Genetic screening for genes in Arabidopsis thaliana that ...€¦ · abundant and high-quality...
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Co-expression analysis of monolignol biosynthetic genes Genes involved in common processes may be transcriptionally coordinated and have similar expression profiles. Our study shows known monolignol biosynthetic genes, such as C3H, 4CL1 and CCR1, have common dominant expression in the developing xylem of Arabidopsis roots where the lignification process occurs (Fig. 1A-1C). Public availability of many abundant and high-quality microarray expression profile data sets and well developed co- expression tools (4) makes it possible to retrieve candidate genes which have a potential function in the lignification processes of Arabidopsis. In order to retrieve the potential candidates involved in the lignification process, we performed a co-expression analysis with known monolignol biosynthetic genes by three co- expression tools: ACT (5), CressExpress (6) and tool developed by Ray Wightman to probe a root microarray dataset (7). In total, 255 genes were retrieved and some were common to different datasets (Fig. 2A); 61 of them showed a dominant expression in developing xylem and were chosen to check for any potential relation with lignin biosynthesis and metabolism. Genetic screening for genes in Arabidopsis thaliana that improve sugar release from plant lignocellulose Christopher McClellan*, Yuguo Xiao, Jason Kam, Abdellah Barakate, Michael Skelly, Kirsty Graham, Dominic Grussu, Gordon Simpson, and Claire Halpin Division of Plant Sciences, University of Dundee, Dundee, United Kingdom Fig.1 - Graphical representation of HCT, CCR1, and 4CL1 expression in Arabidopsis root Stage 1 closest to the root tip; stage 3 furthest from the root tip. Cell layers in the root indicated are lateral root cap (S1and S9), endodermis and cortex (S3 and S7) and stele (S5). (A) At1g15950 (CCR1); (B) At5g48930 (HCT); (C) At1g51680 (4CL1) A B C Expression patterns of monolignol biosynthesis genes ACT CressExpress Ray 61 18 101 4 11 6 54 Preliminary co-expression analysis results A. B C Fig. 2 - Venn diagram of co-expressed genes shared among three gene expression lists and histochemical detection of lignin in loss-of-function lines A. Overview of genes co-expressed with monolignol biosynthetic genes B. Mäule reagent-stained cross sections of inflorescence stems from six-week old wild-type Arabidopsis C. Mäule reagent-stained cross sections of inflorescence stems from six-week old mutant line Gb We have requested T-DNA insertion lines from 61 candidate genes and have identified a homozygous line showing reduced S-lignin accumulation in mature inflorescence stem (Fig. 2B, 2C). Further analysis on these homozygous lines is in progress. REFERENCES 1. Jones, L., et al. (2001) Plant J 26: 205-16. 2. Derikvand, M.M., et al. (2008) Planta 227: 943-56. 3. Hatfield, R.D., et al. (1999) J Agric Food Chem 47: 628-32. 4. Usadel, B., et al. (2009) Plant Cell Environ 32: 1633-51. 5. Manfield, I.W., et al. (2006) Nucleic Acids Res 34: W504-9. 6. Srinivasasainagendra, V., et al. (2008) Plant Physiol 147: 1004-16. 7. Birnbaum, K., et al. (2003) Science 302: 1956–1960 INTRODUCTION Lignin is an abundant polymer in plants that, together with cellulose and hemicellulose, provides the structural framework of the plant cell wall. However, in the production of cellulosic ethanol, lignin limits the availability of cellulose to degrading enzymes, decreasing the amount of ethanol obtained from plant material. The enzymatic pathway for the production of monolignols has been characterised, yet little is known about how monolignols are transported, regulated, or polymerised into lignin. We hope to identify new genes involved in lignification, or genes that improve the yield of sugars from plant cell walls, through a genetic screen. Using both forward and reverse genetics, we are working to identify genotypes that are more useful in the production of bioethanol. Genetic screens of monolignol biosynthesis mutants In Arabidopsis