The effects of different daily irradiance profiles on Arabidopsis growth, with special attention to the role of PsbS.

In nature, light is never constant, while in the controlled environments used for vertical farming, in vitro propagation, or plant production for scientific research, light intensity is often kept constant during the photoperiod. To investigate the effects on plant growth of varying irradiance during the photoperiod, we grew Arabidopsis thaliana under three irradiance profiles: a square-wave profile, a parabolic profile with gradually increasing and subsequently decreasing irradiance, and a regime comprised of rapid fluctuations in irradiance. The daily integral of irradiance was the same for all three treatments. Leaf area, plant growth rate, and biomass at time of harvest were compared. Plants grown under the parabolic profile had the highest growth rate and biomass. This could be explained by a higher average light-use efficiency for carbon dioxide fixation. Furthermore, we compared the growth of wild type plants with that of the PsbS-deficient mutant npq4. PsbS triggers the fast non-photochemical quenching process (qE) that protects PSII from photodamage during sudden increases in irradiance. Based mainly on field and greenhouse experiments, the current consensus is that npq4 mutants grow more slowly in fluctuating light. However, our data show that this is not the case for several forms of fluctuating light conditions under otherwise identical controlled-climate room conditions.

Spectral diversity of photosystem I from flowering plants.

Photosystem I and II (PSI and PSII) work together to convert solar energy into chemical energy. Whilst a lot of research has been done to unravel variability of PSII fluorescence in response to biotic and abiotic factors, the contribution of PSI to in vivo fluorescence measurements has often been neglected or considered to be constant. Furthermore, little is known about how the absorption and emission properties of PSI from different plant species differ. In this study, we have isolated PSI from five plant species and compared their characteristics using a combination of optical and biochemical techniques. Differences have been identified in the fluorescence emission spectra and at the protein level, whereas the absorption spectra were virtually the same in all cases. In addition, the emission spectrum of PSI depends on temperature over a physiologically relevant range from 280 to 298 K. Combined, our data show a critical comparison of the absorption and emission properties of PSI from various plant species.

A kaleidoscope of photosynthetic antenna proteins and their emerging roles.

Photosynthetic light-harvesting antennae are pigment-binding proteins that perform one of the most fundamental tasks on Earth, capturing light and transferring energy that enables life in our biosphere. Adaptation to different light environments led to the evolution of an astonishing diversity of light-harvesting systems. At the same time, several strategies have been developed to optimize the light energy input into photosynthetic membranes in response to fluctuating conditions. The basic feature of these prompt responses is the dynamic nature of antenna complexes, whose function readily adapts to the light available. High-resolution microscopy and spectroscopic studies on membrane dynamics demonstrate the crosstalk between antennae and other thylakoid membrane components. With the increased understanding of light-harvesting mechanisms and their regulation, efforts are focusing on the development of sustainable processes for effective conversion of sunlight into functional bio-products. The major challenge in this approach lies in the application of fundamental discoveries in light-harvesting systems for the improvement of plant or algal photosynthesis. Here, we underline some of the latest fundamental discoveries on the molecular mechanisms and regulation of light harvesting that can potentially be exploited for the optimization of photosynthesis.

Photosynthetic Light Harvesting and Thylakoid Organization in a CRISPR/Cas9 Arabidopsis Thaliana LHCB1 Knockout Mutant.

