Glycosyltransferases• Cell-wall polysaccharides are synthesized by glycosyltransferases (GTs).• GTs are grouped into families (>90 known) based on sequence similarities, particular motifs etc (Carbohydrate Active enZymes (CAZy database).• Arabidopsis has 455 genes in 41 families that encode GTs• Catalyse transfer of glycosyl (sugar) residues from nucleotide sugars to acceptors• Some transfer only a single glycosyl residue.• Others “processive” or “polymerizing” transferases use the product of one addition as the acceptor for the next, producing a chain (polysaccharide synthases)• Specificity is in the enzyme – no template; highly specific for polymer and bond formed
• From genome sequence of Arabidopsis, Somerville identified a family of genes of unknown function with sequence similarity to cellulose synthase (Cutler & Somerville 1997; Richmond & Somerville 2000): cellulose synthase-like (CSL) genes (30 CSL + 10 CESA genes in Arabidopsis)• Six CSL gene subfamilies identified in Arabidopsis• CSL gene subfamilies identified by a letter code: A, B, C etc.• CSL gene subfamilies also identified in the genome sequence of rice (Ozya sativa) (Hazen et al. 2002), but differences.• Unlike Arabidopsis rice has no subfamily B or G• Unlike Arabidopsis rice has two additional subfamilies: F and H
Nine subfamilies of CSL genes Subfamily Arabidopsis Rice A 9 9 B 6 - C 5 6 D 6 5 E 1 3 F - 8 G 3 - H - 2Subfamily J also present in some Poaceae genomes, but not rice (Fincher 2009)
Cellulose synthase/cellulose synthase-like superfamily Fincher, G. B. (2009). Plant Physiology, 149, 27-37.
CSL genes and proteins• Cellulose synthase/cellulose synthase-like superfamily genes encode family GT2 proteins.• Type III integral membrane proteins with 3-6 predicted multiple transmembrane spanning domains towards the COOH teminus and 1-2 (usually) towards the NH2 terminus Burton et al 2008 Plant Physiology 146, 1821-1833Hordeum vulgare CSLF3• All have a D, D, D, QxxRW motif found in all processive family GT2 proteins
• When CSL genes first discovered, all known GT2 glycosyltransferases (cellulose synthases, chitin synthases etc) synthesized polysaccharides with repeating β-glycosyl residues (processive β-glycosyltransferases)Cutler & Somerville (1997) Current Biology 7: R108-R111• Postulated that the different CSL subfamilies function in the synthesis of plant cell-wall polysaccharides with backbones of repeating β-glycosyl residues: – heteromannans – xyloglucans – (13),(14)--glucans (present only in Poaceae & related families) – callose [(1→3)--glucan] – heteroxylans – (1→4)--galactans
Using seeds to discover the functions of CSL gene products• Identifying the genes encoding enzymes involved in the biosynthesis of -glycans in cell walls has been done using particular seeds.• The cotyledons or endosperms of many seeds have thick, non- lignified secondary walls.• These walls usually contain one polysaccharide that functions as a reserve and is metabolised during germination• These polysaccharides include galactomannans, xyloglucans and (13),(14)--glucans
CSLA subfamilyDhugga et al. 2004 [Science 303, 363-366]• Used seeds of guar (Cyamopsis tetragonoloba) (family Fabaceae) to investigate the genes encoding the synthesis of the backbone of galactomannans• The seeds of this and related plants in the Fabaceae have thick cell walls containing galactomannans which are used as additives (thickeners, stabilizers, emulsifiers and gelling agents) in the food industry.
