Snapshot transient absorption spectroscopy: toward in vivo investigations of nonphotochemical quenching mechanisms.

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 S1 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.

Chlorophyll-carotenoid excitation energy transfer and charge transfer in Nannochloropsis oceanica for the regulation of photosynthesis.

Nonphotochemical quenching (NPQ) is a proxy for photoprotective thermal dissipation processes that regulate photosynthetic light harvesting. The identification of NPQ mechanisms and their molecular or physiological triggering factors under in vivo conditions is a matter of controversy. Here, to investigate chlorophyll (Chl)-zeaxanthin (Zea) excitation energy transfer (EET) and charge transfer (CT) as possible NPQ mechanisms, we performed transient absorption (TA) spectroscopy on live cells of the microalga Nannochloropsis oceanica We obtained evidence for the operation of both EET and CT quenching by observing spectral features associated with the Zea S1 and Zea●+ excited-state absorption (ESA) signals, respectively, after Chl excitation. Knockout mutants for genes encoding either violaxanthin de-epoxidase or LHCX1 proteins exhibited strongly inhibited NPQ capabilities and lacked detectable Zea S1 and Zea●+ ESA signals in vivo, which strongly suggests that the accumulation of Zea and active LHCX1 is essential for both EET and CT quenching in N. oceanica.

Chlorophyll-Carotenoid Excitation Energy Transfer in High-Light-Exposed Thylakoid Membranes Investigated by Snapshot Transient Absorption Spectroscopy.

Nonphotochemical quenching (NPQ) provides an essential photoprotection in plants, assuring safe dissipation of excess energy as heat under high light. Although excitation energy transfer (EET) between chlorophyll (Chl) and carotenoid (Car) molecules plays an important role in NPQ, detailed information on the EET quenching mechanism under in vivo conditions, including the triggering mechanism and activation dynamics, is very limited. Here, we observed EET between the Chl Q y state and the Car S1 state in high-light-exposed spinach thylakoid membranes. The kinetic and spectral analyses using transient absorption (TA) spectroscopy revealed that the Car S1 excited state absorption (ESA) signal after Chl excitation has a maximum absorption peak around 540 nm and a lifetime of ∼8 ps. Snapshot TA spectroscopy at multiple time delays allowed us to track the Car S1 ESA signal as the thylakoid membranes were exposed to various light conditions. The obtained snapshots indicate that maximum Car S1 ESA signal quickly rose and slightly dropped during the initial high-light exposure (<3 min) and then gradually increased with a time constant of ∼5 min after prolonged light exposure. This suggests the involvement of both rapidly activated and slowly activated mechanisms for EET quenching. 1,4-Dithiothreitol (DTT) and 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) chemical treatments further support that the Car S1 ESA signal (or the EET quenching mechanism) is primarily dependent on the accumulation of zeaxanthin and partially dependent on the reorganization of membrane proteins, perhaps due to the pH-sensing protein photosystem II subunit S.

Snapshot Transient Absorption Spectroscopy of Carotenoid Radical Cations in High-Light-Acclimating Thylakoid Membranes.

Nonphotochemical quenching mechanisms regulate light harvesting in oxygenic photosynthesis. Measurement techniques for nonphotochemical quenching have typically focused on downstream effects of quenching, such as measuring reduced chlorophyll fluorescence. Here, to directly measure a species involved in quenching, we report snapshot transient absorption (TA) spectroscopy, which rapidly tracks carotenoid radical cation signals as samples acclimate to excess light. The formation of zeaxanthin radical cations, which is possible evidence of zeaxanthin-chlorophyll charge-transfer (CT) quenching, was investigated in spinach thylakoids. Together with fluorescence lifetime snapshot data and time-resolved high-performance liquid chromatography (HPLC) measurements, snapshot TA reveals that Zea•+ formation is closely related to energy-dependent quenching (qE) in nonphotochemical quenching. Quantitative and dynamic information on CT quenching discussed in this work give insight into the design principles of photoprotection in natural photosynthesis.

Modular analysis of hipposin, a histone-derived antimicrobial peptide consisting of membrane translocating and membrane permeabilizing fragments.

Antimicrobial peptides continue to garner attention as potential alternatives to conventional antibiotics. Hipposin is a histone-derived antimicrobial peptide (HDAP) previously isolated from Atlantic halibut. Though potent against bacteria, its antibacterial mechanism had not been characterized. The mechanism of this peptide is particularly interesting to consider since the full hipposin sequence contains the sequences of parasin and buforin II (BF2), two other known antimicrobial peptides that act via different antibacterial mechanisms. While parasin kills bacteria by inducing membrane permeabilization, buforin II enters cells without causing significant membrane disruption, harming bacteria through interactions with intracellular nucleic acids. In this study, we used a modular approach to characterize hipposin and determine the role of the parasin and buforin II fragments in the overall hipposin mechanism. Our results show that hipposin kills bacteria by inducing membrane permeabilization, and this membrane permeabilization is promoted by the presence of the N-terminal domain. Portions of hipposin lacking the N-terminal sequence do not cause membrane permeabilization and function more similarly to buforin II. We also determined that the C-terminal portion of hipposin, HipC, is a cell-penetrating peptide that readily enters bacterial cells but has no measurable antimicrobial activity. HipC is the first membrane active histone fragment identified that does not kill bacterial or eukaryotic cells. Together, these results characterize hipposin and provide a useful starting point for considering the activity of chimeric peptides made by combining peptides with different antimicrobial mechanisms. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.