Systemic signalling of irradiance and CO2 concentration in Arabidopsis (Treatment 2: Elevated CO2 and Ambient Light). Arabidopsis thaliana

We were awarded a BBSRC grant about a year ago to undertake some affymetrix gene chip profiling of light and CO2 systemic signalling in Arabidopsis. The design of the proposed experiment is given below and the appropriate funding has been provided by the BBSRC. The aim of the project is to identify the temporal profile of those genes that respond to light and CO2 systemic signals in developing leaves. Moreover, as thes two signals have opposing effects on leaf development to ascertain whether they involve similar or parallel signalling pathways. The experiment is to examine the effect of exposing mature leaves to high CO2 or low light or both on the gene expression profile of developing leaves. We already have data for maize that changes in gene expression profile occur within 4h and that there are a variety of temporal responses that differ between individual gene transcripts. We have also demonstrated that Arabidopsis leaf development is altered by these systemmic signals and that lesions in the jasmonate and ethylene signalling pathways block these responses. Our experimental design is shown below: We have 4 treatments and 7 timepoints. (0, 2, 4, 12, 24, 48, 96 h) We would sample from 5 individual plants that would be pooled for each RNA preparation. This would require 28 chips and this would include extra replication of the 0 time-point control (deemed by many as nessary). Experimental details: All plants were germinated for 7 days under the following conditions: Humax multi-purpose compost, ambient carbon dioxide (370 ppm) and ambient light (250 µmol/m/s), constant temperature of 20°C and a 10 h photoperiod (8 am until 6 pm). After a week the the seedlings were potted up into 104-cell plug trays for a further 2 weeks and then potted up into 10 cm pots and the bottom part of the signalling cuvette system attached (see Lake et al., Nature 10th May 2001 Vol. 411, pp 154). Twenty four, 4 week old plants, then had the top part of the signalling system attached, trapping leaf insertions 5-13. Humidified, ambient air was passed through them at 500 mls/min via an oil-free air compressor. The three target leaves (19-21) were then marked with non-toxic, acrylic paint. After a 24 h period (the plants were sealed into the cuvettes from 10 am until 10am) of adjustment, the experiment was started by harvesting the target leaves from 4 plants and immediately freezing the tissue in liquid nitrogen to give the 0 h sample before RNA extraction. The remaining 20 plants were divided into 4 groups of five and given one of the following treatments: Ambient carbon dioxide/ambient light (Control) (A) Elevated carbon dioxide (750 ppm)/ambient light (E) Ambient carbon dioxide/low light (50 µmol/m/s) (AS) Elevated carbon dioxide/low light (ES) - For the Elevated CO2, elevated CO2 was pumped in using a CT room next door set to same temperature but with a CO2 cylinder inside and the same pump as used in the ambient room to supply the elevated CO2 laden, humidified air into the signalling room using rubber tubing. - Shade treatment consisted of neutral density filter (Cat. 210 0.6ND, Lee Filters) that had a hole cut in the middle to allow the middle developing leaves to grow through. A timecourse of 2, 4, 12, 24, 48 and 96 h were carried out each using a batch of 24 plants. This whole process was repeated with another batch of 24 plants at the same developmental stage to give a 2, 4, 12, 24, 48 and 96 hour sample from each of the four treatments. The whole timecourse was then repeated 4 times. For the mature leaves: We had 8 chips left over so we devised this little experiment to assess the gene changes that were occurring in the enclosed, treated, mature leaves that were signalling the environment to the young developing leaves. Experimenter name = Simon Coupe Experimenter phone = 0114 222 4115 Experimenter fax = 0114 222 0002 Experimenter institute = University of Sheffield Experimenter address = Animal and Plant Sciences Experimenter address = University of Sheffield Experimenter address = Western Bank Experimenter address = Sheffield Experimenter zip/postal_code = S10 2TN Experimenter country = UK Keywords: development_or_differentiation_design; growth_condition_design Overall design: 26 samples were used in this experiment

