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

大多数生物表现出时间组织相对于昼夜环境振荡，因此，表达昼夜节律。 这些节奏中的许多在恒定的条件下持续存在，表明生物体有能力测量时间，并使用此时间信息来暂时调节其生物学。 昼夜节律是内源性节律的一个子集，其周期约为24小时，在没有环境时间线索的情况下持续存在。 增加对植物对环境输入和内源性时间线索的反应的理解将具有广泛的农业重要性，原因有几个。 环境因子和生物钟都参与了植物开花的光生理决定，对作物生产具有重要意义，此外，生物钟还控制着植物对各种生物和非生物胁迫的反应。也就是说，对压力的反应是昼夜节律的函数昼夜节律相位是昼夜节律性的一个组成部分，但对相位信息的分子机制知之甚少。 拟南芥CAT 2和CAT 3过氧化氢酶基因提供了生物钟的分子指针，用于研究生物钟将基因表达控制在特定昼夜节律阶段的分子基础。 昼夜节律钟将CAT 2和CAT 3的mRNA丰度控制在不同的昼夜节律阶段，并且每个阶段都与CAB mRNA丰度最大的阶段不同。根据定义，从昼夜节律钟到这三个基因中的每一个的输出途径的至少一些组分必须是不同的。 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）照明可以检测CAT 2和CAT 3 mRNA丰度的昼夜振荡。 此外，蓝光信号在长时间黑暗中抑制CAT 3 mRNA振荡中也有作用。 生态型RLD不能阻尼，显然携带了一种突变，这种突变定义了这种阻尼途径的一个新成分。 具体的实验目标是：1. CAT表达所需的顺式作用元件的表征。 比较这些早晨特异性（CAT 2）和早晨特异性（CAT 3）基因将有助于深入了解相位决定的机制。 这将需要构建一系列携带CAT-LUC融合基因的转基因株系，以确定控制CAT表达的昼夜节律反应元件和光反应元件。2. 蓝光光感知系统的特性需要启动CAT 2和CAT 3 mRNA丰度的昼夜振荡，这将需要分析已知的蓝光感知和信号缺陷突变体，以及鉴定新的突变体，在CAT 3-LUC表达的昼夜振荡启动缺陷. 延长黑暗条件下CAT 3 mRNA丰度昼夜节律振荡衰减机制的研究。 如果在RLD生态型中看到的缺陷蛋白阻尼是以遗传上易处理的方式遗传的，那么突变的基因将被克隆和鉴定。 新的loci阻尼振荡所需的CAT 3 mRNA丰度在extendeddark也将被确定。

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.