Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Center for Plant Biology, Purdue University, West Lafayette, Indiana 47907, USA.
Plant Physiol. 2022 Aug 1;189(4):2015-2028. doi: 10.1093/plphys/kiac210.
Lignin contributes substantially to the recalcitrance of biomass toward saccharification. To circumvent this problem, researchers have genetically altered lignin, although, in a number of cases, these efforts have resulted in an undesirable yield penalty. Recent findings have shown that by knocking out two subunits (MED5A and MED5B) of the transcriptional regulatory complex Mediator, the stunted growth phenotype of mutants in p-coumaroyl shikimate 3'-hydroxylase, reduced epidermal fluorescence 8-1 (ref8-1), can be alleviated. Furthermore, these plants synthesize a lignin polymer almost entirely derived from p-coumaryl alcohol. Plants deficient in cinnamyl alcohol dehydrogenase (CAD) are notable in that they primarily incorporate coniferaldehyde and sinapaldehyde into their lignin. We tested the hypothesis that by stacking mutations in the genes encoding for the CAD paralogs C and D on an Arabidopsis (Arabidopsis thaliana) med5a/5b ref8-1 genetic background, the biosynthesis of p-coumaryl alcohol would be blocked, making p-coumaraldehyde available for polymerization into a novel kind of lignin. The med5a/5b ref8-1 cadc cadd plants are viable, but lignin analysis demonstrated that they continue to synthesize p-hydroxyphenyl lignin despite being mutated for the CADs typically considered to be required for monolignol biosynthesis. In addition, enzyme activity tests showed that even in the absence of CADC and CADD, there is high CAD activity in stems. We tested the potential involvement of other CADs in p-coumaraldehyde biosynthesis in the quintuple mutant by mutating them using the CRISPR/Cas9 system. Lignin analysis demonstrated that the resulting hextuple mutant plants continue to deposit p-coumaryl alcohol-derived lignin, demonstrating a route for the synthesis of p-hydroxyphenyl lignin in Arabidopsis independent of four CAD isoforms.
木质素对生物质的糖化有很大的抗性。为了解决这个问题，研究人员已经对木质素进行了基因改造，尽管在许多情况下，这些努力导致了不理想的产量损失。最近的研究结果表明，通过敲除转录调控复合物 Mediator 的两个亚基（MED5A 和 MED5B），突变体 p-香豆酰基莽草酸 3'-羟化酶、减少表皮荧光 8-1（ref8-1）的生长不良表型可以得到缓解。此外，这些植物合成的木质素聚合物几乎完全来自 p-香豆醇。肉桂醇脱氢酶（CAD）缺陷的植物值得注意的是，它们主要将松柏醛和芥子醛掺入到木质素中。我们测试了这样一个假设，即在拟南芥（Arabidopsis thaliana）med5a/5b ref8-1 遗传背景上叠加编码 CAD 同工酶 C 和 D 的基因的突变，p-香豆醇的生物合成将被阻断，使 p-香豆醛可用于聚合形成一种新型木质素。med5a/5b ref8-1 cadc cadd 植物是可行的，但木质素分析表明，尽管它们发生了突变，CADs 通常被认为是木质素单体生物合成所必需的，但它们仍继续合成 p-羟基苯基木质素。此外，酶活性测试表明，即使在缺乏 CADC 和 CADD 的情况下，茎中仍有很高的 CAD 活性。我们通过使用 CRISPR/Cas9 系统对 quintuple 突变体中的其他 CAD 进行突变，测试了它们在 p-香豆醛生物合成中的潜在参与。木质素分析表明，由此产生的 hextuple 突变体植物继续沉积 p-香豆醇衍生的木质素，证明了拟南芥中 p-羟基苯基木质素合成的途径独立于四种 CAD 同工酶。
