Molecular basis of induction and execution of programmed cell death in plants

1.油菜素甾醇（brassinosteroids，BRs）诱导的基因表达谱的微阵列分析：为了了解BRs诱导的气管元件（Tracheary element，TE）细胞死亡机制，本研究利用微阵列技术对BRs诱导的基因表达进行了全面分析。因此，我揭示了大多数参与TE细胞死亡的基因被BR快速诱导，这表明BR是细胞死亡相关基因转录激活的启动子。2.细胞死亡相关基因表达的调控机制以细胞死亡相关基因ZCP 4为标记，鉴定了一个负责TE特异性表达的顺式元件，命名为气管元件调节顺式元件（Tracheary Element Regulating cis Element，TERE）。该元件不仅在细胞死亡相关基因的启动子中，而且在细胞壁相关基因的启动子中是保守的。利用酵母双杂交系统，分离到两个与TERE序列结合的转录因子，均为bZip型转录因子。3.核酸酶的分析对拟南芥S1核酸酶基因的所有成员（BFN 1-BFN 5）进行了启动子分析。BFN 1是一个仅在发育中的TE中表达的S1核酸酶基因。在不同类型的细胞死亡，不同的S1核酸酶基因的表达，这表明每个S1核酸酶基因参与不同的细胞死亡过程。我还鉴定了两个钙依赖性核酸酶的候选基因，它们的活性在TE细胞死亡前瞬时增加。4.核定位脂肪酶的功能分析我发现了一个百日菊脂肪酶基因，其mRNA以TE特异性的方式增加。在这里，我分离了它的拟南芥直向同源物，并分析了拟南芥脂肪酶基因的表达和功能。该基因在发育中的TE中优先表达，过量表达可导致异常的血管形成，提示其参与TE的细胞死亡过程。

1.Microarray analysis of comprehensive gene expression induced by brassinosteroids (BRs) :To understand tracheary element (TE) cell death mechanism induced by BRs, I analyzed gene expression induced by BRs comprehensively using microarray. As a result, I revealed that most genes involved in TE cell death are induced by BRs rapidly, suggesting that BRs are an initiator of transcriptional activation of cell death-related genes. I also identified transcription factors showing transient and rapid changes by BRs, which are candidates of regulators of cell death related genes.2.Regulation mechanism of the expression of cell death-related genesUsing a cell-death related gene, ZCP4 as a marker, I identified a cis-element responsible for TE-specific expression, which is designated Tracheary Element Regulating cis Element (TERE). This element is conserved in promoters not only of cell death-related genes but also of cell wall-related genes. Using yeast two hybrid system, I isolated two factors that bind the TERE sequence, both of which were bZip-type transcription factors.3.Analysis of nucleasesI performed promoter analysis of all the S1 nuclease gene members in Arabidopsis (BFN1-BFN5). BFN1 was only a S1 nuclease gene that was expressed in developing TEs. In various types of cells going to cell death, different S1 nuclease genes were expressed, suggesting that each S1 nuclease gene is involved in distinctive cell death process. I also identified two candidate genes of calcium-dependent nucleases whose activity increases transiently before TE cell death.4.Function analysis of a nucleus-locating lipaseI have indicated a Zinnia lipase gene whose mRNA increases in a TE-specific manner. Here I isolated its Arabidopsis ortholog and analyzed the expression and function of the Arabidopsis lipase gene. This gene is expressed preferentially in developing TEs and overproduction of the gene caused abnormal vascular formation, suggesting its involvement in cell death process of TEs.