Mutant Screen Distinguishes between Residues Necessary for Light-Signal Perception and Signal Transfer by Phytochrome B
Mutant Screen Distinguishes between Residues Necessary for Light-Signal Perception and Signal Transfer by Phytochrome B
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DOI：
10.1371/journal.pgen.1000158
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发表时间：
2008-08-01
期刊：
影响因子：
4.5
通讯作者：
Nagatani, Akira
中科院分区：
文献类型：
--
作者：
Oka, Yoshito;Matsushita, Tomonao;Nagatani, Akira
光敏色素（phyA至phyE）是植物光感受器家族中的一个主要成员，它调节植物对光反应的多种发育过程。phyB的N-末端651个氨基酸结构域（N651）结合开放的四吡咯发色团，其作用是感知和抑制细胞核中的调节光信号。N651结构域包含几个亚结构域：N-末端延伸、Per/Arnt/Sim（PAS）样亚结构域（PLD）、cGMP磷酸二酯酶/腺苷酸环化酶/FhlA（GAF）亚结构域和光敏色素（PHY）亚结构域。为了确定这些亚结构域的功能作用，我们诱变表达N651的拟南芥株系，该株系串联融合绿色荧光蛋白、β-葡萄糖醛酸苷酶和核定位信号。长下胚轴突变体的大规模筛选确定了N651部分中的14个新的基因内错义突变。这些新的突变，沿着8个先前鉴定的突变，分布在整个N651，表明每个亚结构域具有重要的功能。在体外分析这些突变体的光谱特性，使他们能够被分为两个主要类别：光信号感知突变体（那些有缺陷的光谱活动），和信号突变体（那些正常的光感知，但有缺陷的细胞内信号传递）。大多数光谱突变体被发现在GAF和PHY亚结构域。另一方面，信号突变体倾向于位于N-末端延伸和PLD。这些观察结果表明，N-末端延伸和PLD主要参与信号传递，但C-末端GAF和PHY子域负责光感知。在信号转导突变体中，R110 Q、G111 D、G112 D和R325 K特别令人感兴趣。与最近描述的细菌光敏色素的PAS-GAF结构域的三维结构的比对表明，这四个突变位于光敏色素光敏结附近。
The phytochromes (phyA to phyE) are a major plant photoreceptor family that regulate a diversity of developmental processes in response to light. The N-terminal 651-amino acid domain of phyB (N651), which binds an open tetrapyrrole chromophore, acts to perceive and transduce regulatory light signals in the cell nucleus. The N651 domain comprises several subdomains: the N-terminal extension, the Per/Arnt/Sim (PAS)-like subdomain (PLD), the cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) subdomain, and the phytochrome (PHY) subdomain. To define functional roles for these subdomains, we mutagenized an Arabidopsis thaliana line expressing N651 fused in tandem to green fluorescent protein, beta-glucuronidase, and a nuclear localization signal. A large-scale screen for long hypocotyl mutants identified 14 novel intragenic missense mutations in the N651 moiety. These new mutations, along with eight previously identified mutations, were distributed throughout N651, indicating that each subdomain has an important function. In vitro analysis of the spectral properties of these mutants enabled them to be classified into two principal classes: light-signal perception mutants (those with defective spectral activity), and signaling mutants (those normal in light perception but defective in intracellular signal transfer). Most spectral mutants were found in the GAF and PHY subdomains. On the other hand, the signaling mutants tend to be located in the N-terminal extension and PLD. These observations indicate that the N-terminal extension and PLD are mainly involved in signal transfer, but that the C-terminal GAF and PHY subdomains are responsible for light perception. Among the signaling mutants, R110Q, G111D, G112D, and R325K were particularly interesting. Alignment with the recently described three-dimensional structure of the PAS-GAF domain of a bacterial phytochrome suggests that these four mutations reside in the vicinity of the phytochrome light-sensing knot.