Light absorbed by chlorophylls of Photosystems II and I drives oxygenic photosynthesis. Light-harvesting complexes increase the absorption cross-section of these photosystems. Furthermore, these complexes play a central role in photoprotection by dissipating the excess of absorbed light energy in an inducible and regulated fashion. In higher plants, the main light-harvesting complex is trimeric LHCII. In this work, we used CRISPR/Cas9 to knockout the five genes encoding LHCB1, which is the major component of LHCII. In absence of LHCB1, the accumulation of the other LHCII isoforms was only slightly increased, thereby resulting in chlorophyll loss, leading to a pale green phenotype and growth delay. The Photosystem II absorption cross-section was smaller, while the Photosystem I absorption cross-section was unaffected. This altered the chlorophyll repartition between the two photosystems, favoring Photosystem I excitation. The equilibrium of the photosynthetic electron transport was partially maintained by lower Photosystem I over Photosystem II reaction center ratio and by the dephosphorylation of LHCII and Photosystem II. Loss of LHCB1 altered the thylakoid structure, with less membrane layers per grana stack and reduced grana width. Stable LHCB1 knockout lines allow characterizing the role of this protein in light harvesting and acclimation and pave the way for future in vivo mutational analyses of LHCII.

The role of light-harvesting complex I in excitation energy transfer from LHCII to photosystem I in Arabidopsis.

Photosynthesis powers nearly all life on Earth. Light absorbed by photosystems drives the conversion of water and carbon dioxide into sugars. In plants, photosystem I (PSI) and photosystem II (PSII) work in series to drive the electron transport from water to NADP+. As both photosystems largely work in series, a balanced excitation pressure is required for optimal photosynthetic performance. Both photosystems are composed of a core and light-harvesting complexes (LHCI) for PSI and LHCII for PSII. When the light conditions favor the excitation of one photosystem over the other, a mobile pool of trimeric LHCII moves between both photosystems thus tuning their antenna cross-section in a process called state transitions. When PSII is overexcited multiple LHCIIs can associate with PSI. A trimeric LHCII binds to PSI at the PsaH/L/O site to form a well-characterized PSI-LHCI-LHCII supercomplex. The binding site(s) of the "additional" LHCII is still unclear, although a mediating role for LHCI has been proposed. In this work, we measured the PSI antenna size and trapping kinetics of photosynthetic membranes from Arabidopsis (Arabidopsis thaliana) plants. Membranes from wild-type (WT) plants were compared to those of the ΔLhca mutant that completely lacks the LHCI antenna. The results showed that "additional" LHCII complexes can transfer energy directly to the PSI core in the absence of LHCI. However, the transfer is about two times faster and therefore more efficient, when LHCI is present. This suggests LHCI mediates excitation energy transfer from loosely bound LHCII to PSI in WT plants.

Precise estimation of chlorophyll a, b and carotenoid content by deconvolution of the absorption spectrum and new simultaneous equations for Chl determination.

The precise determination of photosynthetic pigment content in green organisms, chlorophylls (Chls) and carotenoids (Cars), is important to investigate many photosynthetic processes such as responses to environmental fluctuations or to gene mutations, as well as to interpret biochemical and structural results obtained on purified membranes and photosynthetic complexes. The most utilized methods for determination by spectrophotometry of Chl content in solution, usually 80% acetone, are based on the use of simultaneous equations. The advantages are the easiness and speed over chromatography, which also requires less common equipment. The disadvantage is that issues in sample preparation or in the measurement are not detectable, which could lead to wrong results. Here we propose a fast, accurate and (almost) error-proof method to measure Chl a, Chl b and also total Car content in a solution of pigments extracted from tissue, membranes or purified complexes. The method is based on the fit of the absorption spectrum of the acetone extract using the spectra of purified pigments as references. We show how this method allows a more precise and accurate estimation of pigment content as compared to classical equations, even in incorrectly prepared acetone solutions. Moreover, the method allows the discovery of artifacts in sample preparation or measurement and thus drastically reduces the risk of mistakes. Examples obtained on purified complexes are also discussed. Based on newly acquired Chl spectra, we also propose a new set of improved simultaneous equations that provide slightly different but more reliable results in comparison with the currently used equations.

Functional analysis of LHCSR1, a protein catalyzing NPQ in mosses, by heterologous expression in Arabidopsis thaliana.

