Callose consists mostly of (1,3)-β-d-glucan and is synthesised in many plant tissues during growt... more Callose consists mostly of (1,3)-β-d-glucan and is synthesised in many plant tissues during growth and development, where it is believed to play a fundamental role in cell plate formation during cell division. Callose deposition also represents an important response to pathogen attack, wounding and to various abiotic stresses. Here, the transcription patterns of members of the callose synthase gene family from barley (Hordeum vulgare) were defined. Thus, fragments of six barley (1,3)-β-d-glucan synthase-like (GSL) cDNAs were obtained by PCR amplification using primers designed to barley expressed sequence tag (EST) sequences. The HvGSL genes, designated HvGSL2 to HvGSL7, were mapped to individual loci that were distributed across the barley genome on chromosomes 3H, 4H, 6H and 7H. The HvGSL1 gene has been isolated and characterised previously. Transcript levels for all the genes were analysed by quantitative real-time PCR in a range of barley tissues and organs, at various developmental stages. High levels of transcript for many of the HvGSL genes were detected in endosperm during the early stages of grain development, when cellularisation of the endosperm was occurring and it is likely that many of the genes participate in this process. Transcripts of HvGSL1 and HvGSL5 mRNAs were significantly more abundant than other GSL mRNAs in the roots of young seedlings, while HvGSL7 mRNA was detected at relatively high levels along the length of two week old shoots.Callose is synthesised in many plant tissues during growth and development. Callose deposition also represents an important response to pathogen attack, wounding and to various abiotic stresses. Here, the transcription patterns of members of the callose synthase gene family from barley (Hordeum vulgare) are defined.
Within the cereal grain, the endosperm and its nutrient reserves are critical for successful germ... more Within the cereal grain, the endosperm and its nutrient reserves are critical for successful germination and in the context of grain utilization. The identification of molecular determinants of early endosperm development, particularly regulators of cell division and cell wall deposition, would allow precise tailoring of grain for defined end-use properties such as yield, quality and nutritional value. Custom microarray data have been generated using RNA isolated from developing barley grain endosperm three to eight days after pollination (DAP). Comparisons of transcript abundance over time revealed 47 'gene expression modules' that can be clustered into 10 broad groups. Superimposing these modules upon cytological data allowed patterns of transcript abundance to be linked with key stages of early grain development. Here, attention was focused on how the microarray datasets could be mined to explore and define the processes of cell wall biosynthesis, remodelling and degradat...
In cereals, the presence of soluble polysaccharides including (1,3;1,4)-β-glucan has downstream i... more In cereals, the presence of soluble polysaccharides including (1,3;1,4)-β-glucan has downstream implications for human health, animal feed and biofuel applications. Sorghum bicolor (L.)Moench is a versatile crop, but there are limited reports regarding the content of such soluble polysaccharides. Here, the amount of (1,3;1,4)-β-glucan present in sorghum tissues was measured using a Megazyme assay. Very low amounts were present in the grain, ranging from 0.16%-0.27% (w/w), while there was a greater quantity in vegetative tissues at 0.12-1.71% (w/w). The fine structure of (1,3;1,4)-β-glucan, as denoted by the ratio of cellotriosyl and cellotetraosyl residues, was assessed by HPLC and ranged from 2.6-3:1 in the grain, whilst ratios in vegetative tissues were lower at 2.1-2.6:1. The distribution of (1,3;1,4)-β-glucan was examined using a specific antibody and observed with fluorescence and transmission electron microscopy. Micrographs showed a variable distribution of (1,3;1,4)-β-glucan influenced by temporal and spatial factors. The sorghum orthologues of genes implicated in the synthesis of (1,3;1,4)-β-glucan in other cereals, such as the Cellulose synthase-like (Csl) F and H gene families were defined. Transcript profiling of these genes across sorghum tissues was carried out using real-time quantitative PCR, indicating that, as in other cereals, CslF6 transcripts dominated.