• Membrane preparations from developing guar seeds showed mannan synthase (ManS) activity which used GDP-mannose as the substrate• Made 3 cDNA libraries at different stages of development• Found 15 ESTs that were similar to CESA (i.e. CSL)• Abundance of these ESTs in different libraries mirrored pattern of ManS activity
• A full-length cDNA was assembled from 12 over-lapping sequence tags• Phylogenetic analysis showed that the gene grouped with the CSLA subfamily of Arabidopsis and rice• To determine the function of the putative CtManS (CSLA) gene, they transformed it into embryogenic soybean suspension-culture cells under the control of a seed-specific promoter (heterologous expression)• The cells were allowed to develop into mature somatic embryos and membrane preparations from these showed ManS enzymic activity (normally did not) and the CtManS (CSLA) gene was expressed (Northern blots)
• Enzyme specifically used GDP-mannose and the product was hydrolysed by an endo--mannanase but not by a cellulose; acid hydrolysis of the product yielded only mannose. Indicated a (1→4)--mannan was formed.• During guar seed development, another membrane-bound enzyme, α-galactosyltransferase (a family GT34 enzyme), which uses UDP-galactose, adds α-galactosyl residues to the mannan backbone
Liepman et al. (2005) PNAS 102, 2221-2226 • Heterologously expressed Arabidopsis CSLA genes (AtCSLA2, 7, and 9) in Drosophila Schneider 2 (S2) cells • Isolated microsomal membranes from S2 cells and incubated these with GDP-mannose, which gave (1→4)--mannan • Also incubated with a mixture of GDP-mannnose and GDP- glucose and a glucomannan was produced • Glucomannans occur in lignified, secondary walls of eudicots (hard woods) and galactoglucomannans occur (in large proportions in lignified, secondary walls of coniferous gymnosperms (softwoods) GlucomannnanGalactoglucomannan
• Also heterogenously expressed AtCSLE1 and OsCSLH1 Plenty of recombinant protein was obtained but was not active in the enzyme assay.Liepman et al. (2007) Plant Phyiology 143, 1881-183• In the same way, heterogenously expressed: From the grass Oryza sativa OsCSLA1 From the gymnosperm Pinus taeda PtCSLA1 From the moss Physcomitrella patens PpCSLA1• Isolated microsomal membranes and incubated with GDP- mannose and formed (1→4)--mannans; incubated with both GDP-mannose and GDP-glucose and formed glucomannans
CSLC subfamilyCocuron et al. (2007) PNAS 104, 8550-8555• Used a similar approach to identify the gene encoding the enzyme that synthesizes the (1→4)-β-glucan backbone of xyloglucans.• Used developing nasturtium (Tropaeolum majus, family Tropaeolaceae) seeds, which at maturity have cotyledons with thick cell walls that contain reserve xyloglucans.
Reserve xyloglucans in seed walls lack the fucosyl residuesfound in xyloglucans in the primary walls of vegetativeorgans of the same plant. Seed xyloglucan Primary wall xyloglucan
• mRNA was isolated from developing seeds at the stage of maximum deposition of the xyloglucans.• A cDNA library was made and partial sequences of 10,000 cDNA clones determined.• A single CSLC gene was overrepresented in the cDNA library.• Heterologously expressed this gene in the yeast Pichia pastoris and analysed the polysaccharides.• P. pastoris usually has large amounts of (1→3)-β-glucan in its cell walls, but only insignificant amounts of (1→4)-β-glucan.• However, transgenic P. pastoris contained (1→4)-β-glucan.• Similar results were obtained with the Arabidopsis CSLC4 gene (AtCSL4), the gene with the highest sequence similarity to the TmCSLC
CSLF, H & J subfamilies• Genes encode enzymes involved in the synthesis of (13),(14)--glucans (-glucans)• In flowering plants, (13),(14)--glucans occur only in cell walls of Poaceae and related families• Barley grains ~4-7% (13),(14)--glucans Oat grains ~3-6% Wheat grains ~0.5-1.0% Barley (Hordeum vulgare)• These glucans occur particularly in cell walls of starchy endosperm and aleurone of cereal grains• Walls of starchy endosperm and aleurone in barley contain 75% and 26%, respectively
Diagram of the barley grain, showing the different organs, tissues and cell types.Reproduced from Briggs (1978) with permission.From: Harris, P.J., and Fincher, G.B. (2009). In: Bacic, A., Fincher, G.B., and Stone, B.A. eds, Chemistry, biochemistry, and biologyof (1→3)-β-glucans and related polysaccharides. San Diego, USA: Academic Press, Elsevier Inc. 621-654.
The caryopsis (C) of wheat and its pericarp (A, B). From: Esau K (1953) Plant anatomy. John Wiley, New York, p 583.
• Linear polysaccharides with (13)-links (~30%) & (14)-links (~70%)• These two different linkages are not randomly distributed but occur as: cellotriosyl units: and cellotetraosyl units:• These units are joined by (13)-links
• In the grains of barley and oats, a high proportion of the (13),(14)--glucans are water soluble• Form viscous solutions: • Cause filtration problems in brewing • Contribute to haze formation in beer • Reduce growth rate of monogastric animals (anti-nutritive effect) • But lower serum cholesterol and reduce glycaemic index• In the grains of wheat <3% water soluble
CLSF subfamilyBurton et al. (2006) Science 311: 1940-1942• Quantitative trait loci (QTL) for (13),(14)--glucan content of grain have been identified for barley• One QTL with a large effect on (13),(14)--glucan content is on barley chromosome 2H• Genetic mapping has shown genome structures of common cereals are similar (synteny)• Used comparative genomics to identify a corresponding region in the rice genome; contains a group of 6CSLF genes (CSLF1,2,3,4,8,&9)• Possible role of CSLF genes in (13),(14)--glucan synthesis tested by expressing them in Arabidopsis (heterologous expression)
• Expressed OsCSLF2 and 4 and examined transgenic Arabidopsis by immunogold microscopy using a monoclonal antibody that specifically recognizes (13),(14)--glucans Epidermal walls of wild type and transgenic plants • Found formation of (13),(14)--glucans in low concentrations
Burton et al. (2008) Plant Physiology 146: 1821-1833• Mapped CSLF genes in barley and found 4 of the 7 in the QTL locus originally identified on chromosome 2H• Spatial and temporal transcription patterns in developing grains determined; transcripts of HvCSLF6 and HvCSLF9 were predominant
CLSH subfamily• Only found so far in Poaceae, so is another candidate for encoding enzymes involved in (13),(14)--glucan synthesis• Barley has only one gene in this subfamilyDoblin et al. (2009) PNAS 106: 5996-6001• Expressed the HvCSLH1 in Arabidopsis and detected (13),(14)--glucans in the transgenic plants using immunogold microscroscopy• Examined expression of HvCSLH1 in barley: only weakly transcribed in developing grain endosperm; it was most highly transcribed in leaf tips
CLSJ subfamily• Been found in some members of Poaceae, including barley, maize, sorghum and wheat, but not in Brachypodium or rice.• “Preliminary association mapping data suggest that the HvCSLJ genes could also be involved in (13),(14)--glucan synthesis” (Fincher 2009).
