Modification of xylan in secondary walls alters cell wall biosynthesis and wood formation programs and improves saccharification

Wood of broad-leaf tree species is a valued source of renewable biomass for biorefinery and a target for genetic improvement efforts to reduce its recalcitrance. Glucuronoxylan (GX) plays a key role in recalcitrance through its interactions with cellulose and lignin. To reduce recalcitrance, we modified wood GX by expressing GH10 and GH11 endoxylanases from Aspergillus nidulans in hybrid aspen (Populus tremula L. x tremuloides Michx.) and targeting the enzymes to cell wall. The xylanases reduced tree height, modified cambial activity by increasing phloem and reducing xylem production, and reduced secondary wall deposition. Xylan molecular weight was decreased, and the spacing between acetyl and MeGlcA side chains was reduced in transgenic lines. The transgenic trees produced hypolignified xylem having thin secondary walls and deformed vessels. Glucose yields of enzymatic saccharification without pretreatment almost doubled indicating decreased recalcitrance. The transcriptomics, hormonomics and metabolomics data provided evidence for activation of cytokinin and ethylene signaling pathways, decrease in ABA levels, transcriptional suppression of lignification and a subset of secondary wall biosynthetic program, including xylan glucuronidation and acetylation machinery. Several candidate genes for perception of impairment in xylan integrity were detected. These candidates could provide a new target for uncoupling negative growth effects from reduced recalcitrance. In conclusion, our study supports the hypothesis that xylan modification generates intrinsic signals and evokes novel pathways regulating tree growth and secondary wall biosynthesis.

阔叶树种木材是生物炼制领域极具价值的可再生生物质来源，同时也是通过遗传改良降低其抗降解性（recalcitrance）的目标对象。葡萄糖醛酸木聚糖（Glucuronoxylan, GX）通过与纤维素和木质素的相互作用，在抗降解性中发挥关键作用。为降低木材的抗降解性，我们在杂交杨（欧洲山杨×美洲山杨，Populus tremula L. × tremuloides Michx.）中表达构巢曲霉（Aspergillus nidulans）来源的GH10和GH11家族内切木聚糖酶，并将酶靶向定位至细胞壁，对木材中的葡萄糖醛酸木聚糖进行了修饰。结果表明，这些木聚糖酶会降低植株高度，通过增加韧皮部数量、减少木质部生成量来改变形成层活性，并抑制次生壁沉积。转基因株系的木聚糖分子量降低，乙酰基与甲基葡萄糖醛酸侧链间的间距也有所缩小。转基因树木产生了低木质化的木质部，其特征为次生壁较薄且导管形态畸形。未经预处理的酶解糖化葡萄糖产率几乎提升了一倍，表明抗降解性得到了改善。转录组学、激素组学（hormonomics）与代谢组学数据表明，细胞分裂素与乙烯信号通路被激活，脱落酸（Abscisic Acid, ABA）水平降低，木质化过程及部分次生壁生物合成程序（包括木聚糖葡萄糖醛酸化与乙酰化机制）受到转录抑制。我们还检测到若干可感知木聚糖完整性受损的候选基因，这些候选基因可为解耦“降低抗降解性”与“负面生长效应”提供新的靶点。综上，本研究支持以下假说：木聚糖修饰可产生内源信号，并激活调控树木生长与次生壁生物合成的全新通路。