Background: (1,3;1,4)-β-Glucan is an important component of the cell walls of barley grain as it ... more Background: (1,3;1,4)-β-Glucan is an important component of the cell walls of barley grain as it affects processability during the production of alcoholic beverages and has significant human health benefits when consumed above recommended threshold levels. This leads to diametrically opposed quality requirements for different applications as low levels of (1,3;1,4)-β-glucan are required for brewing and distilling and high levels for positive impacts on human health. Results: We quantified grain (1,3;1,4)-β-glucan content in a collection of 399 2-row Spring-type, and 204 2-row Winter-type elite barley cultivars originating mainly from north western Europe. We combined these data with genotypic information derived using a 9 K Illumina iSelect SNP platform and subsequently carried out a Genome Wide Association Scan (GWAS). Statistical analysis accounting for residual genetic structure within the germplasm collection allowed us to identify significant associations between molecular markers and the phenotypic data. By anchoring the regions that contain these associations to the barley genome assembly we catalogued genes underlying the associations. Based on gene annotations and transcript abundance data we identified candidate genes.
A genomic DNA fragment encoding (1→3)-β-glucan endohydrolase (EC 3.2.1.39) isoenzyme GI from barl... more A genomic DNA fragment encoding (1→3)-β-glucan endohydrolase (EC 3.2.1.39) isoenzyme GI from barley has been isolated and characterized. The gene has clearly identifiable promoter sequences and a characteristic bias in codon usage but, in contrast to other barley (1→3)-β-glucanase genes, has no introns. The amino acid sequence derived from the nucleotide sequence indicates that, unlike other plant pathogenesis-related proteins, the
Callose consists mostly of (1,3)-b-D-glucan and is synthesised in many plant tissues during growt... more Callose consists mostly of (1,3)-b-D-glucan and is synthesised in many plant tissues during growth and development, where it is believed to play a fundamental role in cell plate formation during cell division. Callose deposition also represents an important response to pathogen attack, wounding and to various abiotic stresses. Here, the transcription patterns of members of the callose synthase gene family from barley (Hordeum vulgare) were defined. Thus, fragments of six barley (1,3)-b-D-glucan synthase-like (GSL) cDNAs were obtained by PCR amplification using primers designed to barley expressed sequence tag (EST) sequences. The HvGSL genes, designated HvGSL2 to HvGSL7, were mapped to individual loci that were distributed across the barley genome on chromosomes 3H, 4H, 6H and 7H. The HvGSL1 gene has been isolated and characterised previously. Transcript levels for all the genes were analysed by quantitative real-time PCR in a range of barley tissues and organs, at various developmental stages. High levels of transcript for many of the HvGSL genes were detected in endosperm during the early stages of grain development, when cellularisation of the endosperm was occurring and it is likely that many of the genes participate in this process. Transcripts of HvGSL1 and HvGSL5 mRNAs were significantly more abundant than other GSL mRNAs in the roots of young seedlings, while HvGSL7 mRNA was detected at relatively high levels along the length of two week old shoots. Crown
An alpha-l-arabinofuranosidase and a beta-d-xylosidase, designated ARA-I and XYL, respectively, h... more An alpha-l-arabinofuranosidase and a beta-d-xylosidase, designated ARA-I and XYL, respectively, have been purified about 1,000-fold from extracts of 5-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, ion exchange chromatography, chromatofocusing, and size-exclusion chromatography. The ARA-I has an apparent molecular mass of 67 kDa and an isoelectric point of 5.5, and its catalytic efficiency during hydrolysis of 4'-nitrophenyl alpha-l-arabinofuranoside is only slightly higher than during hydrolysis of 4'-nitrophenyl beta-d-xyloside. Thus, the enzyme is actually a bifunctional alpha-l-arabinofuranosidase/beta-d-xylosidase. In contrast, the XYL enzyme, which also has an apparent molecular mass of 67 kDa and an isoelectric point of 6.7, preferentially hydrolyzes 4'-nitrophenyl beta-d-xyloside, with a catalytic efficiency approximately 30-fold higher than with 4'-nitrophenyl alpha-l-arabinofuranoside. The enzymes hydrolyze wheat flour arabinoxylan slowly but rapidly hydrolyze oligosaccharide products released from this polysaccharide by (1 --> 4)-beta-d-xylan endohydrolase. Both enzymes hydrolyze (1 --> 4)-beta-d-xylopentaose, and ARA-I can also degrade (1 --> 5)-alpha-l-arabinofuranohexaose. ARA-I and XYL cDNAs encode mature proteins of 748 amino acid residues which have calculated molecular masses of 79.2 and 80.5 kDa, respectively. Both are family 3 glycoside hydrolases. The discrepancies between the apparent molecular masses obtained for the purified enzymes and those predicted from the cDNAs are attributable to COOH-terminal processing, through which about 130 amino acid residues are removed from the primary translation product. The genes encoding the ARA-I and XYL have been mapped to chromosomes 2H and 6H, respectively. ARA-I transcripts are most abundant in young roots, young leaves, and developing grain, whereas XYL mRNA is detected in most barley tissues.
