Yi Xiaoping, McChargue Myriam, Laborde Susan, Frankel Laurie K, Bricker Terry M
Department of Biological Sciences, Biochemistry and Molecular Biology Section, Louisiana State University, Baton Rouge, Louisiana 70803, USA.
J Biol Chem. 2005 Apr 22;280(16):16170-4. doi: 10.1074/jbc.M501550200. Epub 2005 Feb 18.
Interfering RNA was used to suppress the expression of two genes that encode the manganese-stabilizing protein of photosystem II in Arabidopsis thaliana, MSP-1 (encoded by psbO-1, At5g66570), and MSP-2 (encoded by psbO-2, At3g50820). A phenotypic series of transgenic plants was recovered that expressed high, intermediate, and low amounts of these two manganese-stabilizing proteins. Chlorophyll fluorescence induction and decay analyses were performed. Decreasing amounts of expressed protein led to the progressive loss of variable fluorescence and a marked decrease in the fluorescence quantum yield (F(v)/F(m)) in both the absence and the presence of dichloromethylurea. This result indicated that the amount of functional photosystem II reaction centers was compromised in the plants that exhibited intermediate and low amounts of the manganese-stabilizing proteins. An analysis of the decay of the variable fluorescence in the presence of dichlorophenyldimethylurea indicated that charge recombination between Q ((A-)) and the S(2) state of the oxygen-evolving complex was seriously retarded in the plants that expressed low amounts of the manganese stabilizing proteins. This may have indicated a stabilization of the S(2) state in the absence of the extrinsic component. Immunological analysis of the photosystem II protein complement indicated that significant losses of the CP47, CP43, and D1 proteins occurred upon the loss of the manganese-stabilizing proteins. This indicated that these extrinsic proteins were required for photosystem II core assembly/stability. Additionally, although the quantity of the 24-kDa extrinsic protein was only modestly affected by the loss of the manganese-stabilizing proteins, the 17-kDa extrinsic protein dramatically decreased. The control proteins ribulose bisphosphate carboxylase and cytochrome f were not affected by the loss of the manganese-stabilizing proteins; the photosystem I PsaB protein, however, was significantly reduced in the low expressing transgenic plants. Finally, it was determined that the transgenic plants that expressed low amounts of the manganese-stabilizing proteins could not grow photoautotrophically.
干扰RNA被用于抑制拟南芥中编码光系统II锰稳定蛋白的两个基因的表达，即MSP-1（由psbO-1编码，At5g66570）和MSP-2（由psbO-2编码，At3g50820）。获得了一系列转基因植物表型，它们分别高水平、中等水平和低水平表达这两种锰稳定蛋白。进行了叶绿素荧光诱导和衰减分析。表达蛋白量的减少导致可变荧光逐渐丧失，并且在有无二氯甲基脲的情况下，荧光量子产率（F(v)/F(m)）均显著降低。这一结果表明，在中等水平和低水平表达锰稳定蛋白的植物中，功能性光系统II反应中心的数量受到了损害。在二氯苯基二甲基脲存在下对可变荧光衰减的分析表明，在低水平表达锰稳定蛋白的植物中，Q((A-))与放氧复合体的S(2)态之间的电荷复合严重受阻。这可能表明在没有外在成分的情况下S(2)态得到了稳定。对光系统II蛋白质组成的免疫分析表明，随着锰稳定蛋白的丧失，CP47、CP43和D1蛋白显著减少。这表明这些外在蛋白是光系统II核心组装/稳定性所必需的。此外，虽然24 kDa外在蛋白的量仅受到锰稳定蛋白丧失的适度影响，但17 kDa外在蛋白显著减少。对照蛋白核酮糖二磷酸羧化酶和细胞色素f不受锰稳定蛋白丧失的影响；然而，光系统I的PsaB蛋白在低表达转基因植物中显著减少。最后，确定了低水平表达锰稳定蛋白的转基因植物不能进行光合自养生长。
