Functional genomics and molecular evolution of bioactive phenolic conjugates in plants

阐明进化机制是生物科学的核心。基因复制是基因组的丰富来源，因此也是表型多样性的来源。已经开发了许多分子进化模型来解释基因复制如何进化出新的功能。但对于大多数这些模型来说，功能证据很少，因为大多数复杂的适应性特征不能很容易地与单一基因联系起来。 植物次生代谢是一个明显的例外。植物产生各种各样的这些小的生物活性化合物，在化学生态中起到保护剂和引诱剂的作用。次生代谢物的适应性变异经常可以直接与编码其生物合成酶的基因联系在一起，使它们成为验证进化模型的理想测试案例。 这里的重点是一类被称为羟基肉桂酰偶联物(HCCs)的次生代谢物，它广泛分布于植物界，但在一些物种中尤其多样化，如杨树。羟基喜树碱可以是食草动物的威慑剂、抗菌剂或紫外线吸收防晒霜。人类利用它们作为抗氧化剂或药物。在种子植物中发现了一种特殊的肝细胞癌，作为木质素生物合成的中间体。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。 有两种酶负责肝细胞癌的生物合成：一种是羟基肉桂酰基转移酶(HCT)，另一种是属于CYP98A家族的香豆酰结合3-羟基酶。种子植物包含多个成员，而苔藓和番茄科植物只包含一个副本。然而，后者并不参与木质素的生物合成。相反，我们的初步结果表明，HCT/CyP98A的祖先功能是产生一种紫外线吸收和抗微生物的肝癌。我们假设这些酶在正选择下不断扩大它们的拷贝数和底物范围，从而创造出目前的肝细胞癌多样性。在这个过程中，这些重复序列中的一个被招募用于木质素的生物合成，并在纯化选择下保持了其特异性。 为了验证这些假设，我们将把对HCT和CYP98A基因家族的进化分析与生化和化学生态学方法联系起来。 我们将使用整个植物谱系的序列数据来生成系统发育基因树。这些精确定位关键基因复制事件和选择信号，以维持给定的功能(预期在木质素相关基因分支中)或使其功能多样化(在保护相关分支中)。在最近的进化尺度上，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，因为它们进化了特定物种的碳氢化合物多样性，也因为我们确实拥有覆盖30个物种的数千个个体的可用序列数据。 为了在实验中测试分支内的功能多样性(或一致性)，我们将使用在细菌或酵母中重组目标蛋白的生化方法。这将确定整个植物谱系中代表物种的完整HCT/CYP98A家族的底物范围(宽或窄)。我们还将在特定物种中使用反向遗传方法，如苔藓植物Physcomitrella或被子植物拟南芥和杨树。这将有助于使用针对可溶性HC和木质素的分析化学进行代谢组表型分析。这些突变体还将通过控制草食动物和病原体的感染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。总而言之，这将把通过酶多样性实现的适应性化学特征直接与基因复制和选择签名联系起来，从而破译形成肝细胞癌多样性的进化模式。

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.