Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant
Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant
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DOI：
10.1105/tpc.019000
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发表时间：
2004-03-01
期刊：
影响因子：
11.6
通讯作者：
Schroeder, JI
中科院分区：
文献类型：
--
作者：
Leonhardt, N;Kwak, JM;Schroeder, JI
基于寡聚体的DNA亲和基因芯片代表了约三分之一的拟南芥（Arabidopsis thaliana）基因，用于分析单一细胞类型（保卫细胞）中的全局基因表达，鉴定了1309个保卫细胞表达的基因。在存在转录抑制剂的情况下分离高纯度的保卫细胞和叶肉细胞制剂，所述转录抑制剂防止在细胞分离过程中诱导胁迫诱导基因。保卫细胞的表达谱进行了比较与叶肉细胞，导致在识别64个转录表达优先保卫细胞。已知拟南芥基因组中存在许多大的基因家族和基因重复，从而产生冗余，这极大地阻碍了常规的遗传和功能基因组分析。所提出的基因组规模的分析确定冗余表达的特定亚型属于大的基因家族在单细胞水平，这提供了一个强大的工具，功能基因组特征的许多信号转导途径，在保卫细胞中发挥作用。29个基因的逆转录PCR证实了基因芯片结果的可靠性。对脱落酸（阿坝）调控基因启动子区的统计分析揭示了一个过度表达的阿坝响应基序，这是已知的阿坝响应元件。有趣的是，表达谱揭示了许多已知的保卫细胞阿坝信号组分在转录水平的阿坝调制。我们进一步确定了高度ABA诱导的蛋白磷酸酶2C转录，AtP 2C-HA，在保卫细胞。AtP 2C-HA中的T-DNA破坏突变赋予ABA对气孔关闭和种子萌发的超敏调节。所提供的数据为细胞类型特异性基因组规模的基因功能分析提供了基础。
Oligomer-based DNA Affymetrix GeneChips representing about one-third of Arabidopsis (Arabidopsis thaliana) genes were used to profile global gene expression in a single cell type, guard cells, identifying 1309 guard cell-expressed genes. Highly pure preparations of guard cells and mesophyll cells were isolated in the presence of transcription inhibitors that prevented induction of stress-inducible genes during cell isolation procedures. Guard cell expression profiles were compared with those of mesophyll cells, resulting in identification of 64 transcripts expressed preferentially in guard cells. Many large gene families and gene duplications are known to exist in the Arabidopsis genome, giving rise to redundancies that greatly hamper conventional genetic and functional genomic analyses. The presented genomic scale analysis identifies redundant expression of specific isoforms belonging to large gene families at the single cell level, which provides a powerful tool for functional genomic characterization of the many signaling pathways that function in guard cells. Reverse transcription-PCR of 29 genes confirmed the reliability of GeneChip results. Statistical analyses of promoter regions of abscisic acid (ABA)-regulated genes reveal an overrepresented ABA responsive motif, which is the known ABA response element. Interestingly, expression profiling reveals ABA modulation of many known guard cell ABA signaling components at the transcript level. We further identified a highly ABA-induced protein phosphatase 2C transcript, AtP2C-HA, in guard cells. A T-DNA disruption mutation in AtP2C-HA confers ABA-hypersensitive regulation of stomatal closing and seed germination. The presented data provide a basis for cell type-specific genomic scale analyses of gene function.