Alternative Oxidase of Plant Mitochondria

交替氧化酶(AOX)是植物线粒体电子传递链中的一种非能量守恒型末端氧化酶，它可以放松呼吸碳氧化、电子传递和ATP周转之间的耦合。尽管我们对AOX的遗传和生化调控有了很大的了解，但这一辅助呼吸途径在植物代谢和线粒体生理中的作用以及它对植物生长和表现的整体影响仍然知之甚少。正如在有影响力的评论中经常强调的那样，这代表着我们对植物初级新陈代谢的理解存在重大差距。AOX基因的表达对环境信号高度敏感，一些证据表明，该途径在可变和应激条件下可能具有特别重要的作用。从理论上讲，AOX应该会对增长产生负面影响，因为它会降低ATP的产量。然而，它可能在新陈代谢和线粒体生理学方面发挥着关键作用，超过了这一能量成本。 我的研究提案有两个广泛的科学目标。首先是确定AOX在支持叶片新陈代谢中的作用。重点是阐明AOX支持光合作用的机制。我们的初步工作表明，在干旱胁迫期间，缺乏AOX的转基因植物的光合作用受到严重损害。气体交换和叶绿素荧光分析表明，这是缺乏AOX的植物受到严重生化(而不是气孔)限制的结果。详细的生理和生化分析将阐明这种限制的性质。我们还将研究在环境和高二氧化碳环境下生长的植物中AOX和光合作用的相互作用。高二氧化碳有可能提高光合作用速率，但可能会导致光合作用能力的下调，从而无法实现更高来源活性的潜力。这种下调的倾向在很大程度上取决于汇对高二氧化碳的反应。虽然呼吸作用是一种关键的汇活动，但人们对高二氧化碳下AOX的具体情况知之甚少。这是我们在理解未来高二氧化碳条件将如何影响光合作用和生长方面的一个重大差距。 该研究提案的第二个广泛的科学目标是确定AOX在支持线粒体生理学方面的作用。重点将阐明AOX在支持线粒体信号功能中的作用。线粒体充当参与压力适应的信号细胞器，但参与这种反应的线粒体的主要“信号”尚不清楚。我们的建议建立在我们以前工作中的一项重大发现的基础上，其中生化和共聚焦成像分析证实，AOX控制着超氧化物和一氧化氮的ETC生成，这两种活性物质可以在细胞内充当破坏分子或信号分子。这些活性物种以及下游产物(过氧化氢、过氧亚硝酸盐)是压力适应的主要候选信号分子。我们使用改变AOX数量的植物来操纵这些分子水平的能力，提供了一个强大的策略来测试关于来自特定细胞隔间-线粒体的特定活性氧和氮物种的信号传递能力的假说。我们将在两个分子水平上研究这种信号转导能力，以支持应激适应反应(即。核基因表达的变化)和细胞水平(即。守卫细胞运动)。 研究提案的另一个重要总体目标是指导和培训三名博士后研究员、八名研究生和许多本科生。这将包括培养独立性和多学科工作。

Alternative oxidase (AOX) is a non-energy conserving terminal oxidase in the plant mitochondrial electron transport chain that relaxes the coupling between respiratory carbon oxidation, electron transport, and ATP turnover. Despite key advances in our understanding of the genetic and biochemical control of AOX, the role of this auxiliary respiratory pathway in plant metabolism and mitochondrial physiology, as well as its overall impact on plant growth and performance remains poorly understood. As often highlighted in influential reviews, this represents a major gap in our understanding of plant primary metabolism. AOX gene expression is highly responsive to environmental cues and some evidence indicates that the pathway may be of particular importance under variable and stress conditions. Theoretically, AOX should negatively impact growth since it reduces ATP yield. However, it may have key roles in metabolism and mitochondrial physiology that outweigh this energy cost. My research proposal has two broad scientific goals. The first is to establish the role of AOX in supporting leaf metabolism. An emphasis will be to elucidate the mechanisms by which AOX supports photosynthesis. Our preliminary work shows that during drought stress, photosynthesis is strongly compromised in transgenic plants lacking AOX. Gas exchange and chlorophyll fluorescence analyses indicate that this is the result of a severe biochemical (rather than stomatal) limitation in plants lacking AOX. Detailed physiological and biochemical analyses will elucidate the nature of this limitation. We will also investigate the interaction of AOX and photosynthesis in plants grown at ambient versus high CO2. High CO2 has the potential to increase photosynthetic rate but can result instead in a down-regulation of photosynthetic capacity, such that the potential for higher source activity is not met. The propensity for such down-regulation depends strongly upon the response of sinks to high CO2. While respiration is a critical sink activity, little is known regarding specifically AOX at high CO2. This is a major gap in our understanding of how photosynthesis and growth will be impacted by future high CO2 conditions. A second broad scientific goal of the research proposal is to establish the role of AOX in supporting mitochondrial physiology. An emphasis will be elucidating the role of AOX in supporting the signaling function of mitochondria. Mitochondria act as signaling organelles involved in stress acclimation, but the primary “signals” from mitochondria that are involved in such responses are unknown. Our proposal builds upon a major finding from our previous work, where biochemical and confocal imaging analyses established that AOX controls the ETC generation of superoxide and nitric oxide, reactive species that can act as damaging molecules or signaling molecules within the cell. These reactive species, along with downstream products (hydrogen peroxide, peroxynitrite) are lead candidates as signal molecules for stress acclimation. Our ability to manipulate the level of these molecules using plants with altered AOX amount provides a powerful strategy to test hypotheses regarding the signaling capability of specific reactive oxygen and nitrogen species emanating from a specific cell compartment, the mitochondrion. We will investigate this signaling capability in support of stress acclimation responses at both the molecular level (ie. changes in nuclear gene expression) and cell level (ie. guard cell movements). Another important overall goal of the research proposal is the mentorship and training of three postdoctoral fellows, eight graduate students and numerous undergraduates. This will include a fostering of independence and multidisciplinary work.