Background: Endo-(1,4)-β-glucanase (cellulase) glycosyl hydrolase GH9 enzymes have been implicate... more Background: Endo-(1,4)-β-glucanase (cellulase) glycosyl hydrolase GH9 enzymes have been implicated in several aspects of cell wall metabolism in higher plants, including cellulose biosynthesis and degradation, modification of other wall polysaccharides that contain contiguous (1,4)-β-glucosyl residues, and wall loosening during cell elongation.
A family GH5 (family 5 glycoside hydrolase) (1,4)-β-D-mannan endohydrolase or β-D-mannanase (EC 3... more A family GH5 (family 5 glycoside hydrolase) (1,4)-β-D-mannan endohydrolase or β-D-mannanase (EC 3.2.1.78), designated HvMAN1, has been purified 300-fold from extracts of 10-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, followed by ion exchange, hydrophobic interaction and size-exclusion chromatography. The purified HvMAN1 is a relatively unstable enzyme with an apparent molecular mass of 43 kDa, a pI of 7.8 and a pH optimum of 4.75. The HvMAN1 releases Man (mannose or D-mannopyranose)containing oligosaccharides of degree of polymerization 2-6 from mannans, galactomannans and glucomannans. With locust-bean galactomannan and mannopentaitol as substrates, the enzyme has K m constants of 0.16 mg · ml −1 and 5.3 mM and k cat constants of 12.9 and 3.9 s −1 respectively. Product analyses indicate that transglycosylation reactions occur during hydrolysis of (1,4)-β-Dmanno-oligosaccharides. The complete sequence of 374 amino acid residues of the mature enzyme has been deduced from the nucleotide sequence of a near full-length cDNA, and has allowed a three-dimensional model of the HvMAN1 to be constructed. The barley HvMAN1 gene is a member of a small (1,4)-β-Dmannan endohydrolase family of at least six genes, and is transcribed at low levels in a number of organs, including the developing endosperm, but also in the basal region of young roots and in leaf tips. A second barley enzyme that participates in mannan depolymerization through its ability to hydrolyse (1,4)β-D-manno-oligosaccharides to Man is a family GH1 β-Dmannosidase, now designated HvβMANNOS1, but previously identified as a β-D-glucosidase [Hrmova, MacGregor, Biely, Stewart and Fincher (1998) J. Biol. Chem. 273, 11134-11143], which hydrolyses 4NP (4-nitrophenyl) β-D-mannoside three times faster than 4NP β-D-glucoside, and has an action pattern typical of a (1,4)-β-D-mannan exohydrolase.
Following water uptake and germination of a barley grain, hydrolytic enzymes that are mainly secr... more Following water uptake and germination of a barley grain, hydrolytic enzymes that are mainly secreted from the scutellar epithelial layer and the aleurone layer, catalyse the depolymerization of starch and protein reserves in the starchy endosperm. Degradation products are ...
