAQ was used to identify three enzymes involved in siderophore production (Iron Deficiency Sensitive2 [IDS2], IDS3, and a methylthio-ribose kinase) as being elevated in abundance in the B-tolerant plants. Following from this result, we report a potential link between iron, B, and the siderophore hydroxymugineic acid. We believe that this study highlights the potency of the iTRAQ approach to better understand mechanisms of abiotic stress tolerance in cereals, particularly when applied in conjunction with bulked segregant analysis.
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