Size and Fluorescence Properties of Algal Photosynthetic Antenna Proteins Estimated by Microscopy

Antenna proteins play a major role in the regulation of light-harvesting in photosynthesis. However, less is known about a possible link between their sizes (oligomerization state) and fluorescence intensity (number of photons emitted). Here, we used a microscopy-based method, Fluorescence Correlation Spectroscopy (FCS), to analyze different antenna proteins at the particle level. The direct comparison indicated that Chromera Light Harvesting (CLH) antenna particles (isolated from Chromera velia) behaved as the monomeric Light Harvesting Complex II (LHCII) (from higher plants), in terms of their radius (based on the diffusion time) and fluorescence yields. FCS data thus indicated a monomeric oligomerization state of algal CLH antenna (at our experimental conditions) that was later confirmed also by biochemical experiments. Additionally, our data provide a proof of concept that the FCS method is well suited to measure proteins sizes (oligomerization state) and fluorescence intensities (photon counts) of antenna proteins per single particle (monomers and oligomers). We proved that antenna monomers (CLH and LHCIIm) are more “quenched” than the corresponding trimers. The FCS measurement thus represents a useful experimental approach that allows studying the role of antenna oligomerization in the mechanism of photoprotection.

The structure of the Physcomitrium Patens Photosystem I Reveals a Unique Lhca2 Paralogue replacing Lhca4

The moss Physcomitrium patens diverged from green algae shortly after the colonization of land by ancient plants. This colonization posed new environmental challenges which drove evolutionary processes. The photosynthetic machinery of modern flowering plants is adapted to the high light conditions on land. Red shifted Lhca4 antennae are present in the photosystem I light harvesting complex of many green lineage plants but absent from P. patens. The Cryo-EM structure of the P. patens photosystem I light harvesting complex I supercomplex (PSI-LHCI) at 2.8 Å reveals that Lhca4 is replaced by a unique Lhca2 paralogue in moss. This PSI-LHCI supercomplex also retains the PsaM subunit, present in cyanobacteria and several algal species but lost in higher plants, and the PsaO subunit responsible for binding light harvesting complex II. The blue shifted Lhca2 paralogue and chlorophyll b enrichment relative to higher plants make the P. patens PSI-LHCI spectroscopically unique among other green lineage supercomplexes. Overall, the structure represents an evolutionary intermediate PSI with the crescent shaped LHCI common in higher plants and contains a unique Lhca2 paralogue which facilitates the mosses adaptation to low light niches.

Chlamydomonas State Transition Is Quiet Around Pyrenoid and Independent from Thylakoid Deformation

Photosynthetic organisms have developed a rapid regulation mechanism called state transition (ST) to rapidly adjust the excitation balance between two photosystems by light-harvesting complex II (LHCII) movement. Though many researchers have assumed coupling of the ultrastructural dynamics of the thylakoid membrane to the ST mechanism, how ST is related to the ultrastructural dynamic of the thylakoid in Chlamydomonas remains elusive. To clarify the above-mentioned relation, here we used two specialized microscope techniques, observation via the excitation-spectral microscope (ESM) developed recently by us and the super-resolution imaging based on structured illumination microscopy (SIM). The ESM observation revealed a highly reversible rearrangement of LHCII-related fluorescence. More importantly, it clarified lower ST activity in the region surrounding the pyrenoid, which is the specific subcellular compartment associated with the carbon-fixation reaction. On the other hand, the SIM observation resolved partially irreversible fine thylakoid transformations induced by the ST-inducing illumination. Fine irreversible thylakoid transformation was also observed for the Stt7-kinase-lacking mutant. This result, together with the nearly equal structural changes in the less active ST regions around the pyrenoid, suggested the independence of the observed fine structural changes from the LHCII phosphorylation.

