On the evolution of lignin biosynthesis in plants

木质素是所有维管植物的特征。这种酚醛聚合物被包裹在木质次生细胞壁中，为直立生长和长距离水运输提供了必要的机械稳定性和疏水性。尽管木质素的生物合成途径在所有维管植物谱系中都普遍存在，但在不同的植物谱系中，木质素的生物合成途径似乎有所不同。在开花植物中，大多数木质素单体的限速步骤是由酶4- coumaryl -shikimate-3'-羟化酶（C3'H / CYP98）催化的。然而，在早期分化的维管植物谱系石松中，相同木质素单体的生物合成通过不同的途径进行。我们最近的研究表明，在石松类中，CYP98不能代谢4- coumaryl -shikimate，但利用不同的底物，可能产生来自相同途径的与防御相关的次级代谢物。令人惊讶的是，蕨类植物CYP98也不能使用4- coumaryl -shikimate，因此蕨类植物中的单脂素生物合成似乎也使用不同的中间体。即使在针叶树中，CYP98底物谱也与木质素相关的C3'H酶不同。因此，我们假设木质素的生物合成在陆地植物进化过程中多次被显著地独立地修改。我们将通过对针叶树和蕨类植物中单个cyp98的功能表征来验证这一假设。除了详细的生化表征外，我们还需要通过分析大规模基因表达数据，将这些CYP98基因的基因表达谱与已知参与木质素生物合成的基因进行比较。这些也将被挖掘以确定在这些谱系中参与木质素生物合成的新的候选基因。重要的是，我们需要建立在这些非模式物种中产生突变的方法，以功能测试这些基因在木材形成中的直接参与。我们将产生功能丧失突变体，其特征是在开花植物cyp98突变体中通常发现的变化，包括木质素组成的变化。将评估叶片形态和代谢物组成的变化，因为这些变化在与木质素无关的苔藓cyp98突变体中受到影响。这些实验将表明CYP98是否参与蕨类和针叶树的木质素生物合成，或者是否有其他途径进化。如果后者成立，从基因表达数据中鉴定的候选基因将被测试其在模式植物拟南芥中拯救cyp98a3功能表型丧失的能力，如果有必要，然后通过生化和反向遗传方法进一步表征。综上所述，该项目将测试木质素生物合成的进化轨迹，并可能确定生物化学途径的进化优化，这对植物征服土地至关重要，并定义了木材的特性及其对人类的利用。

Lignin is a defining feature of all vascular plants. This phenolic polymer is incrusted into woody secondary cell walls to provide mechanical stability and hydrophobicity necessary for upright growth and long-distance water transport. Despite common occurrence in all vascular plant lineages, the biosynthetic route to lignin appears to be different in different plant lineages. In flowering plants, the rate limiting step towards the majority of lignin monomers is catalyzed by the enzyme 4-coumaroyl-shikimate-3'-hydroxylase (C3'H / CYP98). However, in lycopods, an early diverging vascular plant lineage, biosynthesis to the same lignin monomers proceeds through a different route. Fittingly, we have recently shown that in lycopods CYP98 is unable to metabolize 4-coumaroyl-shikimate, but utilizes different substrates, presumably to produce defense related secondary metabolites derived from the same pathway. Surprisingly, a fern CYP98 was also unable to use 4-coumaroyl-shikimate, and thus monolignol biosynthesis in ferns also appears to employ distinct intermediates. Even in conifers, CYP98 substrate profiles are distinct from lignin related C3'H enzymes. We thus hypothesize that lignin biosynthesis was modified significantly independently multiple times during land plant evolution. We will test this hypothesis by functionally characterizing the single CYP98s from a conifer and from a fern species. Besides detailing biochemical characterizations, we will need to compare gene expression profiles of these CYP98 genes with genes known to be involved in lignin biosynthesis through analysis of large-scale gene expression data. These will also be mined to identify novel candidate genes involved in lignin biosynthesis in these lineages. Importantly, we need to establish approaches to generate mutants in these non-model species to functionally test a direct involvement of these genes in wood formation. We will generate loss-of-function mutants that will be characterized for changes typically found in flowering plant cyp98 mutants, including changes in lignin composition. Alterations in leaf morphology and metabolite composition will be assessed as these are affected in a non-lignin related moss cyp98 mutant. These experiments will show whether CYP98 is involved in lignin biosynthesis in ferns and conifers or if alternative pathways have evolved. If the latter holds true, candidate genes identified from the gene expression data will be tested for their ability to rescue the cyp98a3 loss of function phenotype in the model plant Arabidopsis and, if warranted, then further characterized biochemically and through reverse genetic approaches. Taken together, this program will test the evolutionary trajectory of lignin biosynthesis and may identify evolutionary optimizations of a biochemical pathway that was crucial for plants to conquer land and that defines wood properties and its uses by humans.