Department of Plant Sciences and Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran.
Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran.
Plant Biotechnol J. 2022 Jun;20(6):1197-1212. doi: 10.1111/pbi.13803. Epub 2022 Mar 15.
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.
纤维素是自然界中最丰富的独特生物聚合物，广泛应用于生物能源和高价值生物制品。大型跨膜定位的纤维素合酶（CESA）复合物（CSC）在次生细胞壁（SCW）沉积过程中对排列成平行结晶纤维素微纤丝的生物合成和定向起着关键作用。然而，高潜在同/异二聚化能力的中心 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，我们报道了植物保守区（P-CR）中 PalCESA4 亚基的四个残基 Pro435、Trp436、Pro437 和 Gly438 组成的高潜力结合位点，该位点参与 CESA4-CESA8 异二聚化。在预测的结合位点进行 CRISPR/Cas9 敲除诱变会导致生理异常、生长迟缓和根缺陷。W436Q 和 P437S 的纯合双取代以及 W436 和 P437 残基的杂合双缺失可能降低 CESA4 的结合亲和力，导致正常根、1.5-2 倍的植物生长和细胞壁再生率、1.7 倍的细胞壁变薄、高半纤维素含量、纤维素含量降低 37%-67%、纤维素 DP 升高 1.7 倍、纤维素结晶度降低 25%-37%、糖化效率提高 50%。W436 的杂合缺失增加了约 2 倍的 CESA4 同/异二聚化能力，导致植物生长降低 50%，细胞壁厚度增加，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有前途纤维素应用的商业 CRISPR/Cas9 介导的生物工程化杨树提供了一种策略。我们预计我们的研究结果将在生物能源和纤维素基纳米材料技术方面引发一场工程革命。
