Analyses on the dichroic phytochrome regulating phototropism and photorelocation of chloroplasts in ferns and mosses

在蕨类植物和其他低等植物包括苔藓中，许多光响应都受二色性光敏色素分子的调节。本论文旨在阐明二色性光敏色素分子，并分析其向光性和细胞内叶绿体光迁移的调控过程。主要研究结果如下：1）二色性光敏色素介导的小立碗藓叶绿体光迁移运动：与蕨类植物原丝体一样，小立碗藓原丝体细胞的叶绿体光迁移运动也首次被二色性光敏色素和二色性蓝光受体所调控。使用细胞骨架抑制剂对能动系统的进一步分析表明，光敏色素反应仅利用微管作为轨道，而蓝光反应同时使用微管和肌动蛋白微丝系统。光感受器对运动系统的差异调节以前从未报道过，并且与蕨类植物的情况形成鲜明对比，其中两种光感受器都调节微丝系统而不是微管系统。2）蕨类植物铁线蕨中二色性光敏色素的基因中断：通过将二色性光敏色素基因的候选者PHY3的破坏构建体导入原丝体细胞，我们获得了一个既缺乏向光性又缺乏受光敏色素调节的叶绿体光迁移的菌株。该菌株仍保持蓝光诱导的反应。3）蕨类植物PHY3基因过表达的立碗藓细胞分析：将蕨类植物PHY3基因导入藓类植物细胞中，并在35S启动子的驱动下过表达。但对红光诱导的向光性或叶绿体移位运动没有影响。4）二色性光敏色素定向改变的铁线蕨突变体的分离：筛选了二色性光敏色素胞内定向缺陷的突变体，但至今没有得到突变株。

Many photoresponses are regulated by dichroic phytochrome molecules in ferns and other lower plants including mosses. In the present work, I aimed to elucidate the dichroic phytochrome molecule and to analyze its regulatory processes of phototropism and intracellular chloroplast photorelocation. Results obtained are as follows.1)Dichroic phytochrome-mediate chloroplast relocation movement in the moss, Physcomitrella patens : As in the case of fern protonemata, chloroplast photorelocation movement in Physcomitrella protonemal cells was shown, for the first time, to be regulated by dichroic phytochrome as well as by dichroic blue light receptor. Further analyses on the motile system using cytoskeletal inhibitors revealed that phytochrome responses utilize only microtubules as tracks while blue light responses use both microtubule and actin microfilament systems. The differential regulation of motile systems by photoreceptors has never been reported before and well contrasts with case of fern, where both photoreceptors regulate microfilament system but not microtubule system.2)Gene disruption of dichroic phytochrome in the fern Adiantum capillus-veneris : By introducing a disruption construct of PHY3, a candidate of dichroic phytochrome gene to a protonemal cell, we obtained a strain which lacks both phototropism and chloroplast photorelocation regulated by phytochrome. The strain still retained the blue light-induced responses. Detailed analyses are still on the way.3)Analyses on the Physcomitrella cells over-expressing fern PHY3 gene : Fern PHY3 gene was introduced and over-driven by 35S promoter in the moss cells. However, no effect on the red light-induced phototropism or chloroplast relocation movement was observed.4)Isolation of Adiantum mutant altered in the orientation of dichroic phytochrome : Screening of mutants which are defective in the intracellular orientation of dichroic phytochrome are performed, but so far, no strains are obtained.