RESUMO
In oxygenic photosynthetic organisms, chlorophyll triplets are harmful excited states readily reacting with molecular oxygen to yield the reactive oxygen species (ROS) singlet oxygen. Carotenoids have a photoprotective role in photosynthetic membranes by preventing photoxidative damage through quenching of chlorophyll singlets and triplets. In this work we used mutation analysis to investigate the architecture of chlorophyll triplet quenching sites within Lhcb5, a monomeric antenna protein of Photosystem II. The carotenoid and chlorophyll triplet formation as well as the production of ROS molecules were studied in a family of recombinant Lhcb5 proteins either with WT sequence, mutated into individual chlorophyll binding residues or refolded in vitro to bind different xanthophyll complements. We observed a site-specific effect in the efficiency of chlorophyll-carotenoid triplet-triplet energy transfer. Thus chlorophyll (Chl) 602 and 603 appear to be particularly important for triplet-triplet energy transfer to the xanthophyll bound into site L2. Surprisingly, mutation on Chl 612, the chlorophyll with the lower energy associated and in close contact with lutein in site L1, had no effect on quenching chlorophyll triplet excited states. Finally, we present evidence for an indirect role of neoxanthin in chlorophyll triplet quenching and show that quenching of both singlet and triplet states is necessary for minimizing singlet oxygen formation.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Ligação à Clorofila/química , Clorofila/química , Complexo de Proteína do Fotossistema II/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Carotenoides/química , Proteínas de Ligação à Clorofila/genética , Proteínas de Ligação à Clorofila/metabolismo , Transferência de Energia , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Oxigênio Singlete/química , Oxigênio Singlete/metabolismoRESUMO
The TOR (target of rapamycin) kinase is present in nearly all eukaryotic organisms and regulates a wealth of biological processes collectively contributing to cell growth. The genome of the model plant Arabidopsis contains a single TOR gene and two RAPTOR (regulatory associated protein of TOR)/KOG1 (Kontroller of growth 1) and GßL/LST8 (G-protein ß-subunit-like/lethal with Sec thirteen 8) genes but, in contrast with other organisms, plants appear to be resistant to rapamycin. Disruption of the RAPTOR1 and TOR genes in Arabidopsis results in an early arrest of embryo development. Plants that overexpress the TOR mRNA accumulate more leaf and root biomass, produce more seeds and are more resistant to stress. Conversely, the down-regulation of TOR by constitutive or inducible RNAi (RNA interference) leads to a reduced organ growth, to an early senescence and to severe transcriptomic and metabolic perturbations, including accumulation of sugars and amino acids. It thus seems that plant growth is correlated to the level of TOR expression. We have also investigated the effect of reduced TOR expression on tissue organization and cell division. We suggest that, like in other eukaryotes, the plant TOR kinase could be one of the main contributors to the link between environmental cues and growth processes.
Assuntos
Desenvolvimento Vegetal , Plantas/metabolismo , Serina-Treonina Quinases TOR/fisiologia , Animais , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Humanos , Modelos Biológicos , Filogenia , Plantas/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
The outer antenna system of Chlamydomonas reinhardtii Photosystem I is composed of nine gene products, but due to difficulty in purification their individual properties are not known. In this work, the functional properties of the nine Lhca antennas of Chlamydomonas, have been investigated upon expression of the apoproteins in bacteria and refolding in vitro of the pigment-protein complexes. It is shown that all Lhca complexes have a red-shifted fluorescence emission as compared to the antenna complexes of Photosystem II, similar to Lhca from higher plants, but less red-shifted. Three complexes, namely Lhca2, Lhca4 and Lhca9, exhibit emission maxima above 707 nm and all carry an asparagine as ligand for Chl 603. The comparison of the protein sequences and the biochemical/spectroscopic properties of the refolded Chlamydomonas complexes with those of the well-characterized Arabidopsis thaliana Lhcas shows that all the Chlamydomonas complexes have a chromophore organization similar to that of A. thaliana antennas, particularly to Lhca2, despite low sequence identity. All the major biochemical and spectroscopic properties of the Lhca complexes have been conserved through the evolution, including those involved in "red forms" absorption. It has been proposed that in Chlamydomonas PSI antenna size and polypeptide composition can be modulated in vivo depending on growth conditions, at variance as compared to higher plants. Thus, the different properties of the individual Lhca complexes can be functional to adapt the architecture of the PSI-LHCI supercomplex to different environmental conditions.
