TALEN mediated targeted mutagenesis of the caffeic acid O-methyltransferase in highly polyploid sugarcane improves cell wall composition for production of bioethanol.
Sugarcane (Saccharum spp. hybrids) is a prime crop for commercial biofuel production. Advanced conversion technology utilizes both, sucrose accumulating in sugarcane stems as well as cell wall bound sugars for commercial ethanol production. Reduction of lignin content significantly improves the conversion of lignocellulosic biomass into ethanol. Conventional mutagenesis is not expected to confer reduction in lignin content in sugarcane due to its high polyploidy (x = 10-13) and functional redundancy among homo(eo)logs. Here we deploy transcription activator-like effector nuclease (TALEN) to induce mutations in a highly conserved region of the caffeic acid O-methyltransferase (COMT) of sugarcane. Capillary electrophoresis (CE) was validated by pyrosequencing as reliable and inexpensive high throughput method for identification and quantitative characterization of TALEN mediated mutations. Targeted COMT mutations were identified by CE in up to 74 % of the lines. In different events 8-99 % of the wild type COMT were converted to mutant COMT as revealed by pyrosequencing. Mutation frequencies among mutant lines were positively correlated to lignin reduction. Events with a mutation frequency of 99 % displayed a 29-32 % reduction of the lignin content compared to non-transgenic controls along with significantly reduced S subunit content and elevated hemicellulose content. CE analysis displayed similar peak patterns between primary COMT mutants and their vegetative progenies suggesting that TALEN mediated mutations were faithfully transmitted to vegetative progenies. This is the first report on genome editing in sugarcane. The findings demonstrate that targeted mutagenesis can improve cell wall characteristics for production of lignocellulosic ethanol in crops with highly complex genomes.
Agronomy Department, University of Florida, IFAS, PO Box 110300, Gainesville, FL, 32611, USA.|Institute of Life Science and Natural Resources, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, Republic of Korea.
Je Hyeong
JH
Jung
Agronomy Department, University of Florida, IFAS, PO Box 110300, Gainesville, FL, 32611, USA. altpeter@ufl.edu.|Plant Molecular and Cellular Biology Program, University of Florida, IFAS, PO Box 110300, Gainesville, FL, 32611, USA. altpeter@ufl.edu.|Agronomy Department, University of Florida-IFAS, PO Box 103610, Gainesville, FL, 32611, USA. altpeter@ufl.edu.
Fredy
http://orcid.org/0000-0002-0894-4976
F
Altpeter
D002473:Cell Wall / Q000235:genetics / Q000378:metabolism*; D019075:Electrophoresis, Capillary; D018506:Gene Expression Regulation, Plant; D008031:Lignin / Q000235:genetics / Q000378:metabolism*; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D031786:Saccharum / Q000201:enzymology* / Q000378:metabolism; D000069896:Transcription Activator-Like Effector Nucleases / Q000235:genetics / Q000378:metabolism*
D010940:Plant Proteins; D008031:Lignin; D008780:Methyltransferases; C019608:caffeate O-methyltransferase; D000069896:Transcription Activator-Like Effector Nucleases
Caffeic acid O-methyltransferase; Capillary electrophoresis; Genome editing; Lignin; Sugarcane; Transcription activator-like effector nucleases