BALANCING LIGHT EFFICIENTLY: THE LONG ROAD TO A SUSTAINABLE FUTURE

Photosynthetic organisms harvest light and convert energy into biomass through the process of photosynthesis. Antenna systems are responsible for light harvesting and transfer the energy towards the Reaction Centres (RCs). However, too much light can be damaging and therefore the excess energy is dissipated as heat through a process called Non-Photochemical Quenching (NPQ). NPQ is activated by decreased pH on the luminal side of the thylakoid membranes by either PSBS or LHCSR found in higher plants and green algae respectively. When the antenna systems receive too much light the singlet Chl excited states produced by photon absorption cannot be quenched by photochemistry fast enough, leading to intersystem crossing producing Chl triplet states, which react with O2 and results in the synthesis of Reactive Oxygen Species (ROS). ROS accumulation causes photoinhibition of photosynthesis, drastically limiting growth. Therefore, dissipating light when in excess and using even the last photon under low light is a difficult exercise which is essential to ensure maximal growth. However, plants are more \u201cinterested\u201d in surviving stress and reproducing than in growing big. Therefore, they have developed a hysteretic response to light: in order to avoid damage, plants over-regulate energy dissipation thus growing less than could be afforded under farming conditions, indicating that there is large room for engineering energy dissipation and increase crop production. Furthermore, besides changes in light intensity, plants experience changes in spectral composition as well. However, PSII and PSI have slightly different absorbance spectra and for an efficient linear electron flow between the two photosystems, it is essential that the excitations between the two photosystems are balanced. This is regulated by so-called state transitions a shuttling of antenna proteins between PSII and PSI. In Chapter 1 the differences and similarities between a variety of oxygenic photosynthetic organisms is reviewed. The focus lies on the different sets of antenna systems that evolved during the evolution and how the antenna systems that we currently find in plants and green algae have such an important role in photoprotection. In Chapter 2, PSBS in A. thaliana has been replaced with LHCSR1 from the moss P. patens, an evolutionary intermediate both expressing functional PSBS and LHCSR. The complemented A. thaliana lines showed a partial recovery of NPQ. The partial recovery of NPQ was mainly caused by the reduced capacity to convert violaxanthin into zeaxanthin in A. thaliana in comparison with P. patens. In chapter 3, several different A. thaliana lines lacking one or more PSII-antenna complexes were complemented with LHCSR1 form P. patens in order to identify a possible interaction partner of LHCSR1, where Lhcb5 (CP26) has been identified as the most likely interaction partner of LHCSR1. In chapter 4, the complemented lines from Chapter 2 were grown in different fluctuating light conditions to see whether LHCSR1 could increase the biomass production. However, in all cases WT grew the same or better than the complemented lines. In specific cases the presence of LHCSR1 could partly improve growth in comparison to npq4. In Chapter 5, we looked at the locations of interaction of LHCII with PSI. This is especially interesting since LHCII is the most important protein to induce NPQ, and does perform state-transitions, a process which is essential for an even energy distribution between the two photosystems and therefore necessary to grow properly. A second LHCII-PSI interaction site has been confirmed by looking at the energy transfer in isolated stroma membranes in State I or State II of WT and a mutant devoid of the PSI antenna. We show that the presence of the PSI-antenna (Lhca1-4) increase the rate of energy transfer from LHCII to PSI by 4 times and thus are essential for a proper binding of LHCII to PSI

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

Solid state NMR/Biophysical Organic Chemistr

Data underlying the publication: Spectral Diversity of Photosystem I from Flowering Plants

We investigated the spectroscopic properties of photosystem I from five different plant species (Arabidopsis thaliana, Spinacia oleracea, Zea mays, Spathiphyllum wallisii, Calathea roseopicta). In this dataset we present the absorption spectra, steady state fluorescence spectra at different temperatures (room temperature, 280-298 K, 77 K) and time resolved fluorescence data of the different PSI complexes. We also include the 77 K fluorescence spectra of the thylakoids and the DNA sequences of the lhca proteins that we extracted from the Arabidopsis Information Resource (TAIR)

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

International audienceThe precise determination of photosynthetic pigment content in green organisms, chlorophylls (Chls) andcarotenoids (Cars), is important to investigate many photosynthetic processes such as responses to envi-ronmental fluctuations or to gene mutations, as well as to interpret biochemical and structural resultsobtained on purified membranes and photosynthetic complexes. The most utilized methods for determina-tion by spectrophotometry of Chl content in solution, usually 80% acetone, are based on the use of simulta-neous equations. The advantages are the easiness and speed over chromatography, which also requiresless common equipment. The disadvantage is that issues in sample preparation or in the measurement arenot detectable, which could lead to wrong results. Here we propose a fast, accurate and (almost) error-proof method to measure Chla, Chlband also total Car content in a solution of pigments extracted fromtissue, membranes or purified complexes. The method is based on the fit of the absorption spectrum of theacetone extract using the spectra of purified pigments as references. We show how this method allows amore precise and accurate estimation of pigment content as compared to classical equations, even in incor-rectly prepared acetone solutions. Moreover, the method allows the discovery of artifacts in sample prepa-ration or measurement and thus drastically reduces the risk of mistakes. Examples obtained on purifiedcomplexes are also discussed. Based on newly acquired Chl spectra, we also propose a new set of improvedsimultaneous equations that provide slightly different but more reliable results in comparison with the cur-rently used equation

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

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

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 DLhca 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

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

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

International audienceLight 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

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