Assuntos
Chlamydomonas reinhardtii/genética , Complexos de Proteínas Captadores de Luz/genética , Luz , Complexo de Proteína do Fotossistema I/genética , Sequência de Aminoácidos , Arabidopsis/química , Chlamydomonas reinhardtii/metabolismo , Dicroísmo Circular , Primers do DNA , Dados de Sequência Molecular , Pigmentos Biológicos/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The peripheral light-harvesting complex of photosystem I contains red chlorophylls (Chls) that, unlike the typical antenna Chls, absorb at lower energy than the primary electron donor P700. It has been shown that the red-most absorption band arises from two excitonically coupled Chls, although this interaction alone cannot explain the extreme red-shifted emission (25 nm, approximately 480 cm(-1) for Lhca4 at 4 K) that the red Chls present. Here, we report the electric field-induced absorption changes (Stark effect) on the Q(y) region of the Lhca4 complex. Two spectral forms, centered around 690 nm and 710 nm, were necessary to describe the absorption and Stark spectra. The analysis of the lowest energy transition yields a high value for the change in dipole moment, Deltamu(710nm) approximately 8 Df(-1), between the ground and excited states as compared with monomeric, Deltamu = 1 D, or dimeric, Deltamu = 5 D, Chl a in solution. The high value of the Deltamu demonstrates that the origin of the red-shifted emission is the mixing of the lowest exciton state with a charge-transfer state of the dimer. This energetic configuration, an excited state with charge-transfer character, is very favorable for the trapping and dissipation of excitations and could be involved in the photoprotective mechanism(s) of the photosystem I complex.
Assuntos
Complexos de Proteínas Captadores de Luz/química , Complexo de Proteína do Fotossistema I/química , Proteínas de Plantas/química , Absorção , Proteínas de Ligação à Clorofila , Análise EspectralRESUMO
Lhcb5 is an antenna protein that is highly conserved in plants and green algae. It is part of the inner layer of photosystem II antenna system retained in high light acclimated plants. To study the structure-function relation and the role of individual pigments in this complex, we (i) "knocked out" each of the chromophores bound to multiple (nine total) chlorophyll sites and (ii) exchanged the xanthophylls bound to the three xanthophyll sites. The occupancy and associated energy of the pigment binding sites were determined. The role of the individual pigments in protein folding, stability, energy transfer, and dissipation was studied in vitro. The results indicate that lutein has a primary role in the folding and stability of the complex, whereas violaxanthin and zeaxanthin have a negative effect on folding yield and stability, respectively. The data showed a distinct function for the L1 and L2 carotenoid binding sites, the former preferentially involved in gathering the excitation energy to chlorophyll a (Chl a), whereas the latter modulates the concentration of chlorophyll singlet excited states dependent on the xanthophylls bound to it, likely via an interaction with Chl-603. Our results also underscored the role of zeaxanthin and lutein in quenching the excitation energy, whereas violaxanthin was shown to be very effective in energy transfer. The characteristics of the isolated proteins were consistent with the observed role of Lhcb5 in vivo in catalyzing fluorescence quenching upon zeaxanthin binding.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Clorofila/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Dobramento de Proteína , Xantofilas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sítios de Ligação/fisiologia , Clorofila/genética , Clorofila A , Proteínas de Ligação à Clorofila , Complexos de Proteínas Captadores de Luz/genética , Complexo de Proteína do Fotossistema II/genética , Ligação Proteica/fisiologia , Relação Estrutura-Atividade , Xantofilas/genéticaRESUMO
In bright sunlight, the amount of energy harvested by plants exceeds the electron transport capacity of Photosystem II in the chloroplasts. The excess energy can lead to severe damage of the photosynthetic apparatus and to avoid this, part of the energy is thermally dissipated via a mechanism called non-photochemical quenching (NPQ). It has been found that LHCII, the major antenna complex of Photosystem II, is involved in this mechanism and it was proposed that its quenching site is formed by the cluster of strongly interacting pigments: chlorophylls 611 and 612 and lutein 620 [A.V. Ruban, R. Berera, C. Ilioaia, I.H.M. van Stokkum, J.T.M. Kennis, A.A. Pascal, H. van Amerongen, B. Robert, P. Horton and R. van Grondelle, Identification of a mechanism of photoprotective energy dissipation in higher plants, Nature 450 (2007) 575-578.]. In the present work we have investigated the interactions between the pigments in this cluster not only for LHCII, but also for the homologous minor antenna complexes CP24, CP26 and CP29. Use was made of wild-type and mutated reconstituted complexes that were analyzed with (low-temperature) absorption and circular-dichroism spectroscopy as well as by biochemical methods. The pigments show strong interactions that lead to highly specific spectroscopic properties that appear to be identical for LHCII, CP26 and CP29. The interactions are similar but not identical for CP24. It is concluded that if the 611/612/620 domain is responsible for the quenching in LHCII, then all these antenna complexes are prepared to act as a quencher. This can explain the finding that none of the Lhcb complexes seems to be strictly required for NPQ while, in the absence of all of them, NPQ is abolished.
