Chloroplast photoprotection: face the light or run and hide?

Abstract

Light is both a source of energy and a potential harm for photosynthetic organisms because overexcitation causes photodamage (Mullineaux & Karpinski, 2002). In the highlighted publication, Zubik-Duda et al. focused on two mechanisms that plants have developed to avoid photodamage: chloroplast movement (Gotoh et al., 2018); and excitation quenching (Mullineaux & Karpinski, 2002). Under low light, chloroplasts accumulate along periclinal cell walls, perpendicular to the direction of the light, in order to absorb more photons. Under high light, chloroplasts move to the anticlinal cell walls that are parallel to the light beam direction, in order to avoid excess light (Gotoh et al., 2018). In the process of excitation quenching, excess excitation energy is dissipated either photochemically or non-photochemically. In non-photochemical quenching (NPQ), triplet-state chlorophyll excitation energy is transferred to carotenoids that dissipate the excess energy as heat during their return to a non-excited ground state (Mullineaux & Karpinski, 2002). This excitation quenching can be quantified in intact leaves by measuring chlorophyll a (Chla) fluorescence (Cazzaniga et al., 2013). However, both chloroplast movement and excitation quenching can act simultaneously and are both triggered by similar light intensities and wavelengths, therefore it is difficult to separate them on the basis of fluorescence intensity measurements. Monika Zubik-Duda, assistant professor in the Department of Biophysics, of the Maria Curie-Skłodowska University in Lublin, Poland, together with Wieslaw Gruszecki, head of the department, and their teams, raised the question of whether these parallel processes interfere with the results of the analyses of the individual mechanisms. They tackled the problem by using fluorescence lifetime imaging microscopy (FLIM) to measure both chloroplast movement and excitation quenching at the same time (Zubik-Duda et al., 2023). NPQ measurements based on the average fluorescent lifetime can be performed at very low light intensity, and have the advantage that Chla fluorescence lifetime is independent of the position of chloroplasts in the cell, whereas previous measurements quantified total Chla fluorescence intensity in a whole leaf, and were therefore subject to chloroplast position and orientation (Cazzaniga et al., 2013). For these measurements, Zubik-Duda et al. focused on spongy mesophyll cells in Arabidopsis leaves and used a pulsed blue laser to scan the images, activating excitation quenching and phototropins, the blue light photoreceptors that control chloroplast movements. Measurements of photosynthetic activity under different light intensities showed that at higher light, the photosynthesis rate measured by CO2 fixation increased and reached saturation. At the same time, photochemical quenching decreased and NPQ increased, probably as a mechanism to avoid photodamage due to high light intensities. Determination of the Chla fluorescence lifetime by FLIM and the relative chloroplast-free area deduced from the FLIM image showed that there were three distinguishable ranges of the blue light intensities based on different photoprotective responses (Figure 1a,b). The initial increase in light intensity to 100 μmol m−2 sec−1 led to movement of the chloroplasts to the central area of the cell, accompanied by an increase in the average lifetime of Chla fluorescence. This ensures greater absorption of light quanta combined with a relatively low rate of excitation quenching and therefore optimal conditions for photosynthesis. In the second light intensity range between 100 and 1000 μmol m−2 sec−1, the photosynthetic rate increased and the Chla excitation quenching followed the same trend, while no chloroplast movement was observed. In light intensities above 1000 μmol m−2 sec−1, the photosynthetic rate reached saturation and the chloroplasts moved toward the cell walls parallel to the light direction in a light avoidance response, while Chla lifetime decreased and enabled photoprotection via excitation quenching. Chloroplast movement and excitation quenching measured by fluorescence lifetime imaging microscopy (FLIM). (a) FLIM images of chloroplasts in Arabidopsis leaf cells under different light intensities. (b) Chloroplast movement quantified as chloroplast-free area and chlorophyll a (Chla) excitation quenching measured by the fluorescence lifetime of Chla under different light intensities. (c) Overview of chloroplast photoprotection measures (modified from Zubik-Duda et al., 2023). The authors then tested their system in different photosynthesis-related mutants. In the npq1 mutant that is impaired in excitation quenching, the Chla lifetime measurements showed that it was not possible to perform photoprotective excitation quenching at high light. However, a much greater chloroplast-free area suggested that the leaves could compensate for the lack of the excitation quenching by a stronger light avoidance response of chloroplasts. Mutants impaired in chloroplast movements showed that the lack of chloroplast relocation did not influence the Chla excitation quenching. Using fluorescence lifetime measurements of Chla, Zubik-Duda et al. have addressed the dilemma of which mechanism should be deployed for photoprotection – excitation quenching or chloroplast movement. It seems there may not be a dilemma after all: both photoprotective measures are able to operate effectively over a wide range of light intensities, and interplay to protect chloroplasts from photodamage. Chla excitation quenching steadily increases with photosynthetic activity, indicating that plants maintain a balance in the number of absorbed photons that is low enough to prevent photodamage, but high enough for efficient photosynthesis. When excitation quenching at high light intensities reaches saturation, chloroplast movement is facilitated to ensure maximal photoprotection (Figure 1c). If the photoprotective excitation quenching at high light intensities is blocked, the light avoidance movement of the chloroplasts is enhanced, suggesting a direct link between the level of excitation energy in the photosynthetic apparatus and chloroplast movement. Although the study was conducted in a single plant species (Arabidopsis), there is no reason to think that the results cannot be generalized and that these two photoprotective mechanisms may operate in parallel in other species.