Gilmore A M, Shinkarev V P, Hazlett T L, Govindjee G
Photobioenergetics Group, Australian National University Research School of Biological Sciences, Canberra, ACT.
Biochemistry. 1998 Sep 29;37(39):13582-93. doi: 10.1021/bi981384x.
The xanthophyll cycle-dependent dissipation of excitation energy in higher plants is one of the most important regulatory and photoprotective mechanisms in photosynthesis. Using parallel time-resolved and pulse-amplitude modulation fluorometry, we studied the influence of the intrathylakoid pH and the xanthophyll cycle carotenoids on the PSII chlorophyll (Chl) a fluorescence yield in thylakoids of Arabidopsis, spinach, and barley. Increases in concentrations of dithiothreitol in thylakoids, which have a trans-thylakoid membrane pH gradient and are known to have decreased conversion of violaxanthin (V) to zeaxanthin (Z), lead to (1) decreases in the fractional intensity of the approximately 0.5 ns Chl a fluorescence lifetime (tau) distribution component and simultaneous increases in a 1.6-1.8 ns fluorescence component and (2) increases in the maximal fluorescence intensity. These effects disappear when the pH gradient is eliminated by the addition of nigericin. To quantitatively explain these results, we present a new mathematical model that describes the simultaneous effects of the chloroplast trans-thylakoid membrane pH gradient and xanthophyll cycle pigments on the PSII Chl a fluorescence tau distributions and intensity. The model assumes that (1) there exists a specific binding site for Z (or antheraxanthin, A) among or in an inner antenna complex (primarily CP29), (2) this binding site is activated by a low intrathylakoid pH (pK approximately 4.5) that increases the affinity for Z (or A), (3) about one Z or A molecule binds to the activated site, and (4) this binding effectively "switches" the fluorescence tau distribution of the PSII unit to a state with a decreased fluorescence tau and emission intensity (a "dimmer switch" concept). This binding is suggested to cause the formation of an exciton trap with a rapid intrinsic rate constant of heat dissipation. Statistical analysis of the data yields an equilibrium association constant, Ka, that ranges from 0.7 to 3.4 per PSII for the protonated/activated binding site for Z (or A). The model explains (1) the relative fraction of the approximately 0.5 ns fluorescence component as a function of both Z and A concentration and intrathylakoid pH, (2) the dependence of the ratio of F'm/Fm on the fraction of the 0.5 ns fluorescence tau component (where F'm and Fm are maximal fluorescence intensities in the presence and the absence of a pH gradient), and (3) the dependence of the ratio of F'm/Fm on the concentration of Z and A and the intrathylakoid pH.
高等植物中叶黄素循环依赖的激发能耗散是光合作用中最重要的调节和光保护机制之一。我们使用并行时间分辨和脉冲幅度调制荧光测定法，研究了类囊体腔内pH值和叶黄素循环类胡萝卜素对拟南芥、菠菜和大麦类囊体中PSII叶绿素（Chl）a荧光产量的影响。类囊体中二硫苏糖醇浓度增加时，其具有跨类囊体膜pH梯度且已知紫黄质（V）向玉米黄质（Z）的转化减少，这会导致：（1）约0.5 ns Chl a荧光寿命（tau）分布成分的分数强度降低，同时1.6 - 1.8 ns荧光成分增加；（2）最大荧光强度增加。当通过添加尼日利亚菌素消除pH梯度时，这些效应消失。为了定量解释这些结果，我们提出了一个新的数学模型，该模型描述了叶绿体跨类囊体膜pH梯度和叶黄素循环色素对PSII Chl a荧光tau分布和强度的同时影响。该模型假设：（1）在内周天线复合物（主要是CP29）中或其内部存在Z（或中间型叶黄素，A）的特定结合位点；（2）该结合位点在低类囊体腔内pH（pK约为4.5）下被激活，从而增加对Z（或A）的亲和力；（3）约一个Z或A分子与激活位点结合；（4）这种结合有效地将PSII单元的荧光tau分布“切换”到荧光tau和发射强度降低的状态（“调光开关”概念）。这种结合被认为会导致形成具有快速固有热耗散速率常数的激子陷阱。对数据的统计分析得出平衡缔合常数Ka，对于Z（或A）的质子化/激活结合位点，每个PSII的Ka范围为0.7至3.4。该模型解释了：（1）约0.5 ns荧光成分的相对分数作为Z和A浓度以及类囊体腔内pH值的函数；（2）F'm/Fm比值对0.5 ns荧光tau成分分数的依赖性（其中F'm和Fm分别是存在和不存在pH梯度时的最大荧光强度）；（3）F'm/Fm比值对Z和A浓度以及类囊体腔内pH值的依赖性。