本研究于约一年前获得英国生物技术与生物科学研究理事会（Biotechnology and Biological Sciences Research Council, BBSRC）的项目资助，将开展拟南芥（Arabidopsis）中光与CO₂系统性信号转导的Affymetrix基因芯片分析实验，实验设计如下，相关经费已由BBSRC拨付。

本项目的研究目标为：鉴定发育叶片中响应光与CO₂系统性信号的基因的时序表达谱；鉴于这两种信号对叶片发育具有相反的调控效应，同时明确二者是否共享或平行运作的信号通路。本实验旨在探究：将成熟叶片暴露于高CO₂、低光照或二者联合处理后，对发育叶片基因表达谱的影响。

此前我们已获得玉米相关实验数据：基因表达谱的变化可在4小时内发生，且不同基因转录本的时序响应模式存在多样性。此外我们已证实：拟南芥的叶片发育可被上述系统性信号调控，且茉莉酸与乙烯信号通路的功能缺失突变体会阻断此类响应。

本实验设计如下：共设置4种处理组与7个采样时间点（0、2、4、12、24、48、96小时）。每个RNA样本将混合5株独立植株的组织；本次实验共需28张芯片，其中包含0小时对照组的额外生物学重复（该设置已被广泛认可为必要操作）。

### 实验细节
所有植株均在以下条件下发芽培养7天：采用Humax通用营养土，环境CO₂浓度为370 ppm，光照强度250 μmol·m⁻²·s⁻¹，恒温20℃，光周期为10小时（早8时至晚18时）。
发芽培养1周后，将幼苗移栽至104孔穴盘继续培养2周，随后定植于10 cm口径花盆中，并安装信号比色皿系统的底部组件（详见Lake等人于2001年5月10日发表于《Nature》第411卷第154页的研究）。
选取24株4周龄植株，安装信号比色皿系统的顶部组件，固定第5至13片叶位的叶片；通过无油空气压缩机以500 mL/min的流速向体系中通入加湿后的环境空气。随后用无毒丙烯酸涂料标记第19至21片叶作为目标叶片。
经过24小时的适应期（植株于当日早10时至次日早10时密封于比色皿中），采集4株植株的目标叶片并立即置于液氮中冷冻，获得0小时时间点的样本用于后续RNA提取，至此实验正式启动。
剩余20株植株分为4组，每组5株，分别接受以下一种处理：
1. 环境CO₂/正常光照（对照组，记为A）
2. 高CO₂（750 ppm）/正常光照（记为E）
3. 环境CO₂/低光照（50 μmol·m⁻²·s⁻¹，记为AS）
4. 高CO₂/低光照（记为ES）

#### 处理细节：
- 高CO₂处理：通过隔壁恒温CT室中的CO₂钢瓶与同环境组一致的气泵，经橡胶管路向信号处理室输送加湿后的高浓度CO₂空气，维持体系CO₂浓度为750 ppm。
- 遮阴处理：采用中性密度滤光片（货号210 0.6ND，Lee Filters生产），滤光片中央开孔以允许中部发育叶片正常伸出。

每个时间点（2、4、12、24、48、96小时）的采样均使用一批24株植株完成；使用另一批相同发育阶段的24株植株重复上述实验流程，以获得4种处理组分别对应上述6个时间点的样本。上述完整时间序列实验共重复4次。

针对成熟叶片的补充实验：因剩余8张芯片，我们额外设计了该小型实验，以分析被密封处理的成熟叶片（其正向幼嫩发育叶片传递环境信号）中的基因表达变化。

**实验负责人信息**：
姓名：Simon Coupe
联系电话：[phone]
传真号码：[phone]
所属机构：谢菲尔德大学
通讯地址：动植物科学系，谢菲尔德大学Western Bank校区，谢菲尔德
邮政编码：S10 2TN
国家：英国

**关键词**：发育与分化设计；生长条件设计
**实验总体设计**：本实验共使用26个样本。