Showing papers by "Shiro Suzuki published in 2019"

Recruitment of specific flavonoid B‐ring hydroxylases for two independent biosynthesis pathways of flavone‐derived metabolites in grasses

Abstract: In rice (Oryza sativa), OsF2H and OsFNSII direct flavanones to independent pathways that form soluble flavone C‐glycosides and tricin‐type metabolites (both soluble and lignin‐bound), respectively. Production of soluble tricin metabolites requires CYP75B4 as a chrysoeriol 5′‐hydroxylase. Meanwhile, the close homologue CYP75B3 is a canonical flavonoid 3′‐hydroxylase (F3′H). However, their precise roles in the biosynthesis of soluble flavone C‐glycosides and tricin–lignins in cell walls remain unknown. We examined CYP75B3 and CYP75B4 expression in vegetative tissues, analyzed extractable flavonoid profiles, cell wall structure and digestibility of their mutants, and investigated catalytic activities of CYP75B4 orthologues in grasses. CYP75B3 and CYP75B4 showed co‐expression patterns with OsF2H and OsFNSII, respectively. CYP75B3 is the sole F3′H in flavone C‐glycosides biosynthesis, whereas CYP75B4 alone provides sufficient 3′,5′‐hydroxylation for tricin–lignin deposition. CYP75B4 mutation results in production of apigenin‐incorporated lignin and enhancement of cell wall digestibility. Moreover, tricin pathway‐specific 3′,5′‐hydroxylation activities are conserved in sorghum CYP75B97 and switchgrass CYP75B11. CYP75B3 and CYP75B4 represent two different pathway‐specific enzymes recruited together with OsF2H and OsFNSII, respectively. Interestingly, the OsF2H‐CYP75B3 and OsFNSII‐CYP75B4 pairs appear to be conserved in grasses. Finally, manipulation of tricin biosynthesis through CYP75B4 orthologues can be a promising strategy to improve digestibility of grass biomass for biofuel and biomaterial production.

Lignin characterization of rice CONIFERALDEHYDE 5‐HYDROXYLASE loss‐of‐function mutants generated with the CRISPR/Cas9 system

Abstract: 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.

OsMYB108 loss-of-function enriches p-coumaroylated and tricin lignin units in rice cell walls.

Abstract: Breeding approaches to enrich lignins in biomass could be beneficial to improving the biorefinery process because lignins increase biomass heating value and represent a potent source of valuable aromatic chemicals. However, despite the fact that grasses are promising lignocellulose feedstocks, limited information is yet available for molecular-breeding approaches to upregulate lignin biosynthesis in grass species. In this study, we generated lignin-enriched transgenic rice (Oryza sativa), a model grass species, via targeted mutagenesis of the transcriptional repressor OsMYB108 using CRISPR/Cas9-mediated genome editing. The OsMYB108-knockout rice mutants displayed increased expressions of lignin biosynthetic genes and enhanced lignin deposition in culm cell walls. Chemical and two-dimensional nuclear magnetic resonance (NMR) analyses revealed that the mutant cell walls were preferentially enriched in γ-p-coumaroylated and tricin lignin units, both of which are typical and unique components in grass lignins. NMR analysis also showed that the relative abundances of major lignin linkage types were altered in the OsMYB108 mutants.

OsCAldOMT1 is a bifunctional O-methyltransferase involved in the biosynthesis of tricin-lignins in rice cell walls.

Abstract: Lignin is a phenylpropanoid polymer produced in the secondary cell walls of vascular plants. Although most eudicot and gymnosperm species generate lignins solely via polymerization of p-hydroxycinnamyl alcohols (monolignols), grasses additionally use a flavone, tricin, as a natural lignin monomer to generate tricin-incorporated lignin polymers in cell walls. We previously found that disruption of a rice 5-HYDROXYCONIFERALDEHYDE O-METHYLTRANSFERASE (OsCAldOMT1) reduced extractable tricin-type metabolites in rice vegetative tissues. This same enzyme has also been implicated in the biosynthesis of sinapyl alcohol, a monolignol that constitutes syringyl lignin polymer units. Here, we further demonstrate through in-depth cell wall structural analyses that OsCAldOMT1-deficient rice plants produce altered lignins largely depleted in both syringyl and tricin units. We also show that recombinant OsCAldOMT1 displayed comparable substrate specificities towards both 5-hydroxyconiferaldehyde and selgin intermediates in the monolignol and tricin biosynthetic pathways, respectively. These data establish OsCAldOMT1 as a bifunctional O-methyltransferase predominantly involved in the two parallel metabolic pathways both dedicated to the biosynthesis of tricin-lignins in rice cell walls. Given that cell wall digestibility was greatly enhanced in the OsCAldOMT1-deficient rice plants, genetic manipulation of CAldOMTs conserved in grasses may serve as a potent strategy to improve biorefinery applications of grass biomass.

De novo transcriptome analysis of needles of Thujopsis dolabrata var. hondae.

Abstract: Podophyllotoxin is a starting material of the semisynthetic anticancer medicines etoposide, teniposide, and etopophos. The major plant source of podophyllotoxin is rhizomes of Podophyllum hexandrum, which is a Himalayan endangered species; therefore, alternative sources of podophyllotoxin or bioproduction systems have been pursued to avoid exploiting this limited natural resource. In this paper, we report de novo transcriptome analysis of Thujopsis dolablata var. hondae, which accumulates the podophyllotoxin derivatives (deoxypodophyllotoxin and β-peltatin A methyl ether) in its needles. We analyzed transcriptomes of the T. dolablata var. hondae young needles to obtain the sequences that putatively encode O-methyltransferases, cytochrome P450s, and a 2-oxoglutarate dependent dioxygenase because these protein families are responsible for podophyllotoxin-related compound formation in P. hexandrum. The resulting transcriptomes contained considerable numbers of coding sequences classified into the three protein families. Our results are a genetic basis for identifying genes involved in the biosynthesis of podophyllotoxin and related compounds and also for future metabolic engineering of podophyllotoxin in heterologous hosts.