Cellulose is the most abundant unique biopolymer in nature with widespread applications in bioenergy and high-value bioproducts. The large transmembrane-localized cellulose synthase (CESA) complexes (CSCs) play a pivotal role in the biosynthesis and orientation of the para-crystalline cellulose microfibrils during secondary cell wall (SCW) deposition. However, the hub CESA subunit with high potential homo/heterodimerization capacity and its functional effects on cell wall architecture, cellulose crystallinity, and saccharification efficiency remains unclear. Here, we reported the highly potent binding site containing four residues of Pro435, Trp436, Pro437, and Gly438 in the plant-conserved region (P-CR) of PalCESA4 subunit, which are involved in the CESA4-CESA8 heterodimerization. The CRISPR/Cas9-knockout mutagenesis in the predicted binding site results in physiological abnormalities, stunt growth, and deficient roots. The homozygous double substitution of W436Q and P437S and heterozygous double deletions of W436 and P437 residues potentially reduced CESA4-binding affinity resulting in normal roots, 1.5–2-fold higher plant growth and cell wall regeneration rates, 1.7-fold thinner cell wall, high hemicellulose content, 37%–67% decrease in cellulose content, high cellulose DP, 25%–37% decrease in cellulose crystallinity, and 50% increase in saccharification efficiency. The heterozygous deletion of W436 increases about 2-fold CESA4 homo/heterodimerization capacity led to the 50% decrease in plant growth and increase in cell walls thickness, cellulose content (33%), cellulose DP (20%), and CrI (8%). Our findings provide a strategy for introducing commercial CRISPR/Cas9-mediated bioengineered poplars with promising cellulose applications. We anticipate our results could create an engineering revolution in bioenergy and cellulose-based nanomaterial technologies.

中文翻译：

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CRISPR/Cas9介导的CESA4异二聚化能力的P-CR结构域特异性工程改变了杨树的细胞壁结构并提高了糖化效率

纤维素是自然界中最丰富的独特生物聚合物，在生物能源和高价值生物产品中有着广泛的应用。大型跨膜定位纤维素合酶 (CESA) 复合物 (CSC) 在次生细胞壁 (SCW) 沉积过程中准晶纤维素微纤维的生物合成和定向中发挥着关键作用。然而，具有高潜力同源/异源二聚化能力的中心CESA亚基及其对细胞壁结构、纤维素结晶度和糖化效率的功能影响仍不清楚。在这里，我们报道了PalCESA4亚基植物保守区（P-CR）中包含Pro435、Trp436、Pro437和Gly438四个残基的高效结合位点，这些残基参与CESA4-CESA8异二聚化。预测结合位点的 CRISPR/Cas9 敲除突变会导致生理异常、生长迟缓和根部缺陷。W436Q 和 P437S 的纯合双取代以及 W436 和 P437 残基的杂合双缺失可能会降低 CESA4 结合亲和力，从而导致正常根，植物生长和细胞壁再生率提高 1.5-2 倍，细胞壁薄 1.7 倍，高半纤维素含量降低37%~67%，纤维素DP高，纤维素结晶度降低25%~37%，糖化效率提高50%。W436 的杂合缺失使 CESA4 同源/异源二聚化能力增加约 2 倍，导致植物生长下降 50%，细胞壁厚度、纤维素含量 (33%)、纤维素 DP (20%) 和 CrI (8%) 增加）。我们的研究结果为引入具有前景纤维素应用的商业 CRISPR/Cas9 介导的生物工程杨树提供了策略。我们预计我们的结果可能会在生物能源和纤维素纳米材料技术领域引发一场工程革命。