Functional genomics and molecular evolution of bioactive phenolic conjugates in plants

植物生物活性酚类缀合物的功能基因组学和分子进化

• 批准号: RGPIN-2014-04960
• 负责人: Ehlting, Juergen
• 金额: $ 1.89万
• 依托单位: University of Victoria
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=611347
• 关键词: Functional; genomics; molecular; evolution; bioactive

Elucidating evolutionary mechanism is central to biological sciences. Gene duplications are a rich source of genome and in consequence phenotypic diversity. Many molecular evolutionary models have been developed to explain how gene duplicates may evolve new functions. But for most these models functional evidence is scarce, because most complex adaptive traits cannot be easily linked to a single gene. Plant secondary metabolism provides a notable exception. Plants produce an immense diversity of these small bioactive compounds functioning as protectants and attractants in chemical ecology. Adaptive variations of secondary metabolites can frequently be linked directly to genes encoding their biosynthetic enzymes, making them ideal test cases to validate evolutionary models. The focus here lies on a class of secondary metabolites called hydroxycinnamoyl conjugates (HCCs), which are wide spread across the plant kingdom but are particularly diverse in some species such as poplar trees. HCCs can be herbivore deterrents, antimicrobials, or UV-absorbing sunscreens. Humans exploit them as antioxidants or pharmaceuticals. One particular HCC has been recruited in seed plants as an intermediate in lignin biosynthesis. Lignin is a rigid polymer encrusted into secondary cell walls and enables upright growth. Two enzymes are responsible for HCC biosynthesis: a hydroxycinnamoyl-transferase (HCT) and a coumaroyl-conjugate 3-hydroxylase belonging to the CYP98A family. Seed plants contain multiple members, while mosses and lycopods contain only a single copy. However, the latter are not involved in lignin biosynthesis. Instead, our preliminary results suggest that the ancestral function of HCT/CYP98A was to produce a UV-absorbing and anti-microbial HCC. We hypothesize that these enzymes then continuously expanded their copy number and substrate range under positive selection to create the current HCC diversity. During this process, one of these duplicates was recruited for lignin biosynthesis and has maintained its specificity under purifying selection. To test these hypotheses, we will link evolutionary analyses of the HCT and CYP98A gene families with biochemical and chemical ecological approaches. We will use sequence data from across the plant lineage to generate phylogenetic gene trees. These pinpoint key gene duplication events and signatures of selection to either maintain a given function (expected in the lignin-related gene clade) or to diversify their functions (in protection-related clades). On a more recent evolutionary scale, poplars and related tree species provide a unique system to test ongoing adaptive radiations, because they evolved a species-specific diversity of HCCs and because we do have available sequence data from thousands of individuals covering thirty species. To experimentally test functional diversity (or uniformity) within clades, we will employ biochemical approaches using recombinant production of target proteins in bacteria or yeasts. This will determine substrate ranges (wide or narrow) of complete HCT/CYP98A families from representative species across the plant lineage. We will also use reverse genetic approaches in select species, such as the moss Physcomitrella, or the angiosperms Arabidopsis and poplar. This will facilitate metabolomic phenotyping using analytical chemistry targeting both soluble HCCs and lignin. These mutants will also allow testing the roles of these genes in chemical ecology through controlled infestations with herbivores and pathogens, and through UV exposure experiments. Together, this will link adaptive chemical traits realized through enzymatic diversity directly to gene duplications and selection signatures, thereby deciphering the modes of evolution that shaped HCC diversity.

阐明进化机制是生物科学的核心。基因重复是基因组和表型多样性的丰富来源。许多分子进化模型已经被开发出来，以解释基因重复如何进化出新的功能。但是对于大多数这些模型来说，功能性证据是稀缺的，因为大多数复杂的适应性特征不能轻易地与单个基因联系起来。 植物次生代谢是一个显著的例外。植物产生的这些小的生物活性化合物作为化学生态学中的保护剂和引诱剂的巨大多样性。次级代谢产物的适应性变异通常与编码其生物合成酶的基因直接相关，使其成为验证进化模型的理想测试案例。 这里的重点在于一类称为羟基肉桂酰缀合物（HCCs）的次级代谢产物，其广泛分布于植物界，但在某些物种（如白杨）中特别多样化。HCCs可以是食草动物威慑剂，抗菌剂或紫外线吸收防晒剂。人类利用它们作为抗氧化剂或药物。一种特定的HCC已在种子植物中作为木质素生物合成的中间体被募集。木质素是一种坚硬的聚合物，包裹在次生细胞壁中，使其能够直立生长。 两种酶负责HCC生物合成：属于CYP 98 A家族的羟基肉桂酰转移酶（HCT）和香豆酰缀合物3-羟化酶。种子植物含有多个成员，而苔藓和石松只含有一个副本。然而，后者不参与木质素的生物合成。相反，我们的初步结果表明，HCT/CYP 98 A的祖先功能是产生紫外线吸收和抗微生物HCC。我们假设这些酶在正选择下不断扩大其拷贝数和底物范围，以创造当前的HCC多样性。在此过程中，这些复制品之一被招募用于木质素生物合成，并在纯化选择下保持其特异性。 为了验证这些假设，我们将联系HCT和CYP 98 A基因家族的进化分析与生物化学和化学生态学方法。 我们将使用来自整个植物谱系的序列数据来生成系统发育基因树。这些精确定位了关键的基因复制事件和选择的特征，以维持给定的功能（预期在木质素相关基因进化枝中）或使其功能多样化（在保护相关进化枝中）。在更近的进化尺度上，杨树和相关树种提供了一个独特的系统来测试持续的适应性辐射，因为它们进化出了一种特定物种的HCC多样性，而且我们确实有覆盖30个物种的数千个个体的可用序列数据。 为了实验性地测试进化枝内的功能多样性（或一致性），我们将采用在细菌或酵母中重组产生靶蛋白的生物化学方法。这将确定整个植物谱系中代表性物种的完整HCT/CYP 98 A家族的底物范围（宽或窄）。我们还将在选择的物种中使用反向遗传方法，如苔藓立碗藓，或被子植物拟南芥和白杨。这将有助于使用靶向可溶性HCC和木质素的分析化学进行代谢组学表型分析。这些突变体还将允许通过控制食草动物和病原体的侵扰以及通过紫外线暴露实验来测试这些基因在化学生态学中的作用。总之，这将把通过酶多样性实现的适应性化学性状直接与基因复制和选择特征联系起来，从而破译形成HCC多样性的进化模式。