Seedling transcriptome affected by Norflurazon-induced photobleaching of chloroplasts. Arabidopsis thaliana

Regulation of expression of genes encoding chloroplast components is critical to the autotrophic plant and never more so than in the cotyledons of the de-etiolating seedling. Many chloroplast proteins are nuclear-encoded and a retrograde signal from the chloroplasts (the Plastid Signal) modulates nuclear transcription. However, not all chloroplast-targeted genes are subject to this control and not all plastid-dependent nuclear genes are chloroplast-targeted. We therefore aim to provide the most comprehensive screen yet of which genes are affected by plastid-signalling. To specifically knock-out positive plastid signalling in light-grown cotyledons, the herbicide Norflurazon (NF) is supplied in the growth medium, causing a carotenoid deficiency that leaves the chloroplasts vulnerable to photobleaching. This blocks the expression of a subset of nuclear genes, such as Lhcb and HEMA1. Two pairs of RNAs will directly compare the transcription in seedlings grown under continuous white light with and without NF. A third RNA will also be compared from a mutant that shows a degree of constitutive positive plastid signalling. These two mutants act synergistically to counteract the effect of NF on nuclear transcription. The gun1,gun5 double mutant maintains a significantly higher level of Lhcb and HEMA1 expression in the presence of photobeached chloroplasts than the NF-treated wild-type. This transcriptome set will therefore complement RNA1 (wild-type+NF) and indicate which of the genes identified from the RNA1/RNA2 comparison are subject to the particular gun1/gun5 plastid signalling pathway(s). The growth of conditions of the seedlings (namely on MS medium supplemented with 1.5% sucrose, for 3 days under continuous WL following 2 days germination in darkness) has been chosen from the results of our own recent studies using Northern blotting techniques that show these conditions to maximise the respective NF and gun mutant effects on Lhcb and HEMA1 gene expression. This experiment is part one of a two-part study to compare the transcriptional output of this NF-affected pathway with that of a newly discovered FR-mediated pathway. A subsequent array experiment will assess the nuclear response as affected by this FR/ phyA-input pathway and the two sets of array data will be compared and contrasted. Note: Col-0 wild-type (NASC code N1092). gun1,gun5 double mutant(obtained from Enriquez Lopez-Juez, Royal Holloway, University of London.(Mochizuki et al. 2001 PNAS 98: 2053-2058). This line cannot be donated by us as it is the IP of Joanne Chory (SALK Institute, USA). Treatment = a herbicide (Norflurazon) application which leads to chloroplast photobleaching and hence down regulation of nuclear genes dependent on plastid signalling from intact chloroplasts. Experimenter name = Alex McCormac Experimenter phone = 023 80594297 Experimenter fax = 023 80594319 Experimenter address = School of Biological Sciences Experimenter address = Division of Cell Sciences Experimenter address = University of Southampton Experimenter address = Bassett Crescent East Experimenter address = Southampton Experimenter zip/postal_code = SO16 7PX Experimenter country = UK Keywords: genetic_modification_design Overall design: 6 samples were used in this experiment

编码叶绿体组分的基因表达调控对于自养植物至关重要，而在去黄化幼苗的子叶中，这一调控的重要性尤为突出。诸多叶绿体蛋白质由细胞核编码，而来自叶绿体的逆行信号——质体信号（Plastid Signal）可调控细胞核的转录过程。然而，并非所有靶向叶绿体的基因都受此调控，亦非所有依赖质体的核基因均靶向叶绿体。因此，本研究旨在全面筛查受质体信号通路影响的基因。

为特异性敲除光照生长子叶中的正向质体信号，我们在生长培养基中添加除草剂氟啶酮（Norflurazon, NF），该药剂会引发类胡萝卜素缺乏，使叶绿体极易发生光漂白，进而阻断部分核基因的表达，例如Lhcb与HEMA1。

我们将设置三组RNA样本：其中两组用于直接比较连续白光培养下添加与不添加NF的幼苗的转录组差异；第三组样本取自一株表现出组成型正向质体信号的突变体。其中两种突变体可协同抵消NF对核基因转录的抑制效应：gun1、gun5双突变体在光漂白叶绿体存在的条件下，其Lhcb与HEMA1的表达水平显著高于经NF处理的野生型植株。因此，该转录组数据集将作为RNA1（野生型+NF）的补充数据集，用于明确通过RNA1与RNA2的对比所鉴定出的基因中，哪些受特定的gun1/gun5质体信号通路调控。

本实验选用的幼苗培养条件——即在添加1.5%蔗糖的MS培养基上，先暗培养2天以萌发，随后在连续白光下培养3天——是基于本团队近期采用Northern印迹（Northern blotting）技术获得的研究结果确定的：该条件可最大化NF处理与gun突变体对Lhcb及HEMA1基因表达的调控效果。

本实验是两项系列研究的第一部分，旨在比较受NF影响的质体信号通路与新发现的远红光（Far-Red, FR）介导信号通路的转录输出差异。后续将通过基因芯片实验评估FR/光敏色素A（phyA）信号通路对核基因表达的影响，并将两组芯片数据进行对比分析。

注：Col-0野生型（NASC编号N1092）。gun1、gun5双突变体由伦敦大学皇家霍洛威学院的Enriquez Lopez-Juez提供（Mochizuki等，2001，《美国国家科学院院刊》98: 2053-2058）。由于该株系的知识产权归属于美国SALK研究所的Joanne Chory，本团队无法对外赠送该材料。
处理方式：施用除草剂氟啶酮（Norflurazon），可导致叶绿体光漂白，进而下调依赖完整质体信号的核基因表达。

实验者：Alex McCormac
联系电话：[phone]
传真：[phone]
通讯地址：英国南安普顿大学细胞科学分部生物科学学院，Bassett Crescent East，南安普顿，邮编SO16 7PX

关键词：基因修饰设计（genetic_modification_design）
整体实验设计：本实验共使用6个样本。