Department of Chemistry, University of California, Berkeley, CA, 94720, USA.
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.
Photosynth Res. 2019 Sep;141(3):367-376. doi: 10.1007/s11120-019-00640-x. Epub 2019 Apr 24.
Although the importance of nonphotochemical quenching (NPQ) on photosynthetic biomass production and crop yields is well established, the in vivo operation of the individual mechanisms contributing to overall NPQ is still a matter of controversy. In order to investigate the timescale and activation dynamics of specific quenching mechanisms, we have developed a technique called snapshot transient absorption (TA) spectroscopy, which can monitor molecular species involved in the quenching response with a time resolution of 30 s. Using intact thylakoid membrane samples, we show how conventional TA kinetic and spectral analyses enable the determination of the appropriate wavelength and time delay for snapshot TA experiments. As an example, we show how the chlorophyll-carotenoid charge transfer and excitation energy transfer mechanisms can be monitored based on signals corresponding to the carotenoid (Car) radical cation and Car S excited state absorption, respectively. The use of snapshot TA spectroscopy together with the previously reported fluorescence lifetime snapshot technique (Sylak-Glassman et al. in Photosynth Res 127:69-76, 2016) provides valuable information such as the concurrent appearance of specific quenching species and overall quenching of excited Chl. Furthermore, we show that the snapshot TA technique can be successfully applied to completely intact photosynthetic organisms such as live cells of Nannochloropsis. This demonstrates that the snapshot TA technique is a valuable method for tracking the dynamics of intact samples that evolve over time, such as the photosynthetic system in response to high-light exposure.
虽然非光化学猝灭（NPQ）对光合作用生物量生产和作物产量的重要性已得到充分证实，但导致整体 NPQ 的各个机制的体内运作仍存在争议。为了研究特定猝灭机制的时间尺度和激活动力学，我们开发了一种称为快照瞬态吸收（TA）光谱学的技术，该技术可以以 30 秒的时间分辨率监测参与猝灭响应的分子种类。使用完整的类囊体膜样品，我们展示了常规 TA 动力学和光谱分析如何能够确定快照 TA 实验的适当波长和时间延迟。例如，我们展示了如何根据分别对应于类胡萝卜素（Car）自由基阳离子和 Car S 激发态吸收的信号来监测叶绿素-类胡萝卜素电荷转移和激发能量转移机制。使用快照 TA 光谱学以及之前报道的荧光寿命快照技术（Sylak-Glassman 等人，Photosynth Res 127:69-76, 2016）可以提供有价值的信息，例如特定猝灭物质的同时出现和 Chl 的整体猝灭。此外，我们还表明，快照 TA 技术可以成功应用于完全完整的光合生物体，如 Nannochloropsis 的活细胞。这表明快照 TA 技术是一种跟踪随时间演变的完整样品动力学的有价值的方法，例如光合作用系统对高光暴露的反应。
