Dall'Osto Luca, Caffarri Stefano, Bassi Roberto
Dipartimento Scientifico e Tecnologico, Università di Verona, 37134, Verona, Italy.
Plant Cell. 2005 Apr;17(4):1217-32. doi: 10.1105/tpc.104.030601. Epub 2005 Mar 4.
The regulation of light harvesting in higher plant photosynthesis, defined as stress-dependent modulation of the ratio of energy transfer to the reaction centers versus heat dissipation, was studied by means of carotenoid biosynthesis mutants and recombinant light harvesting complexes (LHCs) with modified chromophore binding. The npq2 mutant of Arabidopsis thaliana, blocked in the biosynthesis of violaxanthin and thus accumulating zeaxanthin, was shown to have a lower fluorescence yield of chlorophyll in vivo and, correspondingly, a higher level of energy dissipation, with respect to the wild-type strain and npq1 mutant, the latter of which is incapable of zeaxanthin accumulation. Experiments on purified thylakoid membranes from all three mutants showed that the major source of the difference between the npq2 and wild-type preparations was a change in pigment to protein interactions, which can explain the lower chlorophyll fluorescence yield in the npq2 samples. Analysis of the xanthophyll binding LHC proteins showed that the Lhcb5 photosystem II subunit (also called CP26) undergoes a change in its pI upon binding of zeaxanthin. The same effect was observed in wild-type CP26 upon treatment that leads to the accumulation of zeaxanthin in the membrane and was interpreted as the consequence of a conformational change. This hypothesis was confirmed by the analysis of two recombinant proteins obtained by overexpression of the Lhcb5 apoprotein in Escherichia coli and reconstitution in vitro with either violaxanthin or zeaxanthin. The V and Z containing pigment-protein complexes obtained by this procedure showed different pIs and high and low fluorescence yields, respectively. These results confirm that LHC proteins exist in multiple conformations, an idea suggested by previous spectroscopic measurements (Moya et al., 2001), and imply that the switch between the different LHC protein conformations is activated by the binding of zeaxanthin to the allosteric site L2. The results suggest that the quenching process induced by the accumulation of zeaxanthin contributes to qI, a component of NPQ whose origin was previously poorly understood.
高等植物光合作用中光捕获的调节，定义为能量向反应中心转移与热耗散比率的应激依赖性调节，通过类胡萝卜素生物合成突变体和具有修饰发色团结合的重组光捕获复合物（LHC）进行了研究。拟南芥的npq2突变体在紫黄质生物合成中受阻，因此积累了玉米黄质，与野生型菌株和npq1突变体相比，其体内叶绿素荧光产量较低，相应地能量耗散水平较高，后者无法积累玉米黄质。对所有三种突变体纯化的类囊体膜进行的实验表明，npq2和野生型制剂之间差异的主要来源是色素与蛋白质相互作用的变化，这可以解释npq2样品中较低的叶绿素荧光产量。对叶黄素结合的LHC蛋白的分析表明，光系统II亚基Lhcb5（也称为CP26）在结合玉米黄质后其pI发生变化。在野生型CP26中，经处理导致膜中玉米黄质积累时也观察到了相同的效果，并被解释为构象变化的结果。通过在大肠杆菌中过表达Lhcb5脱辅基蛋白并在体外与紫黄质或玉米黄质重组获得的两种重组蛋白的分析证实了这一假设。通过该程序获得的含V和Z的色素 - 蛋白质复合物分别显示出不同的pI和高、低荧光产量。这些结果证实了LHC蛋白以多种构象存在，这一观点是先前光谱测量结果所暗示的（Moya等人，2001年），并意味着不同LHC蛋白构象之间的转换是由玉米黄质与变构位点L2的结合激活的。结果表明，玉米黄质积累诱导的猝灭过程有助于qI，NPQ的一个组成部分，其起源以前了解甚少。