thaliana, mutants for several monolignol biosynthetic enzymes have been characterised, including CCR (1, 2). However, the effects of these mutations have differing effects on plant growth and development. The ccr1 mutant has significant defects in lignin content, but has reduced stature and biomass, which reduces the usefulness of any gain in saccharification properties (1,2). Therefore, we performed a suppressor screen in the ccr1 mutant of Arabidopsis thaliana for plants that exhibited increased size. Col-0 ccr1-3 73-3 Col-0 ccr1-3 81-1 Col-0 ccr1-3 81-2 Potential mutants Fig. 1 – Three potential mutants isolated from an EMS-mutagenised population Plants were grown for six weeks under long day conditions (16 h light/ 8 h dark) and photographed. Potential mutants are in the ccr1-3 background. Suppressor mutants still exhibit lignin defective phenotypes Col-0 ccr1-3 73-3 81-2 Fig. 2 – Mäule staining of Arabidopsis stems. Stem sections from six-week old plants stained with Mäule (KMnO 4 :HCl:NH 4 OH). Potential mutants 73-3 and 81-2 still display the irregular xylem (irx) phenotype seen in ccr1-3. 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 Col-0 ccr1-3 73-3 81-1 81-2 Acetyl Bromide Lignin Values Fig. 3 – Acetyl bromide lignin composition assay Dry whole stem samples were ground to powder and three biological replicates per genotype were used for acetyl bromide lignin assays, based on the procedure in Hatfield et al., 1999 (3). Currently, additional mutants are being characterised for lignification phenotypes. Current mutants will also be tested for saccharification properties. Mutants of interest will be used for map-based cloning to identify genes of interest. ACKNOWLEDGEMENTS We would like to thank collaborators Wout Boerjan (University of Gent) for supply of the ccr1-3 genotype, and for sharing lignin mutant data, and Raymond Wightman from Simon Turner’s lab (University of Manchester) for a tool for analysing Arabidopsis root microarray data.
Transcript of Genetic screening for genes in Arabidopsis thaliana that ...€¦ · abundant and high-quality...
Co-expression analysis of monolignol biosynthetic genesGenes involved in common processes may be transcriptionally coordinated and have similar expression profiles. Our study shows known monolignol biosynthetic genes, such as C3H, 4CL1 and CCR1, have common dominant expression in the developing xylem of Arabidopsisroots where the lignification process occurs (Fig. 1A-1C). Public availability of many abundant and high-quality microarray expression profile data sets and well developed co-expression tools (4) makes it possible to retrieve candidate genes which have a potential function in the lignification processes of Arabidopsis. In order to retrieve the potential candidates involved in the lignification process, we performed a co-expression analysis with known monolignol biosynthetic genes by three co-expression tools: ACT (5), CressExpress (6) and tool developed by Ray Wightman to probe a root microarray dataset (7). In total, 255 genes were retrieved and some were common to different datasets (Fig. 2A); 61 of them showed a dominant expression in developing xylem and were chosen to check for any potential relation with lignin biosynthesis and metabolism.
Genetic screening for genes in Arabidopsis thaliana that improve sugar release from plant lignocellulose
Christopher McClellan*, Yuguo Xiao, Jason Kam, Abdellah Barakate, Michael Skelly, Kirsty Graham, Dominic Grussu, Gordon Simpson, and Claire Halpin
Division of Plant Sciences, University of Dundee, Dundee, United Kingdom
Fig.1 - Graphical representation of HCT, CCR1, and 4CL1 expression in Arabidopsis rootStage 1 closest to the root tip; stage 3 furthest from the root tip. Cell layers in the root indicated are lateral root cap (S1and S9), endodermis and cortex (S3 and S7) and stele (S5). (A) At1g15950 (CCR1); (B) At5g48930 (HCT); (C) At1g51680 (4CL1)
A B CExpression patterns of monolignol biosynthesis genes
ACT CressExpress
Ray
61 18 101
411
6
54
Preliminary co-expression analysis resultsA.
