Sun and shade leaves_2x ambient ozone_120906

Tropospheric ozone causes severe oxidative stress in plants. To investigate the transcriptional responsiveness of adult trees to ozone, fully-expanded sun and shade leaves of mature beech trees were harvested at four time points over the entire vegetation period in 2005 and 2006. Microarray analyses were conducted on leaves from trees grown in the field under ambient and twice-ambient ozone concentrations at Kranzberger Forst (Bavarian). Beech trees changed their transcript levels in response to ozone. In the years 2005 and 2006 different transcription patterns were observed; this may have been a result of different weather conditions and ozone uptake. Furthermore, we obtained differences in mRNA expression patterns between shade and sun leaves. In the ozone-treated sun leaves of 2005, slightly up- and down-regulated transcript levels were detected, particularly in the spring and autumn, whereas shade leaves clearly exhibited reduced mRNA-levels, particularly at the end of the vegetation period. In 2006, this pattern could not be confirmed, and in the autumn, four other transcripts were slightly up-regulated in ozone-treated shade leaves. In addition, two additional transcripts were found to be influenced in sun leaves in the spring/summer. While we detected changes in the levels of only a few transcripts, the observed effects were not identical in both years. In conclusion, elevated ozone exhibited very small influence on the transcription levels of genes of mature beech trees. The study was carried out at the Kranzberger Forst research site (near Freising, Germany: 48°25’08’’N, 11°39’41’””E, 485m (Pretzsch et al., 1998) in a mixed 60-year old stand (closed canopy) with about 30m high European beech (Fagus sylvatica) and Norway spruce (Picea abies) trees. Free-air ozone fumigation started in May 2000 at double the ambient ozone concentrations with a cut-off at 150 nl l-1 (Werner and Fabian, 2002), thereby avoiding acute damage to the leaves. The ozone concentrations were measured at four heights within the fumigated space and were additionally monitored with 200 passive samplers (Werner and Fabian, 2002). In 2005 the AOT40 value under twice ambient ozone was 64.3 μmol mol-1 h and in 2006 69.0 μmol mol-1 h. Detailed ozone concentration data over the growing seasons have been reported elsewhere (Gielen et al., 2007; Kitao et al., 2009). Sun and shade leaves from 60-year-old European beech trees were harvested in a total of 8 sampling campaigns in 2005 and 2006. Using scaffolding, five leaves of sun crown (height of about 25 m) and five shade (height of about 19 m) leaves were taken from each of five control and ozone-treated trees. The sampling was carried out in May, June, August, and September of 2005 and in June, August, September and October of 2006. To avoid diurnal effects, the samples were always taken around 11 a.m. For each tree, the four leaves (sun or shade) were combined, frozen in liquid nitrogen and stored at -80 °C until RNA isolation. For one time point we had five microarrays and five dye-swaps for each of sun and shade leaves. The probes of the trees under ambient ozone were labelled with Cy3 and probes of the trees under 2x ambient were labelled with Cy5. For every pair of trees a dye control were carried out, where the control trees were labelled with Cy5 and the ozone-treated one with Cy3. For statistical analysis a coefficient of variation about all microarrays of one time point was calculated using Acuity 4.0 microarray informatics software [Axon Instruments]. We used only those spots that had a coefficient of variation < 50 and were present on at least half of identical slides. The ozone-changed transcript level of genes were expressed in the median values as log2 ratios.

对流层臭氧（Tropospheric ozone）会对植物造成严重的氧化胁迫。为探究成年树木对臭氧的转录响应，本研究于2005和2006年的整个生长季内的四个时间点，采集了成熟山毛榉树（mature beech trees）完全展开的阳生叶与阴生叶。研究在德国巴伐利亚州的克兰茨贝格森林（Kranzberger Forst）开展，对田间种植于环境及两倍环境浓度臭氧条件下的山毛榉叶片进行了基因芯片分析（microarray analyses）。

山毛榉树会通过改变转录本水平响应臭氧胁迫。2005与2006年观测到了不同的转录模式，这可能与当年的气象条件及臭氧吸收量差异有关。此外，阴生叶与阳生叶间的mRNA表达模式也存在差异。2005年经臭氧处理的阳生叶中，仅在春季和秋季检测到轻微上调与下调的转录本水平；而阴生叶则明显表现出mRNA水平降低，尤其在生长季末期。2006年该模式未被重复，且在秋季经臭氧处理的阴生叶中有另外4种转录本被轻微上调；此外，春季/夏季的阳生叶中另有2种转录本受到影响。尽管仅检测到少量转录本的水平变化，且两年间观测到的效应并不完全一致。综上，臭氧浓度升高对成熟山毛榉树的基因转录本水平影响极小。

本研究于克兰茨贝格森林研究站（德国弗赖辛附近，北纬48°25′08″，东经11°39′41″，海拔485米；Pretzsch等，1998）开展，实验林为60年生的混交林（林分封闭），包含约30米高的欧洲山毛榉（Fagus sylvatica）与挪威云杉（Picea abies）。开放式空气臭氧熏蒸（free-air ozone fumigation）于2000年5月启动，以两倍环境臭氧浓度进行熏蒸，当浓度达到150 nl l-1时停止（Werner与Fabian，2002），以避免对叶片造成急性损伤。熏蒸空间内设置了四个高度的臭氧浓度监测点，同时辅以200个被动采样器进行监测（Werner与Fabian，2002）。2005年两倍环境臭氧条件下的AOT40值（AOT40 value）为64.3 μmol mol-1 h，2006年为69.0 μmol mol-1 h。生长季内的详细臭氧浓度数据已在其他文献中报道（Gielen等，2007；Kitao等，2009）。

2005和2006年共开展8次采样，采集60年生欧洲山毛榉的阳生叶与阴生叶。通过脚手架，从5株对照树与5株臭氧处理树中分别采集5片阳生冠层叶片（高度约25米）及5片阴生冠层叶片（高度约19米）。采样时间为2005年的5月、6月、8月、9月，以及2006年的6月、8月、9月、10月。为避免日节律影响，所有样本均于上午11点左右采集。每棵树的4片阳生叶或阴生叶合并后，用液氮速冻并保存于-80℃环境中，直至RNA提取（RNA isolation）。

每个时间点的阳生叶与阴生叶各设置5个基因芯片重复及5次染料互换（dye-swaps）。环境臭氧处理组的样本探针用Cy3荧光染料标记，两倍环境臭氧处理组的样本探针用Cy5荧光染料标记。每对树木均设置染料对照：对照树探针用Cy5标记，臭氧处理树探针用Cy3标记。统计分析阶段，使用Acuity 4.0基因芯片信息学软件（Acuity 4.0 microarray informatics software，Axon Instruments公司）计算单个时间点所有基因芯片的变异系数（coefficient of variation）。仅保留变异系数<50%且在至少半数平行芯片上存在的探针点。臭氧处理后基因的转录本变化水平以中位数形式的以2为底的对数比值（log2 ratios）表示。