Non-photochemical quenching, NPQ, of chlorophyll fluorescence regulates the heat dissipation of chlorophyll excited states and determines the efficiency of the oxygenic photosynthetic systems. NPQ is regulated by a pH-sensing protein, responding to the chloroplast lumen acidification induced by excess light, coupled to an actuator, a chlorophyll/xanthophyll subunit where quenching reactions are catalyzed. In plants, the sensor is PSBS, while the two pigment-binding proteins Lhcb4 (also known as CP29) and LHCII are the actuators. In algae and mosses, stress-related light-harvesting proteins (LHCSR) comprise both functions of sensor and actuator within a single subunit. Here, we report on expressing the lhcsr1 gene from the moss Physcomitrella patens into several Arabidopsis thaliana npq4 mutants lacking the pH sensing PSBS protein essential for NPQ activity. The heterologous protein LHCSR1 accumulates in thylakoids of A. thaliana and NPQ activity can be partially restored. Complementation of double mutants lacking, besides PSBS, specific xanthophylls, allowed analyzing chromophore requirement for LHCSR-dependent quenching activity. We show that the partial recovery of NPQ is mostly due to the lower levels of Zeaxanthin in A. thaliana in comparison to P. patens. Complemented npq2npq4 mutants, lacking besides PSBS, Zeaxanthin Epoxidase, showed an NPQ recovery of up to 70% in comparison to A. thaliana wild type. Furthermore, we show that Lutein is not essential for the folding nor for the quenching activity of LHCSR1. In short, we have developed a system to study the function of LHCSR proteins using heterologous expression in a variety of A. thaliana mutants.

ZraP, the most prominent zinc protein under zinc stress conditions has no direct role in in-vivo zinc tolerance in Escherichia coli.

Escherichia coli ZraP (zinc resistance associated protein) is the major Zn containing soluble protein under Zn stress conditions. ZraP is the accessory protein of a bacterial two-component, Zn2+ sensitive signal transduction system ZraSR. ZraP has also been reported to act as a Zn2+ dependent molecular chaperone. An explanation why ZraP is the major Zn protein under the stress condition of Zn2+ overload (0.2 mM) has remained elusive. We have recombinantly produced E. coli ZraP and measured Zn2+ and Cu2+ affinity in-vitro using Isothermal Titration Calorimetry. ZraP has a significantly higher affinity for Cu2+ than for Zn2+. Mutation of the conserved Cys102 to Ala or Ser resulted in a change of the oligomeric state of the protein. Mutation of the conserved His107 to Ala did not affect the zinc binding affinity or the oligomeric state of the protein. Deletion of the ZraP coding gene from the E. coli genome resulted in a phenotype with tolerance to very high zinc concentrations (up to 2.5 mM) that were lethal to wild type E. coli. These results exclude a direct role for ZraP in Zn2+ tolerance in E. coli.

In vivo NMR as a tool for probing molecular structure and dynamics in intact Chlamydomonas reinhardtii cells.

We report the application of NMR dynamic spectral editing for probing the structure and dynamics of molecular constituents in fresh, intact cells and in freshly prepared thylakoid membranes of Chlamydomonas reinhardtii (Cr.) green algae. For isotope labeling, wild-type Cr. cells were grown on 13C acetate-enriched minimal medium. 1D 13C J-coupling based and dipolar-based MAS NMR spectra were applied to distinguish 13C resonances of different molecular components. 1D spectra were recorded over a physiological temperature range, and whole-cell spectra were compared to those taken from thylakoid membranes, evaluating their composition and dynamics. A theoretical model for NMR polarization transfer was used to simulate the relative intensities of direct, J-coupling, and dipolar-based polarization from which the degree of lipid segmental order and rotational dynamics of the lipid acyl chains were estimated. We observe that thylakoid lipid signals dominate the lipid spectral profile of whole algae cells, demonstrating that with our novel method, thylakoid membrane characteristics can be detected with atomistic precision inside intact photosynthetic cells. The experimental procedure is rapid and applicable to fresh cell cultures, and could be used as an original approach for detecting chemical profiles, and molecular structure and dynamics of photosynthetic membranes in vivo in functional states.