CSLD subfamily• Genes of this subfamily are most similar to CESA genes• Been suggested it is involved in cellulose synthesis but the evidence is inconclusive• Expression of CSLD genes is associated with tip-growing cells• Analysis of CSLD mutants also indicated a role in tip-growing cells• Recent indications that CSLD proteins may be involved in the synthesis of heteromannans (Yin et al. 2011 Molecular Plant, In Press)
Other subfamilies• Arabidopsis, but not rice CSLB and CSLG functions unknown• Rice & Arabidopsis CSLE function unknown
Evidence for CSL genes involvement in cell-wall polysaccharide synthesis – Heteromannans YES – Xyloglucans YES – (13),(14)--glucans (present only in Poaceae & related families) YES – callose [(13)--glucan] NO (other genes found) – Heteroxylans NO (other genes found) – (14)--galactans NO (but other genes not found so far) – Also possibly involved (with CESA genes) in cellulose synthesis
Key papers for discussionBurton RA, Wilson SM, Hrmova M, Harvey AJ, Shirley NJ, Medhurst A, Stone BA,Newbigin EJ, Bacic A, Fincher GB (2006) Cellulose synthase-like CslF genes mediate thesynthesis of cell wall (1,3;1,4)-β-D-glucans. Science 311: 1940-1942Cocuron J-C, Lerouxel O, Drakakaki G, Alonso AP, Liepman AH, Keegstra K, Raikhel N,Wilkerson CG (2007) A gene from the cellulose synthase-like C family encodes a β-1,4glucan synthase. Proceedings of the National Academy of Science USA 104: 8550-8555Doblin MS, Pettolino FA, Wilson SM, Campbell R, Burton RA, Fincher GB, Newbigin E,Antony Bacic A (2009) A barley cellulose synthase-like CSLH gene mediates (1,3;1,4)-β-D-glucan synthesis in transgenic Arabidopsis. Proceedings of the National Academy ofScience USA 106: 5996-6001Dhugga KS, Barreiro R, Whitten B, Stecca K, Hazebroek J, Randhawa GS, Dolan M, KinneyAJ, Tomes D, Nichols S, Anderson P (2004) Guar seed β-mannan synthase is a member ofthe cellulose synthase super gene family. Science 303: 363-366Liepman AH, Wilkerson CG, Keegstra K (2005) Expression of cellulose synthase-like (Csl)genes in insect cells reveal that CslA family members encode mannan synthases.Proceedings of the National Academy of Science USA 102: 2221-2226
Papers for seminarsBurton RA, Jobling SA, Harvey AJ, Shirley NJ, Mather DE, Bacic A, Fincher GB (2008)The genetics and transcriptional profiles of the cellulose synthase-like HvCslF genefamily in barley. Plant Physiology 146: 1821-1833Burton RA, Collins HM, Kibble NAJ, Smith JA, Shirley NJ, Jobling SA, Henderson M,Singh RR, Pettolino F, Wilson SM, Bird AR, Topping DL, Bacic A, Fincher GB (2011) Over-expression of specific HvCslF cellulose synthase-like genes in transgenic barleyincreases the levels of cell wall (1,3;1,4)-β-D-glucans and alters their fine structure.Plant Biotechnology Journal 9: 117-135Davis J, Brandizzi F, Liepman A, Keegstra K (2010) Arabidopsis mannan synthase CSLA9and glucan synthase CSLC4 have opposite orientations in the Golgi membrane. PlantJournal 64: 1028-1037Dwivany FM, Yulia D, Burton RA, Shirley NJ, Wilson SM, Fincher GB, Bacic A, NewbiginE, Doblin MS (2009) The CELLULOSE-SYNTHASE LIKE C (CSLC) family of barley includesmembers that are integral membrane proteins targeted to the plasma membrane.Molecular Plant 2: 1025-1039Liepman AH, Nain CJ, Willats WGT, Sorensen I, Roberts AW, Keegstra K (2007)Functional genomic analysis supports conservation of function among cellulose-synthase-like A gene family members and suggests diverse roles of mannans in plants.Plant Phyiology 143: 1881-1893
