Studies of cell-cell communication during vascular formation

1.拟南芥维管突变体的分析。van 1-van 7：我们克隆了拟南芥血管突变体的致病基因，van 1-van 6显示了我们筛选的片段化的血管模式。VAN 3编码一种调节小G蛋白活化的蛋白。VAN 4编码一种未知的蛋白质，但可能在膜运输中起作用。该基因在血管组织中优先表达.百日菊属离体系统中叶肉细胞向木质部细胞分化的维管细胞分化分析。(1)We分离的ZePSK 1，来自百日菊属的植物磺因子（PSK）的同源物。ZePSK 1 mRNA在木质部细胞分化的早期和晚期积累。用PSK生物合成和PSK的抑制剂进行脉冲处理表明，PSK对于早期和晚期过程的进展都是必要的。(2)为了确定在血管细胞发育过程中控制细胞间通讯的因素，我们试图分离识别这些因素的抗体。为此，我们使用了我们开发的噬菌体展示消减方法。最后成功地分离了约8000个抗体克隆。用随机选择的90种抗体进行的组织染色显示，这些抗体以不同的方式识别血管组织中的质外体因子。因此，我们可以得到工具，找出参与细胞间通讯的因素。(3)我们成功地分离出编码“木质素”的基因，并将其鉴定为一种导管因子诱导因子.未成熟韧皮部特异性HD-Zip同源框基因的分离和表征。我们分离到一个百日菊HD-Zip I类基因，其mRNA在未成熟韧皮部特异性积累.利用该基因作为韧皮部标记，分析了受伤茎的韧皮部再生。

1. Analysis with Arabidopsis vascular mutants. Van1-van7: We performed cloning of causal genes of Arabidqpsis vascular mutants, vanl-van6 showing fragmented vascular patterns that we have screened. VAN3 encoded a protein regulating the activation of small G-protein. VAN4 encoded a unknown protein, but which may function in membrane traffic. This gene was expressed preferentially hi vascular tissues.2. Analysis of vascular cell differentiation with an in vitro Zinnia system in which mesophyll cells differentiate into xylem cells.(1)We isolated ZePSKl, a homologue of phytosulfokine (PSK) from Zinnia. ZePSKl mRNA accumulated in early and late stages of xylem cell differentiation. Pulse treatment with an inhibitor of PSK biosynthesis and PSK revealed that PSK is necessary for progress of both early and late processes.(2) To identify factors controlling cell-cell communication during vascular cell development, we tried to isolate antibodies recognizing such factors. For such purpose, we used phage display subtraction method that we have developed. Finally we succeeded hi the isolation of about 8000 antibody clones. Tissue staining with randomly selected 90 antibodies revealed that these antibodies recognize apoplastic factors in vascular tissues differently. Thus we could get tools to find out factors involved of cell-cell communication.(3) We succeeded in the isolation encoding "xylogen" which we have identified as a tracheary element-inducing factor.2. Isolation and characterization of a ommature phloem-specific HD-Zip homeobox gene. We isolated a Zinnia HD- Zip class I gene whose mRNA accumulates specifically in immature phloem. Using this gene as a phloem marker, phloem regeneration was analyzed in wounded stems.