Photoprotective qH in Arabidopsis occurs in the light-harvesting complex II trimer

Excess light can induce photodamage to the photosynthetic machinery, therefore plants have evolved photoprotective mechanisms such as non-photochemical quenching (NPQ). Different NPQ components have been identified and classified based on their relaxation kinetics and molecular players. The NPQ component qE is induced and relaxed rapidly (seconds to minutes), whereas the NPQ component qH is induced and relaxed slowly (hours or longer). Molecular players regulating qH have recently been uncovered, but the photophysical mechanism of qH and its location in the photosynthetic membrane have not been determined. Using time-correlated single-photon counting analysis of the Arabidopsis thaliana suppressor of quenching 1 mutant (soq1), which displays higher qH than the wild type, we observed shorter average lifetime of chlorophyll fluorescence in leaves and thylakoids relative to wild type. Comparison of isolated photosynthetic complexes from plants in which qH was turned ON or OFF revealed a chlorophyll fluorescence decrease specifically in the trimeric light-harvesting complex II (LHCII) fraction when qH was ON. LHCII trimers are composed of Lhcb1, 2 and 3 proteins, so CRISPR-Cas9 edited and T-DNA insertion lhcb1, lhcb2 and lhcb3 mutants were crossed with soq1. In soq1 lhcb1, soq1 lhcb2, and soq1 lhcb3, qH was not abolished, indicating that no single major Lhcb isoform is necessary for qH. Using transient absorption spectroscopy of isolated thylakoids, no spectral signatures for chlorophyll-carotenoid excitation energy quenching or charge transfer quenching were observed, suggesting that qH may occur through chlorophyll-chlorophyll excitonic interaction.

Plant and Algal PSII–LHCII Supercomplexes: Structure, Evolution and Energy Transfer

Photosynthesis is the process conducted by plants and algae to capture photons and store their energy into a chemical form. The light-harvesting, excitation transfer, charge separation, and electron transfer in photosystem II (PSII) are the critical initial reactions of photosynthesis and thereby largely determine its overall efficiency. In this review, we outline the rapidly accumulating knowledges about the architectures and assemblies of plant and green algal PSII–light harvesting complex II (LHCII) supercomplexes with a particular focus on new insights provided by the recent high-resolution cryo-electron microscopy (cryo-EM) map of the supercomplexes from a green alga Chlamydomonas reinhardtii. We make pair-wise comparative analyses between the supercomplexes from plants and green algae to gain insights about the evolution of the PSII–LHCII supercomplexes involving the peripheral small PSII subunits that might have been acquired during the evolution, and about the energy transfer pathways that define their light-harvesting and photoprotective properties.

Temperature-mediated shifts in chlorophyll biosynthesis in leaves of chlorophyll b-lacking rice (Oryza sativa L.)

Extreme temperatures have become a threat to crop yields. To maintain plant growth and yield, chlorophyll (Chl) biosynthesis plays a crucial role in adaptation to temperature stress. This study investigated the influence of temperature on the biosynthesis and characteristics of pigments (Chl a, Chl b, and carotenoids) in the leaves of Chl b-lacking mutant rice (Chlorina 1, ch1) and wild-type rice (Norin No.8, wt). The ch1 showed thinner stacked grana caused by a decrease in thylakoid membranes per granum at 15 °C, whereas the destacked grana were observed at 35 °C after 12 h incubation. However, the grana are stacked normally, along with the absence of Chl b, and a significantly decreased amount of Chl a in both wt and ch1 were observed after heat stress exposure, demonstrating that light-harvesting complex II proteins are involved in grana stacking. Ch1 was sensitive to 15 °C during the first 4 h of incubation but it subsequently adapted to the cold environment. In addition, there were no significant differences in the photosynthesis between wt and ch1 after 12 h incubation at 35 °C. Differentially expressed gene (DEGs) analysis revealed that GluRS expression decreased, which resulted in a decline in Chl biosynthesis in wt and ch1 at 35 °C. At 8 h and 12 h, there were no significant differences in the expression of DEGs involved in Chl biosynthesis and degradation between wt and ch1 at 15 °C. ALAD expression in wt and ch1 at 15 °C decreased until it was undetectable. These findings suggested that ch1 may adapt to temperatures ranging from 15 °C to 35 °C.