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

认为种子的干燥耐受性是由母源产生的ABA （abscisic acid， ABA）通过积累Lea蛋白引起的，而Lea蛋白的表达受ABA信号转导介质ABI3/VP1因子的控制。然而，由于很难收集到相同发育阶段的合子胚胎，aba诱导种子耐干燥的分子机制尚不清楚。然后，我利用胡萝卜体细胞胚胎进行了研究，以阐明其机制，因为我们可以很容易地收集到许多处于相同发育阶段的胚胎。首先阐明了外源外用ABA对体胚具有较强的干燥耐受性。接下来，我克隆了一个与ABI3/VP1基因同源的胡萝卜基因（C-ABI3），并检测了其表达模式。C-ABI3在体细胞和合子胚中均有胚胎特异性表达。该基因在胡萝卜种子中的表达先于种子中内源ABA的短暂增加。此外，我还制作了转基因胡萝卜植株，并培养了在叶片或细胞中表达C-ABI3的细胞。利用这些植物和经ABA处理的培养细胞，我检测了胡萝卜ECP基因的表达模式，其表达具有胚胎特异性和ABA诱导性，属于Lea基因家族。结果表明，在ABA处理的叶片和细胞中有部分ECP基因表达，而在未处理和未转化ABA的叶片和细胞中没有表达。此外，aba处理的转基因培养细胞表现出明显的干燥耐受性。这些结果表明，C-ABI3是一种胚胎特异性转录因子，通过ABA诱导ECP基因的表达和ECP蛋白的积累获得种子的干燥耐受性，而ECP基因的表达和积累是由C-ABI3因子作为ABA信号转导的中介来控制的。

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.