Studies on molecular mechanisms of induction of seed desiccation tolerance using somatic embryos as a model

种子的脱水耐性被认为是由母体产生的脱落酸(ABA)通过积累Lea蛋白诱导的，其表达受ABA信号转导的中介ABI3/VP1因子调控。然而，ABA诱导种子脱水耐受的分子机制尚不清楚，因为在同一发育阶段收集合子胚胎是非常困难的。然后，我研究了利用胡萝卜体细胞胚胎的机制，因为我们可以很容易地在同一发育阶段收集许多胚胎。首先，我明确了体细胞胚胎通过外源ABA获得了很强的脱水耐性。其次，我克隆了一个与ABI3/VP1基因同源的胡萝卜基因(C-ABI3)，并对其表达模式进行了检测。C-ABI3在体细胞胚胎和合子胚胎中均有胚胎特异性表达。在胡萝卜种子中观察到内源ABA的表达先于种子内源ABA的瞬时增加。此外，我还培育了转基因胡萝卜植株和培养细胞，其中C-ABI3在叶片或细胞中表达。利用这些植株和经ABA处理的培养细胞，我研究了胡萝卜ECP基因的表达模式，该基因是胚胎特异的，ABA诱导的，属于LeA基因家族。结果表明，在ABA处理的叶片和细胞中观察到一些ECP基因的表达，而在ABA处理的未处理和未转化的叶片和细胞中则没有表达。此外，经ABA处理的转基因培养细胞表现出明显的耐脱水能力。这些结果表明，C-ABI3是一种胚特异性转录因子，种子的脱水耐性是通过ABA诱导ECP基因的表达和ECP蛋白的积累获得的，这些ECP基因和ECP蛋白的积累是由C-ABI3因子作为ABA信号转导的中介调控的。

It is thought that seed desiccation tolerance is induced by maternally produced abscisic acid (ABA) through accumulation of Lea proteins of which the expression is controlled by ABI3/VP1 factor, a mediator of ABA signal transduction. However, the molecular mechanisms of the ABA-induced seed desiccation tolerance remains to be clarified, because it is very difficult to collect zygotic embryos at the same developmental stages. Then, I had investigated to clarify the mechanisms using carrot somatic embryos, because we can easily collect many embryos at the same developmental stages. At first, I clarified that somatic embryos acquire strong desiccation tolerance by exgenously appied ABA.Next, I had cloned a carrot gene (C-ABI3) homologous to ABI3/VP1 gene and examined the expression pattern. C-ABI3 showed embryo-specific expression in both somatic and zygotic embryos. The expression in carrot seeds was observed prior to the transient increase of endogenous ABA in the seeds. Furthermore, I had produced transgenic carrot plants and cultured cells in which C-ABI3 was expressed in the leaves or cells. Using these plants and cultured cells treated with ABA, I had examined the expression pattern of carrot ECP genes, the expression being embryo-specific and ABA-inducible and belonging to Lea gene family. The results showed that the expression of some ECP genes was observed in ABA-treated leaves and cells, but not in ABA non-treated ones and non-transformed ones irrespective of ABA treatment. Moreover, ABA-treated transgenic cultured cells showed clearly, desiccation tolerance. These results indicate that C-ABI3 is an embryo-specific transcription factor and that seed desiccation tolerance is acquired by ABA-inducibIe expression of ECP genes and accumulation of ECP proteins which are controlled by C-ABI3 factor as a mediator of ABA signal transduciton.