Background: An important step in characterising the function of a gene is identifying the cells i... more Background: An important step in characterising the function of a gene is identifying the cells in which it is expressed. Traditional methods to determine this include in situ hybridisation, gene promoter-reporter fusions or cell isolation/purification techniques followed by quantitative PCR. These methods, although frequently used, can have limitations including their time-consuming nature, limited specificity, reliance upon well-annotated promoters, high cost, and the need for specialized equipment. In situ PCR is a relatively simple and rapid method that involves the amplification of specific mRNA directly within plant tissue whilst incorporating labelled nucleotides that are subsequently detected by immunohistochemistry. Another notable advantage of this technique is that it can be used on plants that are not easily genetically transformed.
The physiological role and mechanism of nutrient storage within vacuoles of specific cell types i... more The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from Arabidopsis thaliana leaf cells differing in calcium concentration ([Ca], epidermis <10 mM versus mesophyll >60 mM) were compared using a microarray screen and single-cell quantitative PCR. Three tonoplast-localized Ca 2+ transporters, CAX1 (Ca 2+ /H + -antiporter), ACA4, and ACA11 (Ca 2+ -ATPases), were identified as preferentially expressed in Ca-rich mesophyll. Analysis of respective loss-of-function mutants demonstrated that only a mutant that lacked expression of both CAX1 and CAX3, a gene ectopically expressed in leaves upon knockout of CAX1, had reduced mesophyll [Ca]. Reduced capacity for mesophyll Ca accumulation resulted in reduced cell wall extensibility, stomatal aperture, transpiration, CO 2 assimilation, and leaf growth rate; increased transcript abundance of other Ca 2+ transporter genes; altered expression of cell wall-modifying proteins, including members of the pectinmethylesterase, expansin, cellulose synthase, and polygalacturonase families; and higher pectin concentrations and thicker cell walls. We demonstrate that these phenotypes result from altered apoplastic free [Ca 2+ ], which is threefold greater in cax1/cax3 than in wild-type plants. We establish CAX1 as a key regulator of apoplastic [Ca 2+ ] through compartmentation into mesophyll vacuoles, a mechanism essential for optimal plant function and productivity.
Callose consists mostly of (1,3)-β-d-glucan and is synthesised in many plant tissues during growt... more Callose consists mostly of (1,3)-β-d-glucan and is synthesised in many plant tissues during growth and development, where it is believed to play a fundamental role in cell plate formation during cell division. Callose deposition also represents an important response to pathogen attack, wounding and to various abiotic stresses. Here, the transcription patterns of members of the callose synthase gene family from barley (Hordeum vulgare) were defined. Thus, fragments of six barley (1,3)-β-d-glucan synthase-like (GSL) cDNAs were obtained by PCR amplification using primers designed to barley expressed sequence tag (EST) sequences. The HvGSL genes, designated HvGSL2 to HvGSL7, were mapped to individual loci that were distributed across the barley genome on chromosomes 3H, 4H, 6H and 7H. The HvGSL1 gene has been isolated and characterised previously. Transcript levels for all the genes were analysed by quantitative real-time PCR in a range of barley tissues and organs, at various developmental stages. High levels of transcript for many of the HvGSL genes were detected in endosperm during the early stages of grain development, when cellularisation of the endosperm was occurring and it is likely that many of the genes participate in this process. Transcripts of HvGSL1 and HvGSL5 mRNAs were significantly more abundant than other GSL mRNAs in the roots of young seedlings, while HvGSL7 mRNA was detected at relatively high levels along the length of two week old shoots.Callose is synthesised in many plant tissues during growth and development. Callose deposition also represents an important response to pathogen attack, wounding and to various abiotic stresses. Here, the transcription patterns of members of the callose synthase gene family from barley (Hordeum vulgare) are defined.
Within the cereal grain, the endosperm and its nutrient reserves are critical for successful germ... more Within the cereal grain, the endosperm and its nutrient reserves are critical for successful germination and in the context of grain utilization. The identification of molecular determinants of early endosperm development, particularly regulators of cell division and cell wall deposition, would allow precise tailoring of grain for defined end-use properties such as yield, quality and nutritional value. Custom microarray data have been generated using RNA isolated from developing barley grain endosperm three to eight days after pollination (DAP). Comparisons of transcript abundance over time revealed 47 'gene expression modules' that can be clustered into 10 broad groups. Superimposing these modules upon cytological data allowed patterns of transcript abundance to be linked with key stages of early grain development. Here, attention was focused on how the microarray datasets could be mined to explore and define the processes of cell wall biosynthesis, remodelling and degradat...
