Studies of cell-cell communication during vascular formation

1. 拟南芥维管突变体分析。Van1-van7：我们克隆了拟南芥维管突变体的致病基因，van1 -van6显示了我们筛选的碎片化维管模式。VAN3编码一种调节小g蛋白活化的蛋白。VAN4编码了一种未知的蛋白，但可能在膜运输中起作用。该基因在维管组织中优先表达。百日草叶肉细胞向木质部细胞分化的维管细胞分化分析。(1)从百日草中分离到植物磺酸素（phytosulfokine， PSK）同源物ZePSKl。ZePSKl mRNA在木质部细胞分化的早期和晚期积累。PSK生物合成抑制剂和PSK的脉冲治疗表明，PSK对早期和晚期过程的进展都是必要的。(2)为了鉴定血管细胞发育过程中控制细胞间通讯的因子，我们尝试分离识别这些因子的抗体。为此，我们采用了自己开发的噬菌体展示减法。最后，我们成功地分离出了约8000个抗体克隆。用随机选择的90种抗体进行组织染色，发现这些抗体对维管组织中的脂质体因子有不同的识别。因此，我们可以找到工具，找出参与细胞间通讯的因素。(3)成功分离编码“xylogen”，并鉴定其为气管元件诱导因子。成熟韧皮部HD-Zip同源box基因的分离与鉴定。我们分离到了一个百日草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，对伤茎韧皮部再生进行了分析。

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.