Zea mays Transcriptome or Gene expression. Zea mays

During Zea mays (maize) C4 differentiation, mesophyll (M) and bundle sheath (BS) cells accumulate distinct sets of photosynthetic enzymes, with very low photosystem II (PSII) content in BS chloroplasts. Consequently, there is little linear electron transport in the BS and ATP is generated by cyclic electron flow. In contrast, M thylakoids are very similar to those of C3 plants and produce the ATP and NADPH that drive metabolic activities. Regulation of this differentiation process is poorly understood but involves expression and coordination of nuclear and plastid genomes. Here, we identify a recessive allele of the maize Hcf136 homologue that in Arabidopsis thaliana functions as a PSII stability or assembly factor located in the thylakoid lumen. Proteome analysis of the thylakoids and electron microscopy reveal that Zm hcf136 lacks PSII complexes and grana thylakoids in M chloroplasts, consistent with the previously defined Arabidopsis function. Interestingly, hcf136 is also defective in processing the full-length psbB-psbT-psbH-petB-petD polycistron specifically in M chloroplasts. To determine whether the loss of PSII in M cells affects C4 differentiation, we performed cell-type specific transcript analysis of hcf136 and wild-type seedlings. The results indicate that M and BS cells respond uniquely to the loss of PSII, with little overlap in gene expression changes between data sets. These results are discussed in the context of signals that may drive differential gene expression in C4 photosynthesis. Keywords: cell type comparison Overall design: To explore the disruption of PSII activity on gene expression, transcript profiles from separated M and BS cells were examined using two-label microarray analysis. Total RNA was isolated from the second leaves of mutant and wild-type silbings. Six biological replicates were used to compare wild-type and mutant transcript profiles in separate M and BS experiments. To maximize biological replication, different seedling pools were used for each of the 12 hybridizations. Microarray experiments and analyses were performed using the Genisphere MPX900 kit and the Maize Array Consortium oligonucleotide platform (GPL5439; GPL5440). Feature intensity values were log-transformed and corrected for local background signal, and a LOWESS procedure (Dudoit et al., 2002) was used to normalize between channels. Features with either low or saturating signal intensity were discarded from further analysis. High expression filtering was less stringent to avoid elimination of previously characterized, high abundance, C4 cell-specific transcripts. After filtering, features that were not assigned an MZ number by the Maize Array Consortium were discarded from further analysis. The moderated t-test (Smyth, 2004) using the R package limma was applied to identify differentially expressed genes. The p-values for each test (gene) were converted to q-values for false discovery rate analysis as described by Storey et al. (2004). To avoid confounding treatment effects associated with direct comparisons of M and BS transcriptomes (Sawers et al., 2007), comparisons were only made using the same cell type across the hcf136 and wild-type sibling genotypes. Bundle sheath (BS) Samples: GSM245063-GSM245164 Mesophyll (M) Samples: GSM245165 - GSM245206

在玉蜀黍（Zea mays，玉米）C4光合途径分化过程中，叶肉细胞（mesophyll, M）与维管束鞘细胞（bundle sheath, BS）会积累不同的光合酶组，且维管束鞘叶绿体中的光系统II（photosystem II, PSII）含量极低。因此，维管束鞘内几乎无线性电子传递，ATP通过环式电子流生成。与之相对，叶肉类囊体与C3植物的类囊体极为相似，可产生驱动代谢活动的ATP与NADPH。目前对该分化过程的调控机制尚不清楚，但该过程涉及核基因组与质体基因组的表达与协调。本研究鉴定到玉米Hcf136同源基因的隐性等位基因，该基因在拟南芥（Arabidopsis thaliana）中作为定位在类囊体腔的PSII稳定性或组装因子发挥功能。对类囊体的蛋白质组分析与电子显微镜观察显示，Zm hcf136突变体的叶肉叶绿体中缺乏PSII复合物与基粒类囊体，这与此前在拟南芥中确定的功能一致。有趣的是，hcf136突变体还存在特异性缺陷，无法在叶肉叶绿体中加工全长psbB-psbT-psbH-petB-petD多顺反子。为探究叶肉细胞中PSII的缺失是否会影响C4分化，我们对hcf136突变体与野生型同系幼苗开展了细胞类型特异性转录组分析。结果表明，叶肉细胞与维管束鞘细胞对PSII缺失的响应存在显著差异，两组数据集间的基因表达变化重叠度极低。本研究结果结合可能驱动C4光合作用中差异基因表达的信号通路进行了讨论。
关键词：细胞类型比较
整体实验设计：为探究PSII活性紊乱对基因表达的影响，我们采用双标记微阵列分析对分离得到的叶肉与维管束鞘细胞的转录谱进行检测。从突变体与野生型同系植株的第二片叶中提取总RNA。我们设置6个生物学重复，分别对叶肉细胞与维管束鞘细胞开展野生型与突变体转录谱的比较实验。为最大化生物学重复的有效性，12次杂交实验分别使用不同的幼苗混合样本。微阵列实验与分析采用Genisphere MPX900试剂盒与玉米芯片联盟（Maize Array Consortium）寡核苷酸平台（GPL5439; GPL5440）完成。将探针信号强度进行对数转换，并对局部背景信号进行校正，随后采用LOWESS方法（Dudoit等，2002）完成通道间标准化。信号强度过低或饱和的探针将被排除在后续分析之外。为避免剔除此前已被鉴定的高丰度C4细胞特异性转录本，高表达过滤的标准设置得较为宽松。过滤完成后，未被玉米芯片联盟分配MZ编号的探针将被排除在后续分析之外。采用R包limma中的限制性t检验（Smyth，2004）鉴定差异表达基因。将每个基因检验得到的p值转换为q值，以进行错误发现率分析，具体方法参照Storey等（2004）的描述。为避免直接比较叶肉与维管束鞘转录组时产生的混杂处理效应（Sawers等，2007），我们仅在hcf136突变体与野生型同系基因型间，针对同一细胞类型开展比较分析。
维管束鞘细胞（BS）样本：GSM245063-GSM245164
叶肉细胞（M）样本：GSM245165 - GSM245206