In cereals, the presence of soluble polysaccharides including (1,3;1,4)-β-glucan has downstream i... more In cereals, the presence of soluble polysaccharides including (1,3;1,4)-β-glucan has downstream implications for human health, animal feed and biofuel applications. Sorghum bicolor (L.)Moench is a versatile crop, but there are limited reports regarding the content of such soluble polysaccharides. Here, the amount of (1,3;1,4)-β-glucan present in sorghum tissues was measured using a Megazyme assay. Very low amounts were present in the grain, ranging from 0.16%-0.27% (w/w), while there was a greater quantity in vegetative tissues at 0.12-1.71% (w/w). The fine structure of (1,3;1,4)-β-glucan, as denoted by the ratio of cellotriosyl and cellotetraosyl residues, was assessed by HPLC and ranged from 2.6-3:1 in the grain, whilst ratios in vegetative tissues were lower at 2.1-2.6:1. The distribution of (1,3;1,4)-β-glucan was examined using a specific antibody and observed with fluorescence and transmission electron microscopy. Micrographs showed a variable distribution of (1,3;1,4)-β-glucan influenced by temporal and spatial factors. The sorghum orthologues of genes implicated in the synthesis of (1,3;1,4)-β-glucan in other cereals, such as the Cellulose synthase-like (Csl) F and H gene families were defined. Transcript profiling of these genes across sorghum tissues was carried out using real-time quantitative PCR, indicating that, as in other cereals, CslF6 transcripts dominated.
Background: (1,3;1,4)-β-Glucan is an important component of the cell walls of barley grain as it ... more Background: (1,3;1,4)-β-Glucan is an important component of the cell walls of barley grain as it affects processability during the production of alcoholic beverages and has significant human health benefits when consumed above recommended threshold levels. This leads to diametrically opposed quality requirements for different applications as low levels of (1,3;1,4)-β-glucan are required for brewing and distilling and high levels for positive impacts on human health. Results: We quantified grain (1,3;1,4)-β-glucan content in a collection of 399 2-row Spring-type, and 204 2-row Winter-type elite barley cultivars originating mainly from north western Europe. We combined these data with genotypic information derived using a 9 K Illumina iSelect SNP platform and subsequently carried out a Genome Wide Association Scan (GWAS). Statistical analysis accounting for residual genetic structure within the germplasm collection allowed us to identify significant associations between molecular markers and the phenotypic data. By anchoring the regions that contain these associations to the barley genome assembly we catalogued genes underlying the associations. Based on gene annotations and transcript abundance data we identified candidate genes.
