1. Cellulose synthase-like (CSL)
family of genes

2. 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

3. • 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

4. 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 - 2
Subfamily J also present in some Poaceae genomes, but not rice (Fincher 2009)

5. Cellulose synthase/cellulose synthase-like
superfamily
Fincher, G. B. (2009).
Plant Physiology, 149, 27-37.

6. 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, 1821-1833
Hordeum vulgare CSLF3
• All have a D, D, D, QxxRW motif found in all processive
family GT2 proteins

7. Richmond & Somerville 2000 Plant Physiology 124, 495-498

8. • 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
– (13),(14)--glucans (present only in Poaceae & related families)
– callose [(1→3)--glucan]
– heteroxylans
– (1→4)--galactans

9. 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 (13),(14)--glucans

10. CSLA subfamily
Dhugga 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.

11. • 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

12. • 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)

13. • 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

14. 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)
Glucomannnan
Galactoglucomannan

15. • 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

16. CSLC subfamily
Cocuron 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.

17. Reserve xyloglucans in seed walls lack the fucosyl residues
found in xyloglucans in the primary walls of vegetative
organs of the same plant.
Seed xyloglucan
Primary wall
xyloglucan

18. • 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

19. CSLF, H & J subfamilies
• Genes encode enzymes involved in the synthesis of
(13),(14)--glucans (-glucans)
• In flowering plants, (13),(14)--glucans occur only in cell
walls of Poaceae and related families
• Barley grains ~4-7% (13),(14)--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

20. 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 biology
of (1→3)-β-glucans and related polysaccharides. San Diego, USA: Academic Press, Elsevier Inc. 621-654.

21. The caryopsis (C) of wheat and its pericarp (A, B).
From: Esau K (1953) Plant anatomy. John Wiley, New York, p 583.

22. • Linear polysaccharides with (13)-links (~30%) & (14)-links
(~70%)
• These two different linkages are not randomly distributed but
occur as:
cellotriosyl units:
and cellotetraosyl units:
• These units are joined by (13)-links

23. • In the grains of barley and oats, a high proportion of the
(13),(14)--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

24. CLSF subfamily
Burton et al. (2006) Science 311: 1940-1942
• Quantitative trait loci (QTL) for (13),(14)--glucan content
of grain have been identified for barley
• One QTL with a large effect on (13),(14)--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 (13),(14)--glucan synthesis
tested by expressing them in Arabidopsis (heterologous
expression)

25. • Expressed OsCSLF2 and 4 and examined transgenic
Arabidopsis by immunogold microscopy using a monoclonal
antibody that specifically recognizes (13),(14)--glucans
Epidermal walls of wild type and transgenic plants
• Found formation of (13),(14)--glucans in low
concentrations

26. 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

27. CLSH subfamily
• Only found so far in Poaceae, so is another candidate for
encoding enzymes involved in (13),(14)--glucan
synthesis
• Barley has only one gene in this subfamily
Doblin et al. (2009) PNAS 106: 5996-6001
• Expressed the HvCSLH1 in Arabidopsis and detected
(13),(14)--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

28. 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 (13),(14)--glucan
synthesis” (Fincher 2009).

29. 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)

30. Other subfamilies
• Arabidopsis, but not rice
CSLB and CSLG functions unknown
• Rice & Arabidopsis
CSLE function unknown

31. Evidence for CSL genes involvement in cell-wall
polysaccharide synthesis
– Heteromannans YES
– Xyloglucans YES
– (13),(14)--glucans (present only in Poaceae & related
families) YES
– callose [(13)--glucan] NO (other genes found)
– Heteroxylans NO (other genes found)
– (14)--galactans NO (but other genes not found so far)
– Also possibly involved (with CESA genes) in cellulose
synthesis

32. Key papers for discussion
Burton 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 the
synthesis of cell wall (1,3;1,4)-β-D-glucans. Science 311: 1940-1942
Cocuron 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-1,4
glucan synthase. Proceedings of the National Academy of Science USA 104: 8550-8555
Doblin 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 of
Science USA 106: 5996-6001
Dhugga KS, Barreiro R, Whitten B, Stecca K, Hazebroek J, Randhawa GS, Dolan M, Kinney
AJ, Tomes D, Nichols S, Anderson P (2004) Guar seed β-mannan synthase is a member of
the cellulose synthase super gene family. Science 303: 363-366
Liepman 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

33. Papers for seminars
Burton 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 gene
family in barley. Plant Physiology 146: 1821-1833
Burton 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 barley
increases the levels of cell wall (1,3;1,4)-β-D-glucans and alters their fine structure.
Plant Biotechnology Journal 9: 117-135
Davis J, Brandizzi F, Liepman A, Keegstra K (2010) Arabidopsis mannan synthase CSLA9
and glucan synthase CSLC4 have opposite orientations in the Golgi membrane. Plant
Journal 64: 1028-1037
Dwivany FM, Yulia D, Burton RA, Shirley NJ, Wilson SM, Fincher GB, Bacic A, Newbigin
E, Doblin MS (2009) The CELLULOSE-SYNTHASE LIKE C (CSLC) family of barley includes
members that are integral membrane proteins targeted to the plasma membrane.
Molecular Plant 2: 1025-1039
Liepman 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

34. 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: 1209-1218
Tonooka 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: 47-54
van Erp H, Walton JD (2009) Regulation of the cellulose synthase-like gene family by
light in the maize mesocotyl. Planta 229: 885-897
Wang 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

35. 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: 171-184
Doblin MS, Pettolino F, Bacic A (2010) Plant cell walls: the skeleton of the plant world.
Functional Plant Biology 37: 357-381
Fincher 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: 140-147
Fincher GB (2009) Revolutionary times in our understanding of cell wall biosynthesis
and remodeling in the grasses. Plant Physiology 149: 27-37

36. Other resource papers
Cutler S, Somerville C (1997) Cellulose synthesis: cloning in silico. Current Biology 7:
R108-R111
Harris 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 621-654
Hazen SP, Scott-Craig JS, Walton JD (2002) Cellulose synthase-like genes of rice. Plant
Phyiology 128: 336-340
Richmond TA, Somerville CR (2000) The cellulose synthase superfamily. Plant
Physiology 124: 495-498
Richmond TA, Somerville CR (2001) Integrative approaches to determining Csl
function. Plant Phyiology 47: 131-143

38. Families of CSL genes
Family 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

39. • 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