Escola Superior de Tecnologia da Saúde de Lisboa, Av. D. João II, Lt 4.69.01 Parque das Nações 1990-096 Lisboa, Portugal.
Physiol Plant. 2010 Mar;138(3):301-11. doi: 10.1111/j.1399-3054.2009.01335.x. Epub 2009 Nov 23.
Highly purified, intact chloroplasts were prepared from pea (Pisum sativum L.) and spinach (Spinacia oleracea L.) following an identical procedure, and were used to investigate the cupric cation inhibition on the photosynthetic activity. In both species, copper inhibition showed a similar inhibitor concentration that decreases the enzyme activity by 50% (IC(50) approximately 1.8 microM) and did not depend on the internal or external phosphate (Pi) concentration, indicating that copper did not interact with the Pi translocator. Fluorescence analysis suggested that the presence of copper did not facilitate photoinhibition, because there were no changes in maximal fluorescence (F(m)) nor in basal fluorescence (F(o)) of copper-treated samples. The electron transport through the photosystem II (PSII) was also not affected (operating efficiency of PSII-F'v/F'm similar in all conditions). Yet, under Cu(2+) stress, the proportion of open PSII reaction centers was dramatically decreased, and the first quinone acceptor (Q(A)) reoxidation was fully inhibited, as demonstrated by the constant photochemical quenching (q(P)) along experiment time. The quantum yield of PSII electron transport (Phi(PSII)) was also clearly affected by copper, and therefore reduced the photochemistry efficiency. Manganese, when added simultaneously with copper, delayed the inhibition, as measured by oxygen evolution and chlorophyll fluorescence, but neither reversed the copper effect when added to copper-inhibited plastids, nor prevented the inhibition of the Hill activity of isolated copper-treated thylakoids. Our results suggest that manganese competed with copper to penetrate the chloroplast envelope. This competition seems to be specific because other divalent cations e.g. magnesium and calcium, did not interfere with the copper action in intact chloroplasts. All results do suggest that, under these conditions, the stroma proteins, such as the Calvin-Benson cycle enzymes or others are the most probable first target for the Cu(2+) action, resulting in the total inhibition of chloroplast photosynthesis and in the consequent unbalanced rate of production and consumption of the reducing power.
从豌豆（Pisum sativum L.）和菠菜（Spinacia oleracea L.）中按照相同的程序制备高度纯化的完整叶绿体，并用于研究铜离子对光合作用的抑制作用。在这两种植物中，铜的抑制作用具有相似的抑制剂浓度，即降低酶活性 50%（IC 50 约为 1.8 μM），并且不依赖于内部或外部磷酸盐（Pi）浓度，表明铜不与 Pi 转运蛋白相互作用。荧光分析表明，铜的存在不会促进光抑制，因为铜处理样品的最大荧光（F m ）和基础荧光（F o ）没有变化。通过光系统 II（PSII）的电子传递也不受影响（所有条件下 PSII 的操作效率 F'v/F'm 相似）。然而，在 Cu 2+胁迫下，开放 PSII 反应中心的比例显著降低，第一醌受体（Q A ）的再氧化完全受到抑制，这表现为实验过程中恒定的光化学猝灭（q P ）。PSII 电子传递的量子产率（Phi PSII ）也明显受到铜的影响，从而降低了光化学效率。当同时添加锰时，正如氧释放和叶绿素荧光所测量的那样，会延迟抑制作用，但当添加到铜抑制的类囊体时，既不能逆转铜的作用，也不能防止铜处理的类囊体中单离的希尔活性的抑制。我们的结果表明，锰与铜竞争穿透叶绿体包膜。这种竞争似乎是特异性的，因为其他二价阳离子，例如镁和钙，不会干扰完整叶绿体中的铜作用。所有结果都表明，在这些条件下，基质蛋白，如卡尔文-本森循环酶或其他蛋白，可能是铜作用的第一个靶标，导致叶绿体光合作用的完全抑制，以及还原力的产生和消耗之间的不平衡速率。