A genomic DNA fragment encoding (1→3)-β-glucan endohydrolase (EC 3.2.1.39) isoenzyme GI from barl... more A genomic DNA fragment encoding (1→3)-β-glucan endohydrolase (EC 3.2.1.39) isoenzyme GI from barley has been isolated and characterized. The gene has clearly identifiable promoter sequences and a characteristic bias in codon usage but, in contrast to other barley (1→3)-β-glucanase genes, has no introns. The amino acid sequence derived from the nucleotide sequence indicates that, unlike other plant pathogenesis-related proteins, the
Callose consists mostly of (1,3)-b-D-glucan and is synthesised in many plant tissues during growt... more Callose consists mostly of (1,3)-b-D-glucan and is synthesised in many plant tissues during growth and development, where it is believed to play a fundamental role in cell plate formation during cell division. Callose deposition also represents an important response to pathogen attack, wounding and to various abiotic stresses. Here, the transcription patterns of members of the callose synthase gene family from barley (Hordeum vulgare) were defined. Thus, fragments of six barley (1,3)-b-D-glucan synthase-like (GSL) cDNAs were obtained by PCR amplification using primers designed to barley expressed sequence tag (EST) sequences. The HvGSL genes, designated HvGSL2 to HvGSL7, were mapped to individual loci that were distributed across the barley genome on chromosomes 3H, 4H, 6H and 7H. The HvGSL1 gene has been isolated and characterised previously. Transcript levels for all the genes were analysed by quantitative real-time PCR in a range of barley tissues and organs, at various developmental stages. High levels of transcript for many of the HvGSL genes were detected in endosperm during the early stages of grain development, when cellularisation of the endosperm was occurring and it is likely that many of the genes participate in this process. Transcripts of HvGSL1 and HvGSL5 mRNAs were significantly more abundant than other GSL mRNAs in the roots of young seedlings, while HvGSL7 mRNA was detected at relatively high levels along the length of two week old shoots. Crown
An alpha-l-arabinofuranosidase and a beta-d-xylosidase, designated ARA-I and XYL, respectively, h... more An alpha-l-arabinofuranosidase and a beta-d-xylosidase, designated ARA-I and XYL, respectively, have been purified about 1,000-fold from extracts of 5-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, ion exchange chromatography, chromatofocusing, and size-exclusion chromatography. The ARA-I has an apparent molecular mass of 67 kDa and an isoelectric point of 5.5, and its catalytic efficiency during hydrolysis of 4&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-nitrophenyl alpha-l-arabinofuranoside is only slightly higher than during hydrolysis of 4&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-nitrophenyl beta-d-xyloside. Thus, the enzyme is actually a bifunctional alpha-l-arabinofuranosidase/beta-d-xylosidase. In contrast, the XYL enzyme, which also has an apparent molecular mass of 67 kDa and an isoelectric point of 6.7, preferentially hydrolyzes 4&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-nitrophenyl beta-d-xyloside, with a catalytic efficiency approximately 30-fold higher than with 4&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-nitrophenyl alpha-l-arabinofuranoside. The enzymes hydrolyze wheat flour arabinoxylan slowly but rapidly hydrolyze oligosaccharide products released from this polysaccharide by (1 --&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 4)-beta-d-xylan endohydrolase. Both enzymes hydrolyze (1 --&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 4)-beta-d-xylopentaose, and ARA-I can also degrade (1 --&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 5)-alpha-l-arabinofuranohexaose. ARA-I and XYL cDNAs encode mature proteins of 748 amino acid residues which have calculated molecular masses of 79.2 and 80.5 kDa, respectively. Both are family 3 glycoside hydrolases. The discrepancies between the apparent molecular masses obtained for the purified enzymes and those predicted from the cDNAs are attributable to COOH-terminal processing, through which about 130 amino acid residues are removed from the primary translation product. The genes encoding the ARA-I and XYL have been mapped to chromosomes 2H and 6H, respectively. ARA-I transcripts are most abundant in young roots, young leaves, and developing grain, whereas XYL mRNA is detected in most barley tissues.
Background: Endo-(1,4)-β-glucanase (cellulase) glycosyl hydrolase GH9 enzymes have been implicate... more Background: Endo-(1,4)-β-glucanase (cellulase) glycosyl hydrolase GH9 enzymes have been implicated in several aspects of cell wall metabolism in higher plants, including cellulose biosynthesis and degradation, modification of other wall polysaccharides that contain contiguous (1,4)-β-glucosyl residues, and wall loosening during cell elongation.
