The physiological network regulating sulfite detoxification in plants

在此之前，我们已经在植物中发现了亚硫酸盐氧化酶（SO），克隆了其基因，并对其生物化学（原子结构、光谱、反应机理）、定位和功能等方面有了一定的了解。亚硫酸盐的底物对植物细胞具有高活性和高毒性。亚硫酸盐可以在硫代谢过程中内源性产生，也可以来自空气污染物二氧化硫等外部来源。我们发现，在拟南芥和杨树中，SO对于排毒过量的亚硫酸盐（安全阀功能）是必不可少的。本项目的目的是分析(a) so活性的转录和翻译后调控以及(b)与另一种亚硫酸盐解毒机制的相互作用。对于转录调控而言，特定的转录因子对SO启动子的激活/失活尤为重要：特定的dna结合位点强烈表明受RAV1控制。此外，还将研究SO蛋白的翻译后修饰（sumo化、泛素化和磷酸化）。此外，mRNA深度测序揭示了植物中的第二种亚硫酸盐解毒机制：外胞体过氧化物酶（Apoplastic peroxidase），这也是Pfanz及其同事（1990）的研究成果。SO2在so敲除植物中诱导外胞体过氧化物酶表明独立的外胞体和共塑解毒机制。我们已经完成了第一个定位实验，并获得了与底物瓜木酚和亚硫酸盐酶催化反应的第一个初步数据。现在，我们将从分子、细胞生物学和生化水平（亚硫酸盐解毒的反应机制）对细胞外胞体过氧化物酶进行研究，并计划研究这些细胞外胞体过氧化物酶及其与过氧化物酶体的共调控的重要性。此外，我们打算在自然栖息地和受控熏蒸实验中研究植物对二氧化硫水平增加的适应性。我们计划在火山岛（伊奥利亚群岛，意大利）收集植物材料，以二氧化硫浓度在µL L-1水平而闻名。我们想要回答的问题是，在自然环境中，植物是如何对高浓度二氧化硫做出反应的。我们是否发现植物对自然界中二氧化硫的长期适应是通过更高的二氧化硫蛋白量（转录或转录后修饰）或通过适应的翻译后调节来实现的？

Previously, we have discovered the enzyme sulfite oxidase (SO) in plants, cloned its gene, and provided knowledge about its biochemistry (atomic structure, spectroscopy, and reaction mechanism), localization and function. The substrate of SO - sulfite - is highly reactive and highly toxic for the plant cell. Sulfite can be produced endogenously during sulfur metabolism and can also derive from external sources like the air pollutant sulfur dioxide. We showed that SO is essential for detoxifying excessive amounts of sulfite (safety-valve function) in Arabidopsis and poplar. The aim of the present project is to analyze (a) the transcriptional and posttranslational regulation of SO-activity and (b) the interaction with an additional sulfite detoxification mechanism. For transcriptional regulation specific transcription factors for activation/inactivation of the SO promoter are of particular importance: specific DNA-binding-sites strongly suggest a control by RAV1. Moreover, posttranslational modifications of SO protein (SUMOylation, ubiquitinilation and phosphorylation) will be studied. Moreover, mRNA deep-sequencing revealed a second sulfite detoxification mechanism in plants: Apoplastic peroxidase, also known from work of Pfanz and colleagues (1990). Induction of the apoplastic peroxidases by SO2 in SO-knock out plants indicates independent apoplastic and symplastic detoxification mechanisms. We have done first localization experiments and have first preliminary data of the enzymatic reaction with the substrates guajacol and sulfite. Now, we will to investigate the apoplastic peroxidases on molecular, cell-biological and biochemical level (reaction mechanism for sulfite detoxification) and plan to study the importance of these apoplastic peroxidases and their co-regulation with peroxisomal SO. In addition, we intend to study plant adaptations to increased levels of SO2 both, in natural habitats and in controlled fumigation experiments. We plan to collect plant material at Volcano Island (Aeolian Archipelago, Italy) known for SO2-concentration in the µL L-1-level. We want to answer the question, how do plants react to higher SO2-concentrations in natural environments. Do we find a long-term adaptation of plants to SO2 achieved in nature by higher SO protein amounts (transcriptional or post-transcriptional modification) or by an adapted post-translational regulation?