Nemeth C, Freeman J, Jones HD, Sparks C, Pellny TK, Wilkinson MD, Dunwell J, Andersson AAM, Åman P, Guillon F, Saulnier L, Mitchell RAC, Shewry PR (2010) Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-β-D-glucan in endosperm of wheat. Plant Physiology 152: 1209-1218Tonooka T, Aokil E, Yoshioka T, Taketa S (2009) A novel mutant gene for (1-3, 1-4)-β-D- glucanless grain on barley (Hordeum vulgare L.) chromosome 7H. Breeding Science 59: 47-54van Erp H, Walton JD (2009) Regulation of the cellulose synthase-like gene family by light in the maize mesocotyl. Planta 229: 885-897Wang W, Wang L, Chen C, Xiong G, Tan X-Y, Yang K-Z, Wang Z-C, Zhou Y, Ye D, Chen L-Q (2011) Arabidopsis CSLD1 and CSLD4 are required for cellulose deposition and normal growth of pollen tubes. Journal of Experimental Botany (In press) (NB pdf is available from journal website)Yin L, Verhertbruggen Y, Oikawa A, Manisseri C, Knierim B, Prak L, Jensen JK, Knox JP, Auer M, Willats WGT, Scheller HV (2011) The cooperative activities of CSLD2, CSLD3, and CSLD5 are required for normal Arabidopsis development. Molecular Plant (In press) (NB pdf is available from journal website)Yin Y, Huang J, Xu Y (2009) The cellulose synthase superfamily in fully sequenced plants and algae. BMC Plant Biology 9: 99
Reviews(NB only parts of these reviews are about CSL genes and their products; you need to read selectively)Carpita NC (2011) Update on mechanisms of plant cell wall biosynthesis: how plants make cellulose and other (1→4)-β-D-glycans. Plant Physiology 155: 171-184Doblin MS, Pettolino F, Bacic A (2010) Plant cell walls: the skeleton of the plant world. Functional Plant Biology 37: 357-381Fincher GB (2009) Exploring the evolution of (1,3;1,4)-β-D-glucans in plant cell walls: comparative genomics can help! Current Opinion in Plant Biology 12: 140-147Fincher GB (2009) Revolutionary times in our understanding of cell wall biosynthesis and remodeling in the grasses. Plant Physiology 149: 27-37
Other resource papersCutler S, Somerville C (1997) Cellulose synthesis: cloning in silico. Current Biology 7: R108-R111Harris PJ, Fincher GB (2009) Distribution, fine structure and function of (1,3;1,4)-β- glucans in the grasses and other taxa. In: Bacic A, Fincher GB, Stone BA (eds) Chemistry, biochemistry, and biology of (1→3)-β-glucans and related polysaccharides. Academic Press, Elsevier Inc., San Diego, USA, pp 621-654Hazen SP, Scott-Craig JS, Walton JD (2002) Cellulose synthase-like genes of rice. Plant Phyiology 128: 336-340Richmond TA, Somerville CR (2000) The cellulose synthase superfamily. Plant Physiology 124: 495-498Richmond TA, Somerville CR (2001) Integrative approaches to determining Csl function. Plant Phyiology 47: 131-143
Families of CSL genesFamily Arabidopsis Rice Function A 9 9 Heteromannan synthesis B 6 - ? C 5 6 Synthesis of xyloglucan main chain D 6 5 Cellulose synthesis in tip- growing cells? E 1 3 ? F - 8 Synthesis of (1→3)(1→4)-β-glucans G 3 - ? H - 2 Synthesis of (1→3)(1→4)-β-glucans
• From genome sequence of Arabidopsis, Somerville identified a family of genes of unknown function with sequence similarity to cellulose synthase (Cutler & Somerville 1997; Richmond & Somerville 2000): cellulose synthase-like (CSL) genes (30 CSL + 10 CESA genes in Arabidopsis)• Postulated they encode enzymes that synthesize non- cellulosic polysaccharides.• Six CSL gene subfamilies identified in Arabidopsis and Somerville speculated each responsible for biosynthesis: Callose Xyloglucan Heteroxylan Homogalacturonan (HG) Rhamnogalacturonan I Pectic polysaccharides Rhamnogalacturonan II