Ecophysiology and genetic analysis of plant adaptation to soil metal contamination

土壤金属含量高可能严重威胁生态系统的可持续性。调控金属在植物体内积累和抗性的生理和遗传机制是复杂的。尽管最近使用了高通量方法，但我们对植物对金属毒性的反应的理解仍然很差。该研究项目将为关键的非模式植物物种（在生物修复中有用）如何应对高土壤金属水平提供新的见解。这四个部分（目标）解决了四个相关的问题：1)植物如何利用遗传和比较转录组分析来应对金属；2)细胞和细胞外的位置以及所涉及的转运蛋白；3)金属污染对胞嘧啶甲基化水平和分布的影响；4)金属中毒反应中DNA甲基化与基因调控之间的联系。我们首先证明，为了应对金属：1)槭糖（银枫）排除它们；2) tremuloides（颤杨）、Betula papyrifera（白桦树）和Quercus rubra（红橡树）聚集；3)槭（红枫）避免它们。重要的是，我们完成并鉴定了纸莎草杆菌的第一个转录组，并鉴定了其中的6个候选耐镍基因。最近，我们研究了桦树属植物抗镍基因的水平转移。我们相信，结合这些方法将为阐明植物的金属应对机制提供最有力的方法。通过遗传分析、转录组测序和RT-qPCR，对红曲霉（A. rubrum）、糖化曲霉（A. saccharinum）、纸叶曲霉（B. papyrifera）、银耳曲霉（P. tremuloides）和红曲霉（Q. rubra）进行比较分析，将确定在高镍（Ni）和高铜（Cu）环境下差异调节的功能基因集。与Ni/Cu抗性相关的新基因在加拿大和其他地方的其他植物中的分布将被确定。原位荧光、蛋白质表征和电子显微镜技术将建立细胞和细胞外分布图，并阐明Ni和Cu及其相关转运蛋白的主要作用。甲基化敏感扩增多态性（MSAP）分析和亚硫酸盐测序将评估表观遗传学在植物金属反应中的作用。不仅在甲基组/转录组尺度上DNA甲基化与基因表达之间存在相关性，而且还将建立DNA甲基化与基因对Ni/Cu污染反应调控之间的联系。我们的研究计划将有助于了解重金属稳态是如何实现的，以及不同基因在不同物种的转运体运作中的作用，这些转运体在金属积累和排除中可能存在很大差异。我们实现的目标将解决几个基本的植物：金属相互作用问题，直接适用于重要的生物工程或植物选择策略。在这一阶段，将培训六名研究生，四名本科生和暑期学生，以填补加拿大生物技术方面的空白。

High soil metal levels may seriously threaten ecosystem sustainability. Physiological and genetic mechanisms regulating metal accumulation and resistance in plants are complex. Our understanding of plant responses to metal toxicity is poor despite recent uses of high-throughput methods. This research program will provide new insights on how key non-model plant species (useful in bioremediation) cope with high soil metal levels. The four parts (objectives) address four linked issues: 1) how plants cope with metals using genetic and comparative transcriptome analyses; 2) the cellular and extracellular locations and the transport proteins involved; 3) the levels and distribution of cytosine methylation in response to metal contamination; and 4) the link between DNA methylation and gene regulation in response to metal toxicity. We first demonstrated that, to cope with metals: 1) Acer saccharinum (silver maple) excludes them; 2) Populus tremuloides (trembling aspen), Betula papyrifera (white birch), and Quercus rubra (red oak) accumulate them; and 3) Acer rubrum (red maple) avoids them. Importantly, we completed and characterized the first transcriptome of B. papyrifera and identified six candidate nickel resistance genes therein. Recently, we characterized horizontal gene transfer of nickel resistance in the Genus Betula. We believe combining approaches will provide the most robust way of elucidating plant:metal coping mechanisms. Comparative analysis of A. rubrum, A. saccharinum, B. papyrifera, P. tremuloides, and Q. rubra, using genetic analysis, transcriptome sequencing, and RT-qPCR will identify functional gene-sets differentially regulated in response to high nickel (Ni) and copper (Cu). Distribution of novel genes associated with Ni/Cu resistance in other plants in Canada and elsewhere will be established. Fluorescence in situ, protein characterization, and electron microscopy techniques will establish cellular and extracellular distribution maps and clarify the main effects of Ni and Cu and involved transport proteins. Methylation sensitive amplified polymorphism (MSAP) analysis and bisulfite sequencing will assess the role of epigenetics in plant:metal responses. Not only correlations between DNA methylation and gene expression at the methylome/transcriptome scale, but also the link between DNA methylation and regulation of gene response to Ni/Cu contamination, will be established. Our research program will help understanding of how heavy metal homeostasis is achieved, and the role of different genes, in the operation of transporters in different species that may differ greatly in metal accumulators and excluders. Our achieved goals will address several fundamental plant:metal interaction issues directly applicable to important bioengineering or plant selection strategies. Six graduate, four undergraduate, and summer students will be trained, to fill this void in biotechnology in Canada, in this phase.