Developing New Spectroscopic Techniques to Study Carotenoid-Dependent Non-Photochemical Quenching in Plants and Algae
Photosynthetic organisms live in environments with highly variable light conditions. In order to survive, they must maximize photosynthetic production while at the same time minimizing photodamage caused by excess absorbed sunlight. To prevent this photodamage, photosynthetic organisms have developed a suite of mechanisms called non-photochemical quenching (NPQ) mechanisms which dissipate excess excitation energy as heat. However, little is known about the specific molecular mechanisms that convert excitation energy to heat or how these mechanisms are activated and de-activated.
NPQ mechanisms have previously been characterized according to their timescale of activation, with the fastest group, referred to as qE mechanisms, responding within 30 s to a few minutes to increases in light intensity. As the fastest responders, these mechanisms are responsible for the largest portion of the energy quenching. Previous work has shown that these mechanisms require the carotenoid zeaxanthin (Zea) in order to fully function. There have been two types of proposed molecular mechanisms by which this carotenoid could be directly involved in excitation energy quenching, and this thesis describes the development of new spectroscopic techniques that are capable of tracking intermediate species associated with each of these mechanisms as samples acclimate to high light conditions. These techniques were then used to determine the role of each of these quenching mechanisms in several photosynthetic organisms.
The first molecular mechanism of interest is called charge transfer (CT) quenching. This mechanism involves a chlorophyll (Chl) –Zea dimer, which accepts excitation energy from light-harvesting chlorophylls, then undergoes charge separation, and finally charge recombination to complete the cycle. Previous transient absorption (TA) experiments found that an intermediate Zea cation species formed selectively in light-acclimated plant thylakoid samples, but did not determine at what point during the light acclimation process the Zea cation began forming. The difficulty lies in tracking sub-microsecond processes, such as Zea cation absorption, which evolve over seconds to minutes in real time. Combining these two timescales into one measurement has previously been developed in fluorescence lifetime snapshots, which measure quenching by following fast changes in the Chl fluorescence lifetime as a function of high-light acclimation. This thesis will discuss the development of a new transient absorption technique, called snapshot transient absorption, capable of tracking signals that change on the same timescale as qE, seconds to minutes. Development of this technique required numerous changes to the instrumental setup as well as optimization of sample preparation and data collection to obtain a sufficient signal-to-noise ratio to accurately track Zea cation signals in spinach photosynthetic membranes.
This technique, combined with previously established fluorescence lifetime snapshots as well as time-resolved HPLC measurements, determined that the timing and intensity of the Zea cation signal observed in spinach membranes is consistent with CT quenching acting as a qE quenching mechanism. However, results indicated that CT quenching only utilized a small percentage of the overall Zea concentration, implying that Zea may also participate in the second theorized quenching mechanism, excitation energy transfer (EET) quenching. The EET quenching mechanism involves energy transfer from excited Chl to the dark S1 state of Zea, which then rapidly relaxes down to the ground state.
To determine the feasibility of the EET quenching mechanism, snapshot TA technique was adapted to track the population of the Car S1 state in photosynthetic samples. Zea cations absorption occurs at a wavelength where the underlying Chl excited state dynamics are largely constant throughout the experiment, but the same is not true for Car S1 absorption. Therefore in order to collect data on Car S1 absorption, the snapshot TA was adjusted to account for changes in the Chl ESA background. These experiments determined that in spinach photosynthetic membranes, EET quenching likely occurs in tandem with CT quenching, though its activation time is slightly longer. Snapshot TA spectroscopy was also used to study a series of Nannochloropsis oceanica mutants. N. oceanica is a small ocean algae which appears to rely on Zea, as well as its epoxidized forms, for much of its quenching ability. While this research is still in progress, results thus far are discussed. It appears that the EET quenching signal in N. oceanica is highly dependent on the Zea concentration, and that lhcx1, a suspected quenching protein, does not perform EET quenching. Future work should focus on determining the relationship between the EET and CT quenching mechanisms in N. oceanica mutants.