Mechanisms of Selenate-Specific Transport and Selenium Hyperaccumulation and Tolerance in Stanleya pinnata - Hypothesized Key Genes SpSultr1;2 and SpAPS2

一些植物有一种迷人的特性，它们超积累一种特定的有毒元素，其含量比周围的植被高100-1000倍。来自芥菜科的太子羽（Stanleya pinnata）就是这样一种植物。这种原产于美国西部的植物可以积累高达其干重0.5%的硒。硒是包括人类在内的许多生命形式的微量营养素，但含量过高是有毒的。它与硫非常相似，大多数生命形式无法区分这两种元素。然而，这种超富集体具有独特的能力来区分硒和硫，并优先积累硒酸盐。超富集菌还通过积累有机形式的硒（例如在氨基酸中）来区分自己，而非超富集菌的近亲则积累无机硒。这种能力可能解释了它们对硒的耐受性。在这个项目中解决的问题是：S. pinnata进化出硒特异性转运体了吗？这对任何生物体来说都是第一次。这种转运体有哪些不同之处，可以让我们了解它的进化？哪些基因是产生有机硒的基础？根据目前的实验结果，确定了一个硒酸盐转运体候选基因，以及一个可能影响有机硒积累的关键基因。该项目将通过在酵母和模式植物拟南芥（一种来自芥菜科的非积累植物）中的表达来研究该基因和其他基因。该项目的研究结果有望更好地了解硒的超积累机制：硒酸盐/硫酸盐区分以及硒的吸收、同化和耐受性增强。此外，来自超积累体的基因可用于培育具有较强的硒污染土壤或水体（植物修复）清理能力的植物，也可作为高营养价值的硒强化作物。从更广泛的角度来看，该项目可以作为研究植物对极端土壤条件适应的进化模型，也可以作为研究转运体和酶特异性控制机制的生化模型。这些机制是代谢和膜运输的基础。在对硒（Se）高富集物石柱和非富集物石柱的转录组测序研究中，有两个基因在高富集物中表现突出，因为它们的转录水平要高得多：与拟南芥Sultr1同源的高亲和力硫酸盐/硒酸盐转运蛋白；2（高44倍）和一个与AtAPS2同源的ATP硫化酶，AtAPS2是硫/硒同化的关键酶（高479倍）。这些发现导致了以下假设：(1)S. pinnata (Sp) Sultr1；(2) SpAPS2负责叶参硒的过量积累和硒盐的特异性吸收；(2)SpAPS2是硒盐同化为有机形式的关键，因此具有硒耐受性。本课题的研究目的是：(1)确定SpSultr1基因表达水平的升高是否影响；2和SpAPS2与硒酸盐和/或硫酸盐摄取率升高以及ATP硫酰化酶活性升高有关；(2)分析SpSULTR1在细胞内的定位；2和SpAPS2蛋白及其组织特异性表达模式；(3)对桄榔子和叶参SULTR1的硫酸盐和硒酸盐转运特性进行功能分析；酵母（酿酒酵母）中的2个转运体；(4)确定桄榔子和桄榔子Sultr1基因表达的生理效应；2和APS2在拟南芥中的表达。将测量的参数包括硒酸盐和硫酸盐（单独和组合）的摄取动力学，硒积累的浓度，形式和位置（使用ICP-OES， LC-MS， XAS）和硒耐受性。在酵母中，还将进行结构-功能研究，以研究选定的氨基酸对硒酸盐特异性的重要性。

Some plants have the fascinating property that they hyperaccumulate a particular toxic element to levels that are 100-1000 fold higher than surrounding vegetation. Prince's plume (Stanleya pinnata), from the mustard family, is such a plant. This native to the Western USA can hyperaccumulate selenium up to 0.5% of its dry weight. Selenium is a micronutrient for many life forms including humans, but toxic at high levels. It is very similar to sulfur, and most life forms cannot discriminate between the two elements. This hyperaccumulator, however, has the unique capacity to distinguish selenium from sulfur, and preferentially accumulates selenate. Hyperaccumulators also distinguish themselves by accumulating organic forms of selenium (in amino acids for example) where non-hyperaccumulator relatives accumulate inorganic selenium. This capacity likely explains their selenium tolerance. Questions addressed in this project are: Has S. pinnata evolved a selenate-specific transporter? That would be a first for any organism. In what ways is such a transporter different that would provide insights about its evolution? And which genes underlie the capacity to produce organic selenium? Based on experiments so far, a candidate selenate transporter has been identified, as well as a possible key gene for the accumulation of organic selenium. This and other genes will be investigated in this project by expressing them in both yeast and in the model plant species Arabidopsis: a nonaccumulator from the mustard family. The results from this project are expected to provide better insight into mechanisms of selenium hyperaccumulation: selenate/sulfate discrimination as well as enhanced selenium uptake, assimilation and tolerance. Also, the genes from the hyperaccumulator may be used to breed plants with superior capacity to clean up selenium-polluted land or water (phytoremediation) and that can also serve as selenium-fortified crops with higher nutritional value. From a broader perspective, this project may serve as an evolutionary model to study plant adaptation to extreme soil conditions, and as a biochemical model to study mechanisms by which specificity of transporters and enzymes is controlled. These mechanisms are at the basis of metabolism and membrane transport.In a transcriptome sequencing study using selenium (Se) hyperaccumulator Stanleya pinnata and non-accumulator Stanleya elata, two genes stood out because their transcript levels were much higher in the hyperaccumulator: a high affinity sulfate/selenate transporter homologous to Arabidopsis thaliana Sultr1;2 (44x higher) and an ATP sulfurylase homologous to AtAPS2, a key enzyme of sulfur/selenium assimilation (479x higher). These findings led to the hypotheses that (I) S. pinnata (Sp) Sultr1;2 is responsible for selenium hyperaccumulation and selenate-specific uptake in S. pinnata, and (II) SpAPS2 is key to the selenate assimilation into organic forms, and thus Se tolerance. The research objectives of this project are: (1) To determine whether the elevated expression levels of SpSultr1;2 and SpAPS2 are associated with elevated uptake rates of selenate and/or sulfate and elevated levels of ATP sulfurylase activity; (2) To analyze the intracellular location of the SpSULTR1;2 and SpAPS2 proteins, as well as their tissue-specific expression patterns; (3) To functionally analyze the sulfate and selenate transport properties of the S. pinnata and S. elata SULTR1;2 transporters in yeast (Saccharomyces cerevisiae); (4) To determine the physiological effects of expression of S. pinnata and S. elata Sultr1;2 and APS2 in Arabidopsis thaliana. Parameters that will be measured include uptake kinetics of selenate and sulfate (alone and in combination), concentration, forms and locations of selenium accumulated (using ICP-OES, LC-MS, XAS), and selenium tolerance. In yeast, structure-function studies will also be carried out, to study the importance of selected aminoacids for selenate specificity.