Catalase Expression to Study Information Flow To and From the Circadian Clock

大多数生物体表现出与昼夜环境振荡相关的时间组织，因此表现出昼夜节律。其中许多节律在持续的条件下持续存在，这表明生物体有能力测量时间，并利用这些时间信息来暂时调节他们的生物学。昼夜节律是内源性节律的一个子集，周期约为24小时，在没有环境时间提示的情况下持续存在。由于几个原因，增加对植物对环境输入和内源时间线索的反应的理解将具有广泛的农业重要性。环境信号和生物钟都有助于光周期决定开花，这对作物生产具有明显的影响。此外，人们越来越清楚的是，昼夜节律控制着植物对各种生物和非生物胁迫的反应，即对胁迫的反应作为昼夜节律的函数而变化。昼夜节律是昼夜节律性的一个组成部分，但人们对昼夜节律信息产生的分子机制知之甚少。拟南芥CAT2和Cat3过氧化氢酶基因提供了生物钟的分子指针，用来研究生物钟将基因表达门控到特定的昼夜节律阶段的分子基础。生物钟将CAT2和Cat3的mRNA丰度门控到不同的昼夜相，每个都不同于CAB mRNA丰度最高的时期，从昼夜钟到这三个基因的每个基因的输出通路中至少有一些成分必须根据定义是不同的。第二个目标是研究生物钟和光之间的相互作用。用连续的蓝光(而不是红光)照射可以检测到CAT2和Cat3mRNA丰度的昼夜振荡。此外，蓝光信号在长时间暗条件下抑制Cat3mRNA的振荡中也有一定的作用。生态型RLD不能抑制，并且显然携带着一种突变，该突变定义了这种抑制途径的新成分。具体的实验目标是：1.CAT表达所需的顺式作用元件的特征。对这些早晨特异的(CAT2)和晚上特异的(Cat3)基因的比较将有助于深入了解阶段决定的机制。这将需要构建一系列携带CAT-LUC融合的转基因株系，以确定控制CAT表达的昼夜反应元件和光响应元件。2.鉴定启动CAT2昼夜节律振荡所需的蓝光接收系统，以及Cat3-Luc表达的昼夜振荡启动突变。这将需要分析已知的蓝光感知和信号缺陷突变以及鉴定新的在启动Cat3-Luc表达昼夜振荡方面存在缺陷的突变体。在长时间黑暗中抑制cat3基因丰度昼夜振荡的机制的研究。如果在生态型RLD中看到的降凝素抑制是在遗传上可遗传的，突变的基因将被克隆和鉴定。还将确定在延长的黑暗中抑制Cat3mRNA丰度振荡所需的新的位置。

Most organisms exhibit temporal organization with respect to theenvironmental oscillation of day and night and, thus, express diurnalrhythms. Many of these rhythms persist in constant conditions, indicatingthat organisms have the capacity to measure time and to use this timeinformation to temporally regulate their biology. Circadian rhythms are asubset of endogenous rhythms with periods of approximately 24 hr thatpersist in the absence of environmental time cues. Increased understandingof plant responses to environmental input and to endogenous temporal cueswill have broad agricultural importance for several reasons. Bothenvironmental cues and the circadian clock contribute to the photoperiodicdecision to flower, with obvious implications for crop production.Additionally, it has become increasingly clear that the circadian clockgates the responses of plants to a variety of biotic and abiotic stresses;that is, the response to stresses varies as a function of circadian phase(time of day).Circadian phase is an integral aspect of circadian rhythmicity, yet littleis known of the molecular mechanisms by which phase information isimparted. The Arabidopsis CAT2 and CAT3 catalase genes provide molecularhands of the circadian clock with which to investigate the molecular basisby which the clock gates gene expression to specific circadian phases. Thecircadian clock gates mRNA abundance of CAT2and CAT3 to distinct circadianphases, and each is distinct from the phase of maximal CAB mRNA abundance.At least some of the components of the output pathway from the circadianclock to each of these three genes must, by definition, be distinct. Asecond goal is to investigate the interaction between the circadian clockand light. Illumination with continuous blue (but not with red) lightallows the detection of circadian oscillations in CAT2 and CAT3 mRNAabundance. In addition, there is a role for blue light signaling in thedamping of CAT3 mRNA oscillations in extended dark. Ecotype RLD fails todamp and apparently carries a mutation that defines a new component of thisdamping pathway. The specific experimental objectives are:1. The characterization of the cis-acting elements required for CATexpression. A comparison of these morning-specific (CAT2) andevening-specific (CAT3) genes will allow insight into the mechanisms ofphase determination. This will entail the construction of series oftransgenic lines carrying CAT-LUC fusionsin order to define circadianresponse elements and light response elements controlling CAT expression. 2. The characterization of the blue light photoperception system requiredfor initiation of circadian oscillation in CAT2 and CAT3 mRNA abundance.This will entail the analysis of known mutants with defects in blue lightperception and signaling as well as the identification of novel mutantsdefective in the initiation of circadian oscillations in CAT3-LUC expression.3. The investigation of the mechanism of damping of the circadianoscillations of CAT3 mRNA abundance seen in extended dark. If the defectin damping seen in ecotype RLD is inherited in a genetically tractablefashion, the mutated gene will be cloned and characterized. New locirequired for damping the oscillation in CAT3 mRNA abundance in extendeddark will also be identified.