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Lignin characterization of rice CONIFERALDEHYDE 5‐HYDROXYLASE loss‐of‐function mutants generated with the CRISPR /Cas9 system
Author(s) -
Takeda Yuri,
Suzuki Shiro,
Tobimatsu Yuki,
Osakabe Keishi,
Osakabe Yuriko,
Ragamustari Safendrri K.,
Sakamoto Masahiro,
Umezawa Toshiaki
Publication year - 2019
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.14141
Subject(s) - lignin , mutant , oryza sativa , chemistry , biochemistry , tilling , gene , botany , food science , biology , genetics , organic chemistry
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 (Os CA ld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized Os CA ld5H1 ‐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 Os CA ld5H1 on rice lignin structure, we generated loss‐of‐function mutants of Os CA ld5H1 using the CRISPR /Cas9‐mediated genome editing system. Homozygous Os CA ld5H1 ‐knockout lines harboring anticipated frame‐shift mutations in Os CA ld5H1 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 Os CA ld5H1 were overexpressed in the mutants. These data suggested that CA ld5H 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.