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Overexpression of ferulate 5-hydroxylase increases syringyl units in Sorghum bicolor

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Abstract

Ferulate 5-hydroxylase (F5H) of the monolignol pathway catalyzes the hydroxylation of coniferyl alcohol, coniferaldehyde and ferulic acid to produce 5-hydroxyconiferyl moieties, which lead to the formation of sinapic acid and syringyl (S) lignin monomers. In contrast, guaiacyl (G) lignin, the other major type of lignin monomer, is derived from polymerization of coniferyl alcohol. In this study, the effects of manipulating S-lignin biosynthesis in sorghum (Sorghum bicolor) were evaluated. Overexpression of sorghum F5H (SbF5H), under the control of the CaMV 35S promoter, increased both S-lignin levels and the ratio of S/G lignin, while plant growth and development remained relatively unaffected. Maüle staining of stalk and leaf midrib sections from SbF5H overexpression lines indicated that the lignin composition was altered. Ectopic expression of SbF5H did not affect the gene expression of other monolignol pathway genes. In addition, brown midrib 12-ref (bmr12-ref), a nonsense mutation in the sorghum caffeic acid O-methyltransferase (COMT) was combined with 35S::SbF5H through cross-pollination to examine effects on lignin synthesis. The stover composition from bmr12 35S::SbF5H plants more closely resembled bmr12 stover than 35S::SbF5H or wild-type (WT) stover; S-lignin and total lignin concentrations were decreased relative to WT or 35S::SbF5H. Likewise, expression of upstream monolignol biosynthetic genes was increased in both bmr12 and bmr12 35S::SbF5H relative to WT or 35S::SbF5H. Overall, these results indicated that overexpression of SbF5H did not compensate for the loss of COMT activity.

Key message

Overexpression of F5H in sorghum increases S-lignin without increasing total lignin content or affecting plant growth, but it cannot compensate for the loss of COMT activity in monolignol synthesis.

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Acknowledgements

We thank John Toy, Coehn Preble, Samantha Timmons and Sara Finnegan for technical assistance, and Dr. Heather Van Buskirk for critically reviewing the manuscript. We thank the Plant Transformation Core Research Facility at the University of Nebraska for creating the overexpression lines. This research was supported by the United States Department of Agriculture: National Institute of Food and Agriculture AFRI Grant Number 2011-67009-30026 (SES and DLF-H) and additional funding from USDA-ARS, CRIS Projects 3042-21220-033-00-D (SES and DLF-H). The University of Nebraska DNA Sequencing Core receives partial support from the National Institute for General Medical Science (NIGMS) INBRE-P20GM103427-14 and COBRE-1P30GM110768-01 Grants as well as The Fred and Pamela Buffett Cancer Center Support Grant: P30CA036727. This publication’s contents are the sole responsibility of the authors and do not necessarily represent the official views of the NIH or NIGMS. The US Department of Agriculture, Agricultural Research Service, is an equal opportunity/affirmative action employer and all agency services are available without discrimination. Mention of commercial products and organizations in this manuscript is solely to provide specific information. It does not constitute endorsement by USDA-ARS over other products and organizations not mentioned.

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HMT, SES and DLFH designed the research; HMT, TG, NAP, SS and ZG performed the experiments; HMT, TG, NAP, DLFH, GS and SES analyzed and interpreted the data; HMT and SES wrote the first draft of the manuscript, and all authors reviewed and revised the manuscript prior to publication.

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Correspondence to Scott E. Sattler.

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Tetreault, H.M., Gries, T., Palmer, N.A. et al. Overexpression of ferulate 5-hydroxylase increases syringyl units in Sorghum bicolor. Plant Mol Biol 103, 269–285 (2020). https://doi.org/10.1007/s11103-020-00991-3

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