Exploration of molecular bases of polyamine's defensive roles against environmental stresses in plants

在植物中，多胺在细胞分裂、生长和分化中起着重要作用，因为在活跃分裂细胞和生殖器官中多胺含量较高。在非生物和生物胁迫条件下，编码多胺生物合成酶的基因的表达往往增强。因此，多胺有望在保护寄主植物免受各种环境胁迫方面发挥作用。在启动本项目之前，申请人在植物多胺研究方面有两个重要发现；即(1)精胺缺失突变体拟南芥对高盐和高脱水胁迫变得敏感，表明精胺对这两种胁迫具有防御作用。(2)精胺具有信号传导作用，可引起病原体防御相关基因子集的表达。2007年12月，一个研究小组报道，先前被认为编码精胺合成酶的ACL5基因编码了一种热精胺（精胺的一种异构体）合成酶。在此背景下，首先建立了分离精胺和热精胺两种四胺的方法。其次，通过监测β-葡萄糖醛酸酶报告基因在拟南芥启动子下的表达情况，分析了精胺合酶基因和热精胺合酶基因在拟南芥中的时空表达。在精胺信号通路中，多胺氧化酶（多胺氧化酶）可能参与其中。第三，对拟南芥和水稻的多胺氧化酶（PAO）基因进行了表征。在拟南芥中，将4个PAO基因克隆到大肠杆菌表达载体中。所有重组PAOs均能催化反转化反应，但不能催化多胺末端分解代谢反应。水稻基因组包含7个PAO基因。其中，至少有3个PAO基因催化了多胺的反转化反应，强烈提示水稻多胺有两条分解代谢途径。第四，精胺在热休克反应中具有防御作用（已提交，准备修订）。我们还证明了bZIP60是未折叠应激反应（UPR）的主基因之一，可被精胺上调。bZIP60的产物在生理状态下存在于内质网（ER）中，一旦UPR被触发，bZIP60转录物就会通过IRE1的RNase活性进行剪接。第五，精胺对bZIP60转录物进行剪接，产生靶向细胞核的活性bZIP60蛋白（手稿正在制备中）。在这个激活过程中，IRE1也参与其中。最后，我们发现热精胺在诱导防御基因和抑制植物病毒颗粒增殖方面具有与精胺相似的信号活性。少

In plants, polyamines function in cell division, growth and differentiation because the polyamine contents were found to be higher in actively dividing cells and in reproductive organs. Expression of the genes encoding polyamine biosynthesis enzymes is often enhanced in abiotic and biotic stress condition. Thus polyamines are expected to function for defending host plants against various environmental stresses. Before starting this project, the applicant did two important findings in plant polyamine research ; i. e.,(1)spermine-deficient mutant Arabidopsis plant became hypersensitive to high salinity and dehydration stresses, suggesting that spermine has a defensive role against these two stresses.(2)spermine has a signaling role to evoke the expression of a subset of genes involved in pathogen defense. In December, 2007, a research group reported that ACL5 gene which was previously thought to encode a spermine synthase, encodes a thermospermine (an isomer of spermine)synthase. With th … More is background, first we established a method to separate those two tetraamines, spermine and thermospermine. Second we analyzed the spatio-temporal expression of spermine synthase gene and thermospermine synthase gene in Arabidopsis by monitoring β-glucuronidase reporter gene expression under the promoters of these genes in Arabidopsis. In spermine-signaling pathway, the involvement of polyamine oxidase(s)is strongly suggested. Third, therefore, characterization of polyamine oxidase (PAO)genes of Arabidopsis thaliana and Oryza sativa was performed. In Arabidopsis, four of the PAO genes were cloned into Escherichia coli expression vector. All of the recombinat PAOs catalyzed the back-conversion reaction but not the polyamine terminal catabolism reaction. Rice genome contains 7 PAO genes. Of them, at least 3 PAO genes catalyzed the polyamine back-conversion reaction, strongly suggesting that rice plant has two catabolic pathways for polyamines. Fourth, spermine has a defensive role in heat shock response (submitted, revision prepared). We also demonstrated that bZIP60, one of the master genes in the unfolded stress response (UPR), is upregulated by spermine. The product of bZIP60 resides in endoplasmic reticulum (ER)in physiological condition, while, once UPR is triggered, it is shown that bZIP60 transcript is spliced by RNase activity of IRE1. Fifth, bZIP60 transcript is spliced by spermine and the resulting transcript produces the active bZIP60 protein which targets into the nucleus (manuscript in preparation). In this activation process, IRE1 is also involved. Lastly we showed that thermospermine has a similar signaling activity to induce the defense genes and inhibits phytoviral particle multiplication as spermine does. Less