Periodic Reporting for period 1 - GrInSun (Harvesting Light for Life: Green Proteins as the Interface between Sun Energy and Biosphere)
Reporting period: 2022-10-01 to 2025-03-31
Life on earth feeds on photons. Photosynthesis of green algae and land plants has been the world’s most successful biological process which has conquered the most diverse environments. Photosynthetic reaction centres are extremely well conserved. Antenna systems, instead, are widely diversified and yet only the Light-Harvesting Complexes (LHCs) have been selected for growth in the stressing land environment. The secrets of LHCs are still concealed for lack of experimental systems allowing for reverse and forward genetic analysis. We will perform an in-depth analysis of LHCII proteins in green algae and plants by deploying a new technology that we have developed. Firstly, we deleted all genes encoding LHCII in model species of both land plants and green algae by genome editing and complemented plant ΔLHCII lines with site-directed, mutated sequences, demonstrating that reverse genetics can reveal the domains involved in the regulation of photon harvesting, photoprotection and growth. Secondly, forward genetics, on the other hand, will enable the identification of protein determinants by selecting specific phenotypes on complementing mixotrophic algal ΔLHCII lines with randomly mutagenized sequences. This will lead to a map of structures and functions that identifies the specific biological role of each component of the antenna system in vivo and in vitro. The project’s outcome will be the ability to design in a rational way the light-harvesting systems of plants and algae in the context of sustainable agriculture and bio-industry.
We have used genome-editing for the analysis of the LHC gene sub-family in higher plants as well as in unicellular algae in order to perform reverse genetics in plants and forward genetics in unicellular algae. In short, we have deleted the genes encoding these proteins and verified the functional changes in the main functions catalyzed by these proteins, which are light energy harvesting and photoprotection against excess light. These seemingly contradictory functions are both needed in order to deal with the earth rotation which continuously changes the intensity of the photon flow which fuels photosynthesis and primary productivity on earth. The advancement state of the WPs is reported here. In WP 1 we have deleted either the monomeric LHC proteins or the trimeric LHCII antenna proteins or both. Functional analysis of these mutant genotypes allowed for localization of different function in each LHC subunits. We then proceeded to detail the structure and function by applying single point mutation to individual LHCs, which allowed for identification of structural domains catalysing each specific function. In the case of LHCB4 three genes were present and expressed in different environmental conditions and we constructed plants with one of then only in order to understand how the plant function was modified by the presence of one gene or the next. To this aim we used cryo-EM structural analysis, which showed that photosystem II (PSII) structure was affected by the incorporation of each of the LHCB4 genes. Different structures entrains change in function which adapt plant to different light conditions.
In WP2,. Identification of structure/function basis of photoprotective energy dissipation in LHCII. we applied single point muations to LHCII and showed that the enrgy dissiipation function is located in a protein domain different from previously hypothesized.
In WP3, we applied genomic editing to three LHC proteins in Chlamysdomonas reinhardtii an complemented the mutation with mutant proteins which change the photon harvesting color properties of the LHCs.
In WP4, we isolated WT and mutant proteins and studieed by fast spectroscopy the fundamental processes that determine the colours of light which is absorbed by the LHC proteins
In WP2,. Identification of structure/function basis of photoprotective energy dissipation in LHCII. we applied single point muations to LHCII and showed that the enrgy dissiipation function is located in a protein domain different from previously hypothesized.
In WP3, we applied genomic editing to three LHC proteins in Chlamysdomonas reinhardtii an complemented the mutation with mutant proteins which change the photon harvesting color properties of the LHCs.
