LACCASE14 is required for the deposition of guaiacyl lignin and affects cell wall digestibility in poplar.

BACKGROUND: The recalcitrance of lignocellulosic biomass provided technical and economic challenges in the current biomass conversion processes. Lignin is considered as a crucial recalcitrance component in biomass utilization. An in-depth understanding of lignin biosynthesis can provide clues to overcoming the recalcitrance. Laccases are believed to play a role in the oxidation of lignin monomers, leading to the formation of higher-order lignin. In plants, functions of only a few laccases have been evaluated, so little is known about the effect of laccases on cell wall structure and biomass saccharification. RESULTS: In this study, we screened a gain-of-function mutant with a significant increase in lignin content from Arabidopsis mutant lines overexpressing a full-length poplar cDNA library. Further analysis confirmed that a Chinese white poplar (Populus tomentosa) laccase gene PtoLAC14 was inserted into the mutant, and PtoLAC14 could functionally complement the Arabidopsis lac4 mutant. Overexpression of PtoLAC14 promoted the lignification of poplar and reduced the proportion of syringyl/guaiacyl. In contrast, the CRISPR/Cas9-generated mutation of PtLAC14 results in increased the syringyl/guaiacyl ratios, which led to integrated enhancement on biomass enzymatic saccharification. Notably, the recombinant PtoLAC14 protein showed higher oxidized efficiency to coniferyl alcohol (precursor of guaiacyl unit) in vitro. CONCLUSIONS: This study shows that PtoLAC14 plays an important role in the oxidation of guaiacyl deposition on cell wall. The reduced recalcitrance of the PtoLAC14-KO lines suggests that PtoLAC14 is an elite target for cell wall engineering, and genetic manipulation of this gene will facilitate the utilization of lignocellulose.

Plant regeneration in Vernicia fordii from apical bud.

Vernicia fordii is a woody plant species with important economic value, its seeds could be used to produce biodiesel and the oil extracted from them serve many kinds of industrial applications. In this study, we established a regeneration system of V. fordii for the first time using apical buds as explants on the Murashige and Skoog (MS) medium supplemented with the plant growth regulators, 6-benzylaminopurine (6-BA) and 3-indolebutyric acid (IBA). The optimal condition for adventitious bud induction was MS medium containing 1.5 mg/L 6-BA and 0.01 mg/L IBA, which produced a satisfactory induction efficiency. In subculture, the MS medium containing 1.0 mg/L 6-BA and 0.01 mg/L IBA was the optimum selection when considering both proliferation rate and tissue growth status. The adventitious shoots inoculated on 1/2 MS medium containing 0.5 mg/L IBA could produce strong roots and 96.4% of the regenerated plants survived after transplanting.

Brassinosteroid overproduction improves lignocellulose quantity and quality to maximize bioethanol yield under green-like biomass process in transgenic poplar.

BACKGROUND: As a leading biomass feedstock, poplar plants provide enormous lignocellulose resource convertible for biofuels and bio-chemicals. However, lignocellulose recalcitrance particularly in wood plants, basically causes a costly bioethanol production unacceptable for commercial marketing with potential secondary pollution to the environment. Therefore, it becomes important to reduce lignocellulose recalcitrance by genetic modification of plant cell walls, and meanwhile to establish advanced biomass process technology in woody plants. Brassinosteroids, plant-specific steroid hormones, are considered to participate in plant growth and development for biomass production, but little has been reported about brassinosteroids roles in plant cell wall assembly and modification. In this study, we generated transgenic poplar plant that overexpressed DEETIOLATED2 gene for brassinosteroids overproduction. We then detected cell wall feature alteration and examined biomass enzymatic saccharification for bioethanol production under various chemical pretreatments. RESULTS: Compared with wild type, the PtoDET2 overexpressed transgenic plants contained much higher brassinosteroids levels. The transgenic poplar also exhibited significantly enhanced plant growth rate and biomass yield by increasing xylem development and cell wall polymer deposition. Meanwhile, the transgenic plants showed significantly improved lignocellulose features such as reduced cellulose crystalline index and degree of polymerization values and decreased hemicellulose xylose/arabinose ratio for raised biomass porosity and accessibility, which led to integrated enhancement on biomass enzymatic saccharification and bioethanol yield under various chemical pretreatments. In contrast, the CRISPR/Cas9-generated mutation of PtoDET2 showed significantly lower brassinosteroids level for reduced biomass saccharification and bioethanol yield, compared to the wild type. Notably, the optimal green-like pretreatment could even achieve the highest bioethanol yield by effective lignin extraction in the transgenic plant. Hence, this study proposed a mechanistic model elucidating how brassinosteroid regulates cell wall modification for reduced lignocellulose recalcitrance and increased biomass porosity and accessibility for high bioethanol production. CONCLUSIONS: This study has demonstrated a powerful strategy to enhance cellulosic bioethanol production by regulating brassinosteroid biosynthesis for reducing lignocellulose recalcitrance in the transgenic poplar plants. It has also provided a green-like process for biomass pretreatment and enzymatic saccharification in poplar and beyond.