Expression data from leaflets of domesticated tomato and wild-related Solanum pennellii plants subjected to water stress. Expression data from leaflets of domesticated tomato and wild-related Solanum pennellii plants subjected to water stress

Transcriptomics study which main goal is to elucidate the programme of gene expression triggered by water stress in leaflets of the drought-tolerant wild-related tomato Solanum pennellii (acc. PE47) compared with domesticated tomato (S. lycopersicum, cv. P73). In this study we used S. lycopersicum (Sl) (cv. P73) and S. pennellii (Sp) (acc. PE47) species displaying remarkable divergences regarding drought tolerance, to investigate the physiological and molecular responses in leaves of plants grown without stress (control) and after four days of water withholding (water stress, WS), when plant water loss was significant but leaves did not show visual dehydration symptoms yet. Significant physiological differences between species were found, showing Sp leaves higher ability to avoid water loss. Leaf transcriptomic analysis showed important constitutive expression differences between Sp and Sl, including genes with unknown function. In relation to the genes specifically induced by drought in Sp, those linked to stomatal closure, cell wall and primary carbohydrate metabolism and, specially, nitrogen metabolism were identified. Thus, genes linked to NH4+ assimilation, GOGAT/GS cycle and the GDH- and GABA-shunt were specifically induced by water stress in leaves of Sp. Our results showed also the up-regulation in Sp of genes involved in JA biosynthesis pathway, which were induced in both conditions, whereas genes involved in ET biosynthesis were specifically induced under WS. Regarding ET signaling, ERF genes were up-regulated by WS in Sp, hinting at the importance of these transcriptional regulators in the drought response of Sp. Overall design: Domesticated tomato (cv. P73) and wild-related tomato S. pennellii (acc. PE47) plants were cultivated by pairs in pots with substrate. Eighteen pots with plants having 2-3 full-developed leaves for domesticated tomato, and 3-4 full-developed leaves for wild-related tomato were selected (18 P73 plants and 18 PE47 plants in total). Nine couples divided into three biological replicates were maintained in standard culture conditions (control) while the other nine, also divided into three biological replicates, were subjected to water stress treatment by withholding watering during four days (water stress, WS). RNA isolated from leaflets coming from three individual pooled plants (biological replicates) was used in hybridization to one chip, resulting in total 12 chips: three replicates of P73 control, three replicates of PE47 control, three replicates of P73 WS and three replicates of PE47 WS. The biotinilated cRNA was hybridized to GeneChip® Tomato Genome Array of Affymetrix interrogating 10209 sequences. The intensity value of each probe in the array were processed and normalized according to the Robust Multichip Average (RMA) method to obtain an individual intensity value for each probeset. First filtering, normalization and second filtering of probes yield a final list of 5050 sequences (working list). A statistical analysis based in multiple test corrections was performed using the FDR test on the working list. Genes with an FDR < 0.05 and a fold change (ratio value) of ≥ 2.0 when comparing genotypes in the same experimental condition were identified as differentially expressed genes. A final list of 535 genes was accomplished with the above conditions. The annotation of probe sets was obtained from Affymetrix and loaded in the Partek Genomics Suite software (Partek Incorporated, St. Louis, USA). For the functional study of DEGs MapMan software was used (http://mapman.gabipd.org). The Slyc_AFFY_SGN_BUILD2_070709 database was loaded and used for this functional analysis. The statistical analysis followed was of Willcoxon Rank Sum test with Benjamini-Hochberg correction.

本转录组学（Transcriptomics）研究的核心目标为解析耐旱野生近缘番茄Solanum pennellii（登录号PE47）小叶中由水分胁迫（water stress, WS）触发的基因表达程序，并与栽培番茄（S. lycopersicum，品种P73）进行对比。
本研究选用在耐旱性上存在显著分化的两个物种——栽培番茄S. lycopersicum（Sl，品种P73）与野生近缘番茄S. pennellii（Sp，登录号PE47），探究两种番茄在无胁迫（对照组）及持续控水4天（水分胁迫组，WS）后的叶片生理与分子响应。此时植株水分流失已较为显著，但叶片尚未出现肉眼可见的脱水症状。
研究发现两物种间存在显著生理差异，Sp叶片具备更强的保水能力。
叶片转录组学分析显示，Sp与Sl之间存在重要的组成型表达差异，其中包含部分功能未知的基因。
针对Sp中特异性受干旱诱导的基因，研究鉴定出与气孔关闭、细胞壁及初级碳水化合物代谢相关的基因，尤以氮代谢相关基因为突出。具体而言，与NH4+同化、GOGAT/GS循环以及GDH和GABA分流相关的基因，在Sp叶片中被水分胁迫特异性诱导。
本研究结果还显示，Sp中参与茉莉酸（JA）生物合成通路的基因在两种处理条件下均被诱导；而参与乙烯（ET）生物合成通路的基因则仅在水分胁迫条件下被诱导。
关于乙烯信号通路，乙烯响应因子（Ethylene Response Factor, ERF）基因在Sp中被水分胁迫上调，提示这类转录调控因子在Sp的干旱响应中发挥关键作用。
【实验总体设计】：将栽培番茄（品种P73）与野生近缘番茄S. pennellii（登录号PE47）植株成对种植于基质盆中。共选取18盆植株：栽培番茄每盆拥有2~3片完全展开叶，野生近缘番茄每盆拥有3~4片完全展开叶，总计18株P73植株与18株PE47植株。其中9盆分为3个生物学重复，维持标准培养条件（对照组）；剩余9盆同样分为3个生物学重复，接受4天控水的水分胁迫处理（水分胁迫组，WS）。
从3株混合取样的植株小叶中分离得到的RNA（生物学重复）用于芯片杂交，总计制备12张芯片：P73对照组、PE47对照组、P73水分胁迫组、PE47水分胁迫组各3个生物学重复。
将生物素标记的互补RNA（cRNA）与Affymetrix公司的GeneChip®番茄基因组芯片进行杂交，该芯片可检测10209条序列。芯片上每个探针的强度值经稳健多芯片平均法（Robust Multichip Average, RMA）处理并标准化，以获得每个探针集的单独强度值。对探针进行首轮过滤、标准化及二次过滤后，最终得到包含5050条序列的工作列表。
基于该工作列表，采用错误发现率（False Discovery Rate, FDR）检验进行多重检验校正后的统计分析。在相同实验条件下比较基因型间差异时，FDR<0.05且倍数变化（比值）≥2.0的基因被鉴定为差异表达基因（Differentially Expressed Genes, DEGs）。最终共得到符合上述条件的535个基因。
探针集的注释信息来自Affymetrix公司，并导入Partek Genomics Suite软件（Partek Incorporated，美国圣路易斯）进行后续分析。对于差异表达基因的功能研究，本研究使用了MapMan软件（http://mapman.gabipd.org），并加载Slyc_AFFY_SGN_BUILD2_070709数据库开展功能分析。统计分析采用Wilcoxon秩和检验，并结合Benjamini-Hochberg校正。