In WP4, we isolated WT and mutant proteins and studieed by fast spectroscopy the fundamental processes that determine the colours of light which is absorbed by the LHC proteins
(1) The finding that the lifetime of Chlorophyll excited state lifetime is controlled by the rate of energy transfer from Chlorophyll a and Xanthophylls and can be modulated experimentally
(2) The identification of the structural changes in the Lhca3 and Lhca4 proteins responsible for the formation of Chl a absorption red-forms in higher plants PSI-LHCI supercomplex
(3) The mechanism through which the expression of alternative isoforms of the antenna protein CP29 controls the organization of the PSII supercomplexes in the photosynthetic membrane and the balance between photon energy conversion and linear electron transport (ref IP4).
(4) The mapping of different regulation and photoprotective mechanisms into individual gene products of the PSII antenna system (ref. R1).
figures loaded refers to the item (2) as an example of structure-function analysis of Light harvesting proteins.
List of papers in preparation. (R) under revision, (IP) In Press, (ID) manuscript submitted.
ID. Z. Guardini, L. Dall’Osto, R. Gomez, R. Caferri, P. Joliot, R. Bassi. Mapping light-harvesting and photoprotection responses in higher plant Photosystem II antenna system. Submitted
ID. Z. Guardini, R. Gomez, R. Caferri, J. Stuttmann, L. Dall’Osto, R. Bassi. A recently evolved member among LHCII encoding genes mediates grana stacking in land plant chloroplasts. Submitted
R. S. Capaldi, Z. Guardini, D. Montepietra, V. Pagliuca, A. Amelii, D. Bonnet, A. Chaves-Sanjuan, L. Dall’Osto, R. Bassi. Structural determinants for red-shifted absorption in higher-plants Photosystem I.
ID. D. Montepietra, A. Amelii, E. Cutolo, C. Battarra, R. Caferri, L. Dall’Osto, R. Bassi. Harnessing Far-Red Light: Evolutionary Adaptations and Bioengineering of Light-Harvesting Complexes.
IP. R. Caferri, L. Dall’Osto, Q. Zhou, A. Amelii, J. Shan, Z. Liu, R. Bassi. The Lhcb8 antenna protein reshapes the functional architecture of the photosystem II supercomplex in Arabidopsis thaliana]
(2) The identification of the structural changes in the Lhca3 and Lhca4 proteins responsible for the formation of Chl a absorption red-forms in higher plants PSI-LHCI supercomplex
(3) The mechanism through which the expression of alternative isoforms of the antenna protein CP29 controls the organization of the PSII supercomplexes in the photosynthetic membrane and the balance between photon energy conversion and linear electron transport (ref IP4).
(4) The mapping of different regulation and photoprotective mechanisms into individual gene products of the PSII antenna system (ref. R1).
figures loaded refers to the item (2) as an example of structure-function analysis of Light harvesting proteins.
List of papers in preparation. (R) under revision, (IP) In Press, (ID) manuscript submitted.
ID. Z. Guardini, L. Dall’Osto, R. Gomez, R. Caferri, P. Joliot, R. Bassi. Mapping light-harvesting and photoprotection responses in higher plant Photosystem II antenna system. Submitted
ID. Z. Guardini, R. Gomez, R. Caferri, J. Stuttmann, L. Dall’Osto, R. Bassi. A recently evolved member among LHCII encoding genes mediates grana stacking in land plant chloroplasts. Submitted
R. S. Capaldi, Z. Guardini, D. Montepietra, V. Pagliuca, A. Amelii, D. Bonnet, A. Chaves-Sanjuan, L. Dall’Osto, R. Bassi. Structural determinants for red-shifted absorption in higher-plants Photosystem I.
ID. D. Montepietra, A. Amelii, E. Cutolo, C. Battarra, R. Caferri, L. Dall’Osto, R. Bassi. Harnessing Far-Red Light: Evolutionary Adaptations and Bioengineering of Light-Harvesting Complexes.
IP. R. Caferri, L. Dall’Osto, Q. Zhou, A. Amelii, J. Shan, Z. Liu, R. Bassi. The Lhcb8 antenna protein reshapes the functional architecture of the photosystem II supercomplex in Arabidopsis thaliana]