B C
Fig. 2 - Venn diagram of co-expressed genes shared among three gene expression lists and histochemical detection of lignin in loss-of-function lines
A. Overview of genes co-expressed with monolignol biosynthetic genesB. Mäule reagent-stained cross sections of inflorescence stems from six-week old wild-type ArabidopsisC. Mäule reagent-stained cross sections of inflorescence stems from six-week old mutant line Gb
We have requested T-DNA insertion lines from 61 candidate genes and have identified a homozygous line showing reduced S-lignin accumulation in mature inflorescence stem (Fig. 2B, 2C). Further analysis on these homozygous lines is in progress.
REFERENCES1. Jones, L., et al. (2001) Plant J 26: 205-16.2. Derikvand, M.M., et al. (2008) Planta 227: 943-56.3. Hatfield, R.D., et al. (1999) J Agric Food Chem 47: 628-32.4. Usadel, B., et al. (2009) Plant Cell Environ 32: 1633-51.5. Manfield, I.W., et al. (2006) Nucleic Acids Res 34: W504-9.6. Srinivasasainagendra, V., et al. (2008) Plant Physiol 147: 1004-16.7. Birnbaum, K., et al. (2003) Science 302: 1956–1960
INTRODUCTION
Lignin is an abundant polymer in plants that, together with cellulose and hemicellulose, provides the structural framework of the plant cell wall. However, in the production of cellulosic ethanol, lignin limits the availability of cellulose to degrading enzymes, decreasing the amount of ethanol obtained from plant material. The enzymatic pathway for the production of monolignols has been characterised, yet little is known about how monolignols are transported, regulated, or polymerised into lignin. We hope to identify new genes involved in lignification, or genes that improve the yield of sugars from plant cell walls, through a genetic screen. Using both forward and reverse genetics, we are working to identify genotypes that are more useful in the production of bioethanol.
Genetic screens of monolignol biosynthesis mutantsIn Arabidopsis thaliana, mutants for several monolignol biosynthetic enzymes have been characterised, including CCR (1, 2). However, the effects of these mutations have differing effects on plant growth and development. The ccr1 mutant has significant defects in lignin content, but has reduced stature and biomass, which reduces the usefulness of any gain in saccharification properties (1,2). Therefore, we performed a suppressor screen in the ccr1mutant of Arabidopsis thaliana for plants that exhibited increased size.
Col-0 ccr1-3 73-3 Col-0 ccr1-3 81-1 Col-0 ccr1-3 81-2
Potential mutants
Fig. 1 – Three potential mutants isolated from an EMS-mutagenised populationPlants were grown for six weeks under long day conditions (16 h light/ 8 h dark) and photographed. Potential mutants are in the ccr1-3 background.
Suppressor mutants still exhibit lignin defective phenotypes
Col-0 ccr1-3 73-3 81-2
Fig. 2 – Mäule staining of Arabidopsis stems.Stem sections from six-week old plants stained with Mäule (KMnO4:HCl:NH4OH). Potential mutants 73-3 and 81-2 still display the irregular xylem (irx) phenotype seen in ccr1-3.
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Col-0 ccr1-3 73-3 81-1 81-2
Acetyl Bromide Lignin Values
Fig. 3 – Acetyl bromide lignin composition assay
Dry whole stem samples were ground to powder and three biological replicates per genotype were used for acetyl bromide lignin assays, based on the procedure in Hatfield et al., 1999 (3).
Currently, additional mutants are being characterised for lignification phenotypes. Current mutants will also be tested for saccharification properties. Mutants of interest will be used for map-based cloning to identify genes of interest.
ACKNOWLEDGEMENTS
We would like to thank collaborators Wout Boerjan (University of Gent) for supply of the ccr1-3 genotype, and for sharing lignin mutant data, and Raymond Wightman from Simon Turner’s lab (University of Manchester) for a tool for analysing Arabidopsis root microarray data.