A family GH5 (family 5 glycoside hydrolase) (1,4)-β-D-mannan endohydrolase or β-D-mannanase (EC 3... more A family GH5 (family 5 glycoside hydrolase) (1,4)-β-D-mannan endohydrolase or β-D-mannanase (EC 3.2.1.78), designated HvMAN1, has been purified 300-fold from extracts of 10-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, followed by ion exchange, hydrophobic interaction and size-exclusion chromatography. The purified HvMAN1 is a relatively unstable enzyme with an apparent molecular mass of 43 kDa, a pI of 7.8 and a pH optimum of 4.75. The HvMAN1 releases Man (mannose or D-mannopyranose)containing oligosaccharides of degree of polymerization 2-6 from mannans, galactomannans and glucomannans. With locust-bean galactomannan and mannopentaitol as substrates, the enzyme has K m constants of 0.16 mg · ml −1 and 5.3 mM and k cat constants of 12.9 and 3.9 s −1 respectively. Product analyses indicate that transglycosylation reactions occur during hydrolysis of (1,4)-β-Dmanno-oligosaccharides. The complete sequence of 374 amino acid residues of the mature enzyme has been deduced from the nucleotide sequence of a near full-length cDNA, and has allowed a three-dimensional model of the HvMAN1 to be constructed. The barley HvMAN1 gene is a member of a small (1,4)-β-Dmannan endohydrolase family of at least six genes, and is transcribed at low levels in a number of organs, including the developing endosperm, but also in the basal region of young roots and in leaf tips. A second barley enzyme that participates in mannan depolymerization through its ability to hydrolyse (1,4)β-D-manno-oligosaccharides to Man is a family GH1 β-Dmannosidase, now designated HvβMANNOS1, but previously identified as a β-D-glucosidase [Hrmova, MacGregor, Biely, Stewart and Fincher (1998) J. Biol. Chem. 273, 11134-11143], which hydrolyses 4NP (4-nitrophenyl) β-D-mannoside three times faster than 4NP β-D-glucoside, and has an action pattern typical of a (1,4)-β-D-mannan exohydrolase.
Following water uptake and germination of a barley grain, hydrolytic enzymes that are mainly secr... more Following water uptake and germination of a barley grain, hydrolytic enzymes that are mainly secreted from the scutellar epithelial layer and the aleurone layer, catalyse the depolymerization of starch and protein reserves in the starchy endosperm. Degradation products are ...
Background: An important step in characterising the function of a gene is identifying the cells i... more Background: An important step in characterising the function of a gene is identifying the cells in which it is expressed. Traditional methods to determine this include in situ hybridisation, gene promoter-reporter fusions or cell isolation/purification techniques followed by quantitative PCR. These methods, although frequently used, can have limitations including their time-consuming nature, limited specificity, reliance upon well-annotated promoters, high cost, and the need for specialized equipment. In situ PCR is a relatively simple and rapid method that involves the amplification of specific mRNA directly within plant tissue whilst incorporating labelled nucleotides that are subsequently detected by immunohistochemistry. Another notable advantage of this technique is that it can be used on plants that are not easily genetically transformed.
The physiological role and mechanism of nutrient storage within vacuoles of specific cell types i... more The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from Arabidopsis thaliana leaf cells differing in calcium concentration ([Ca], epidermis <10 mM versus mesophyll >60 mM) were compared using a microarray screen and single-cell quantitative PCR. Three tonoplast-localized Ca 2+ transporters, CAX1 (Ca 2+ /H + -antiporter), ACA4, and ACA11 (Ca 2+ -ATPases), were identified as preferentially expressed in Ca-rich mesophyll. Analysis of respective loss-of-function mutants demonstrated that only a mutant that lacked expression of both CAX1 and CAX3, a gene ectopically expressed in leaves upon knockout of CAX1, had reduced mesophyll [Ca]. Reduced capacity for mesophyll Ca accumulation resulted in reduced cell wall extensibility, stomatal aperture, transpiration, CO 2 assimilation, and leaf growth rate; increased transcript abundance of other Ca 2+ transporter genes; altered expression of cell wall-modifying proteins, including members of the pectinmethylesterase, expansin, cellulose synthase, and polygalacturonase families; and higher pectin concentrations and thicker cell walls. We demonstrate that these phenotypes result from altered apoplastic free [Ca 2+ ], which is threefold greater in cax1/cax3 than in wild-type plants. We establish CAX1 as a key regulator of apoplastic [Ca 2+ ] through compartmentation into mesophyll vacuoles, a mechanism essential for optimal plant function and productivity.
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Papers by Rachel Burton