生命科学学院知识库

光系统 II(PSII)是由多个亚基组成的色素蛋白复合体，它催化光驱动的水的裂解和醌的氧化。由于其结构的复杂性，PSII 的生物发生和组装需要核基因与叶绿体基因编码的蛋白以一定次序地合成并组装。这是一个多步骤的过程，需要大量核基因编码的辅助因子和调节蛋白的协助。到目前为止，对于 PSII 生物发生的调节因子知之甚少。分离、鉴定拟南芥中这些核基因编码的调节因子以及研究它们的作用机制有助于我们认识高等植物PSII复合物组装和功能调控的分子机理。基于这个原因，我们从 T-DNA 插入的拟南芥突变体库中筛选具有高叶绿素荧光表型的 PSII 突变体并对其中的一个突变体 lpa1 进行了详细的研究。

主要结果如下：1、通过对 62,000 个突变体株系进行高通量的筛选，获得了 136 个高叶绿素荧光的突变体。根据测定的叶绿素荧光慢诱导曲线，我们将突变体进行了简单的分类，其中一类属于 PSII 突变体。对 PSII 突变体进行 SDS-urea-PAGE 和蛋白免疫印记分析发现突变体中 PSII 蛋白的累积量与野生型相比显著降低，而其它复合物含量的变化不大。这些低 PSII 累积的突变体我们称之为lpa(lowphotosystemII accumulation)突变体。2、我们重点刻画了拟南芥突变体 lpa1。在突变体 lpa1 中，其生长量、色素含量与野生型相比均显著降低。叶绿素荧光诱导和 P700 的氧化还原动力学分析表明在突变体中主要是 PSII 的活性受到明显影响。蛋白免疫印记和 BN-PAGE电泳分析表明，突变体中可以形成有功能的 PSII 复合物，但是 PSII 的累积量仅有野生型的 20%左右。进一步用核酸杂交和多聚核糖体实验证明了 PSII 累积量的降低不是由于某个 PSII 亚基在转录和翻译起始水平受到影响而造成的，而可能是由于翻译后的组装或稳定性受到了影响。蛋白质标记实验表明突变体中D1、D2 的合成量显著降低，而其他叶绿体编码蛋白的翻译速率与野生型相似。新合成的PSII蛋白可以组装成有功能的复合物，但是组装效率没有野生型的高。除此之外，突变体中新合成的 PSII 亚基 D1、D2、CP43 和 CP47 的周转速度比野生型的快。3、用 IPCR 方法克隆到的 LPA1 基因编码一个含有 TPR 结构域的叶绿体膜蛋白，但它不是 PSII 的一个组分。LPA1 先于 PSII 复合物而存在于黄花苗中并且 LPA1 结合在核糖体的新生链组分中。酵母双杂交实验证明了 LPA1 与 D1 蛋白特异性的相互作用，而不和 D2、细胞色素 b 6 、Alb3 相互作用。因此，LPA1可能作为一个结合在膜上的分子伴侣通过直接与 PSII 反应中心蛋白 D1 相互作用，对 PSII 的有效组装起重要作用。4、用荧光衰减动力学和热发光分析突变体lpa1中电子在组装好的PSII受体侧和供体侧的传递几乎没有受到影响。在 20 µmol m -2  s -1 到 120 µmol m -2  s -1 光强之间，突变体中 F v /F m 随光强的升高而降低，而野生型则变化不大。将生长光下的突变体转移到弱光下时，F v /F m 有所升高。通过免疫印记分析这些处理过程中 PSII 蛋白累积的变化，发现突变体中 PSII 反应中心蛋白的含量与 F v /F m 是成正相关的。所以突变体中组装好的 PSII 是有功能的，并且与野生型比，突变体有更强的光敏感性。

Photosystem II (PSII) is a multisubunit pigment-protein complex that catalyzesthe light-driven water oxidation and reduction of plastoquinone. Because of the structural complexity of PSII, biogenesis and assembly of PSII requires the coordinated synthesis and assembly of both chloroplast- and nuclear-encoded proteins.These are likely to be a multi-step processes, which are assisted by many nuclear-encoded auxiliary and regulatory proteins. Until recently, the factors involved in regulating the biogenesis of PSII have remained largely unknown. Identification and characterization of these nuclear -encoded chloroplast proteins will increase our knowledge on molecular mechanisms of the PSII complexes biogenesis. To this purpose, we have screened PSII mutants with high chlorophyll fluorescence phenotypes from a collection of pSKI015 T-DNA-mutagenized Arabidopsis thaliana
lines, and we further characterized the lpa1 mutant.

The main results are summarized as follows:1.We have screened 62,000 T-DNA-mutagenized Arabidopsis thaliana lines and obtained 136 mutants with high chlorophyll fluorescence phenotype. Mutants with impaired energy transfer within PSII were identified based on the chlorophyll fluorescence induction kinetics. SDS-PAGE and immunoblot analyses indicated that the PSII subunits were reduced significantly and other complexes content were only affected slightly. These mutants with low photosystem II accumulation were nameded lpa mutants 2. To gain insight into the processes involved in PSII biogenesis and maintenance, we characterized the lpa1 mutant of Arabidopsis thaliana, which generally accumulates lower than wild-type levels of the PSII complex. The pigment contents, relative growth were significantly reduced in the mutant. Analyses of the chlorophyll fluorescence induction and P700 redox kinetics showed that the mutants have defects in energy transfer within PSII. Immunoblot and BN-PAGE studies
showed that the PSII can be assembled in the mutant, but the amounts of the PSII proteins were reduced to about 20% of wild-type levels. No alterations in theabundance or patterns of PSII gene transcripts were detected in the lpa1 mutant
demonstrate that the reduced PSII contents are not due to the absence of transcripts encoding one of these structural PSII proteins. Further more, no significant difference in the association with polysomes between the wild type and the mutant was detected for the psbA and psbD genes. In vivo protein labeling experiments showed that synthesis of the D1 and D2 proteins was greatly reduced in the lpa1 mutant while other plastid-encoded proteins were translated at rates similar to wild type. In addition,turnover rates of the PSII core proteins CP47, CP43, D1 and D2 were higher in lpa1 than in wild-type plants. The newly synthesized PSII proteins were assembled into functional protein complexes, but the assembly was less efficient in the mutant.3．LPA1 encodes a chloroplast protein that contains two tetratricopeptide repeat (TPR) domains and is an intrinsic membrane protein but not an integral subunit of PSII. Yeast two-hybrid studies revealed that LPA1 interacts with D1, but not with D2,Cyt b 6 or Alb3. Thus, LPA1 appears to be an integral membrane chaperone that is required for efficient PSII assembly, probably through direct interaction with the PSII reaction center protein D1.4．Analysis of fluorescence decay kinetics, and thermoluminescence profiles demonstrated that the electron-transfer reaction on either the donor or the acceptor side of the PSII remained largely unaffected in the lpa1 mutant. From 20 µmol m -2  s -1 to 120 µmol m -2  s -1 light intensity, the F v /F m decreased with increasing light intensity in the mutant, and remained almost unchanged in the WT plants under different light conditions. The Fv/Fm values were also increased when the mutant plants were transferred from the standard growth light to low light conditions. Analysis of PSII protein accumulation further confirmed that the amounts of PSII reaction center protein are correlated with the changes of Fv/Fm values in lpa1 plants. Thus, the assembled PSII in the mutant were functional and also showed increased photosensitivity compared with the WT plants.