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Title: Expression of brown-midrib in a spontaneous sorghum mutant is linked to a 5'-UTR deletion in lignin biosynthesis gene SbCAD2

Author
item LI, HUANG - Oklahoma State University
item Huang, Yinghua

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/4/2017
Publication Date: 9/15/2017
Citation: Li, H., Huang, Y. 2017. Expression of brown-midrib in a spontaneous sorghum mutant is linked to a 5'-UTR deletion in lignin biosynthesis gene SbCAD2. Scientific Reports. 7:11664. https://www.nature.com/articles/s41598-017-10119-1#Abs1.

Interpretive Summary: Plant biomass (i.e. lignocellulosic materials) has great potential for use as a feedstock for bioethanol production. But challenges with respect to processing steps in converting biomass to liquid transportation fuel like pretreatment, hydrolysis, microbial fermentation, and separation still exist. Lignin is a major component of plant biomass and negatively affects the saccharification and bioethanol production. This study focused on characterization of the gene that encodes a key enzyme for lignin biosynthesis in plants. Genetic analysis of sorghum bmr6 mutants revealed that mutations in a cinnamyl alcohol dehydrogenase gene (SbCAD2) disrupt the last step of the lignin biosynthesis pathway, resulting in a significant reduction of lignin abundance in biomass tissues and the concomitant enhancement of saccharification efficiency. Here we report that a novel SbCAD2 allele was discovered in this study, which has an 8-bp deletion in its 5'-untranslated region (UTR), thus conferring the spontaneous brown midrib trait and lignin deficiency in sorghum germplasm line PI595743. Moreover, the result of the molecular experiments indicated that the SbCAD2 promoter was functionally conserved in terms of driving a specific expression pattern in lignifying vascular tissues. In summary, the findings from this study will be beneficial to design preferred lignocellulosic feedstocks for bio-ethanol processing to meet the industry's need for cost-effective biofuel production.

Technical Abstract: Brown midrib (bmr) mutants in sorghum (Sorghum bicolor (L.) Moench) and several other C4 grasses are associated with reduced lignin concentration, altered lignin composition and improved cell wall digestibility, which are desirable properties in biomass development for the emerging lignocellulosic biofuel industry. Studying bmr mutants has considerably expanded the understanding of the molecular basis underlying lignin biosynthesis and perturbation in grasses. For example, characterization of sorghum bmr6 mutants revealed that mutations in a cinnamyl alcohol dehydrogenase gene (SbCAD2) disrupt the last step of lignin biosynthesis, resulting in a significant reduction of lignin abundance and the concomitant enhancement of saccharification efficiency. In this study, we identified a novel SbCAD2 allele that has an 8-bp deletion in its 5'-untranslated region (UTR) conferring the spontaneous brown midrib trait and lignin deficiency in the sorghum germplasm line PI595743. Using a recombinant inbred line (RIL) population, a major QTL harboring the SbCAD2 was delimited to a 474 kb region on the short arm of chromosome 4. Complementation test and DNA sequencing revealed that mutations in PI595743 and bmr6 mutations line are two different alleles in the same gene SbCAD2. qRT-PCR results confirmed that the SbCAD2 expression in PI595743 was significantly reduced throughout its growth stages relative to the non-bmr line BTx623. Moreover, promoter-GUS fusion study in transgenic Arabidopsis thaliana plants found that the SbCAD2 promoter was functionally conserved in terms of driving a specific expression pattern in lignifying vascular tissues. This study provides new insights to investigate the regulatory mechanism underlying the natural occurrence of bmr traits in sorghum and other grass species.