Gilmor A M, Itoh S
Photobioenergetics Group, Research School of Biological Sciences, Australian National University, Canberra, ACT.
Philos Trans R Soc Lond B Biol Sci. 2000 Oct 29;355(1402):1371-84. doi: 10.1098/rstb.2000.0699.
This study presents a novel measurement, and simulation, of the time-resolved room temperature chlorophyll a fluorescence emission spectra from leaves of the barley wild-type and chlorophyll-b-deficient chlorina (clo) f2 and f104 mutants. The primary data were collected with a streak-camera-based picosecond-pulsed fluorometer that simultaneously records the spectral distribution and time dependence of the fluorescence decay. A new global spectral-kinetic analysis programme method, termed the double convolution integral (DCI) method, was developed to convolve the exciting laser pulse shape with a multimodal-distributed decay profile function that is again convolved with the spectral emission band amplitude functions. We report several key results obtained by the simultaneous spectral-kinetic acquisition and DCI methods. First, under conditions of dark-level fluorescence, when photosystem II (PS II) photochemistry is at a maximum at room temperature, both the clo f2 and clo f104 mutants exhibit very similar PS II spectral-decay contours as the wild-type (wt), with the main band centred around 685 nm. Second, dark-level fluorescence is strongly influenced beyond 700 nm by broad emission bands from PS I, and its associated antennae proteins, which exhibit much more rapid decay kinetics and strong integrated amplitudes. In particular a 705-720 nm band is present in all three samples, with a 710 nm band predominating in the clo f2 leaves. When the PS II photochemistry becomes inhibited, maximizing the fluorescence yield, both the clo f104 mutant and the wt exhibit lifetime increases for their major distribution modes from the minimal 205-500 ps range to the maximal 1500-2500 ps range for both the 685 nm and 740 nm bands. The clo f2 mutant, however, exhibits several unique spectral-kinetic properties, attributed to its unique PS I antennae and thylakoid structure, indicating changes in both PS II fluorescence reabsorption and PS II to PS I energy transfer pathways compared to the wt and clo f104. Photoprotective energy dissipation mediated by the xanthophyll cycle pigments and the PsbS protein was uninhibited in the clo f104 mutant but, as commonly reported in the literature, significantly inhibited in the clo f2; the inhibited energy dissipation is partly attributed to its thylakoid structure and PS II to PS I energy transfer properties. It is concluded that it is imperative with steady-state fluorometers, especially for in vivo studies of PS II efficiency or photoprotective energy dissipation, to quantify the influence of the PS I spectral emission.
本研究展示了一种对大麦野生型以及叶绿素b缺陷型绿质体（clo）f2和f104突变体叶片的时间分辨室温叶绿素a荧光发射光谱进行测量和模拟的新方法。主要数据是通过基于条纹相机的皮秒脉冲荧光计收集的，该荧光计同时记录荧光衰减的光谱分布和时间依赖性。开发了一种新的全局光谱动力学分析程序方法，称为双卷积积分（DCI）方法，用于将激发激光脉冲形状与多峰分布的衰减轮廓函数进行卷积，该衰减轮廓函数再与光谱发射带幅度函数进行卷积。我们报告了通过同时进行光谱动力学采集和DCI方法获得的几个关键结果。首先，在暗水平荧光条件下，当室温下光系统II（PS II）光化学处于最大值时，clo f2和clo f104突变体与野生型（wt）表现出非常相似的PS II光谱衰减轮廓，主峰位于685 nm左右。其次，700 nm以上的暗水平荧光受到来自PS I及其相关天线蛋白的宽发射带的强烈影响，这些发射带表现出更快的衰减动力学和较强的积分幅度。特别是在所有三个样品中都存在一个705 - 720 nm的波段，在clo f2叶片中710 nm的波段占主导。当PS II光化学受到抑制，荧光产率最大化时，clo f104突变体和wt的主要分布模式的寿命都从最小的205 - 500 ps范围增加到685 nm和740 nm波段的最大1500 - 2500 ps范围。然而，clo f2突变体表现出一些独特的光谱动力学特性，这归因于其独特的PS I天线和类囊体结构，表明与wt和clo f104相比，PS II荧光再吸收和PS II到PS I的能量转移途径都发生了变化。由叶黄素循环色素和PsbS蛋白介导的光保护能量耗散在clo f104突变体中未受抑制，但正如文献中普遍报道的那样，在clo f2中受到显著抑制；能量耗散的抑制部分归因于其类囊体结构和PS II到PS I的能量转移特性。得出的结论是，对于稳态荧光计来说，尤其是在对PS II效率或光保护能量耗散进行体内研究时，量化PS I光谱发射的影响是至关重要的。
