Lignin characterization of rice CONIFERALDEHYDE 5‐HYDROXYLASE loss‐of‐function mutants generated with the CRISPR/Cas9 system. (18th December 2018)

• Record Type: Journal Article
• Authors: Takeda, Yuri; Suzuki, Shiro; Tobimatsu, Yuki; Osakabe, Keishi; Osakabe, Yuriko; Ragamustari, Safendrri K.; Sakamoto, Masahiro; Umezawa, Toshiaki
• Is Part Of: Plant journal. Volume 97:Number 3(2019)
• Page Start: 543
• Page End: 554
• Publication Date: 2018-12-18
• Subjects: lignocellulose -- genome editing -- Oryza sativa -- S/G ratio -- grass -- p‐coumarate -- NMR -- DFRC; Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.14141
• Languages: English
• ISSNs: 0960-7412

Abstract

Summary: The aromatic composition of lignin is an important trait that greatly affects the usability of lignocellulosic biomass. We previously identified a rice ( Oryza sativa ) gene encoding coniferaldehyde 5‐hydroxylase (OsCAld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized OsCAld5H1 ‐knockdown rice lines, which were produced via an RNA‐interference approach, showed augmented G lignin units yet contained considerable amounts of residual S lignin units. In this study, to further investigate the effect of suppression of OsCAld5H1 on rice lignin structure, we generated loss‐of‐function mutants of OsCAld5H1 using the CRISPR/Cas9‐mediated genome editing system. Homozygous OsCAld5H1 ‐knockout lines harboring anticipated frame‐shift mutations in OsCAld5H1 were successfully obtained. A series of wet‐chemical and two‐dimensional NMR analyses on cell walls demonstrated that although lignins in the mutant were predictably enriched in G units all the tested mutant lines produced considerable numbers of S units. Intriguingly, lignin γ‐ p ‐coumaroylation analysis by the derivatization followed by reductive cleavage method revealed that enrichment of G units in lignins of the mutants was limited to the non‐γ‐ p ‐coumaroylated units, whereas grass‐specific γ‐ p ‐coumaroylated lignin units were almost unaffected. Gene expression analysis indicated that no homologous genes of OsCAld5H1 were overexpressed in the mutants. These data suggested that CAld5H is mainly involved in the production of non‐γ‐ p ‐coumaroylated S lignin units, common in both eudicots and grasses, but not in the production of grass‐specific γ‐ p ‐coumaroylated S units in rice. Significance Statement: CRISPR/Cas9‐mediated gene knockout of rice coniferaldehyde 5‐hydroxylase ( OsCAld5H1 ) resulted in lignins enriched in guaiacyl (G) units. However, the mutants still produced considerable numbers of syringyl (S) units. The enrichment of G units was limited to the non‐γ‐ p ‐coumaroylated lignin units, and the grass‐specific γ‐ p ‐coumaroylated lignin units were apparently unchanged, suggesting that OsCAld5H1 is mainly involved in the biosynthesis of non‐γ‐ p ‐coumaroylated monolignols common in both eudicots and grasses, but not in that of grass‐specific γ‐ p ‐coumaroylated monolignols.