Assuntos
Complexos de Proteínas Captadores de Luz/metabolismo , Luz , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Plantas/metabolismo , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Transporte de Elétrons/fisiologia , Complexos de Proteínas Captadores de Luz/química , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida , Complexo de Proteína do Fotossistema II/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Zea mays/química , Zea mays/genética , Zea mays/metabolismoRESUMO
In this work the photoprotective role of all xanthophylls in LHCII, Lhcb4, and Lhcb5 is investigated by laser-induced Triplet-minus-Singlet (TmS) spectroscopy. The comparison of native LHCII trimeric complexes with different carotenoid composition shows that the xanthophylls in sites V1 and N1 do not directly contribute to the chlorophyll triplet quenching. The largest part of the triplets is quenched by the lutein bound in site L1, which is located in close proximity to the chlorophylls responsible for the low energy state of the complex. The lutein in the L2 site is also active in triplet quenching, and it shows a longer triplet lifetime than the lutein in the L1 site. This lifetime difference depends on the occupancy of the N1 binding site, where neoxanthin acts as an oxygen barrier, limiting the access of O(2) to the inner domain of the Lhc complex, thereby strongly contributing to the photostability. The carotenoid triplet decay of monomeric Lhcb1, Lhcb4, and Lhcb5 is mono-exponential, with shorter lifetimes than observed for trimeric LHCII, suggesting that their inner domains are more accessible for O(2). As for trimeric LHCII, only the xanthophylls in sites L1 and L2 are active in triplet quenching. Although the chlorophyll to carotenoid triplet transfer is efficient (95%) in all complexes, it is not perfect, leaving 5% of the chlorophyll triplets unquenched. This effect appears to be intrinsically related to the molecular organization of the Lhcb proteins.
Assuntos
Arabidopsis/metabolismo , Clorofila/metabolismo , Luteína/metabolismo , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Xantofilas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação à Clorofila , Complexos de Proteínas Captadores de Luz/metabolismoRESUMO
In this work, the spectroscopic characteristics of carotenoids associated with the antenna complexes of Photosystem I have been studied. Pigment composition, absorption spectra, and laser-induced triplet-minus-singlet (T-S) spectra were determined for native LHCI from the wild type (WT) and lut2 mutant from Arabidopsis thaliana as well as for reconstituted individual Lhca WT and mutated complexes. All WT complexes bind lutein and violaxanthin, while beta-carotene was found to be associated only with the native LHCI preparation and recombinant Lhca3. In the native complexes, the main lutein absorption bands are located at 492 and 510 nm. It is shown that violaxanthin is able to occupy all lutein binding sites, but its absorption is blue-shifted to 487 and 501 nm. The "red" lutein absorbing at 510 nm was found to be associated with Lhca3 and Lhca4 which also show a second carotenoid, peaking around 490 nm. Both these xanthophylls are involved in triplet quenching and show two T-S maxima: one at 507 nm (corresponding to the 490 nm singlet absorption) and the second at 525 nm (with absorption at 510 nm). The "blue"-absorbing xanthophyll is located in site L1 and can receive triplets from chlorophylls (Chl) 1012, 1011, and possibly 1013. The red-shifted spectral component is assigned to a lutein molecule located in the L2 site. A 510 nm lutein was also observed in the trimers of LHCII but was absent in the monomers. In the case of Lhca, the 510 nm band is present in both the monomeric and dimeric complexes. We suggest that the large red shift observed for this xanthophyll is due to interaction with the neighbor Chl 1015. In the native T-S spectrum, the contribution of carotenoids associated with Lhca2 is visible while the one of Lhca1 is not. This suggests that in the Lhca2-Lhca3 heterodimeric complex energy equilibration is not complete at least on a fast time scale.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Carotenoides/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação , Carotenoides/química , Carotenoides/genética , Proteínas de Ligação à Clorofila , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/genética , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/genética , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Espectrometria de FluorescênciaRESUMO
Lhc proteins constitute a family of transmembrane proteins which share homology in sequence and similarity in the general organisation although members can be strongly differentiated such as in the case of PsbS and ELIPs. In this work, we report on the structure of Lhca3, a pigment-protein subunit component of the antenna system of higher plants Photosystem I, through the effect of point mutations in critical sites. Based on the structure of PSI-LHCI (Ben Shem et al., PDB file 1QZV remark 999) it has been suggested that Lhca3 may have different folding as compared to other members of the Lhc family. In particular, it was proposed that the two central helices may be swapped and chlorophylls in sites 1013 and 1023 are not present. This different folding would imply that the chlorophylls coordinated to the two central helices have different ligands in Lhca3 with respect to the other Lhc complexes. The structural model was tested by substituting the putative binding residues with residues unable to coordinate chlorophyll and the spectroscopic properties of the individual pigments were used as structural probes. The results indicate that Lhca3 folds in the same way as the other antenna proteins. Moreover, the low-energy absorption form originates from interaction between chlorophylls in site 1015 and 1025, like for the other PSI antenna subunits. Evidence is also shown for the presence in Lhca3 of chlorophylls in sites 1013 and 1023.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/genética , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/genética , Substituição de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Sítios de Ligação/genética , Clorofila/química , Proteínas de Ligação à Clorofila , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Dobramento de Proteína , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometria de Fluorescência , EspectrofotometriaRESUMO
The red-most fluorescence emission of photosystem I (733 nm at 4 K) is associated with the Lhca4 subunit of the antenna complex. It has been proposed that this unique spectral feature originates from the low energy absorption band of an excitonic interaction involving chlorophyll A5 and a second chlorophyll a molecule, probably B5 (Morosinotto, T., Breton, J., Bassi, R., and Croce, R. (2003) J. Biol. Chem. 278, 49223-49229). Because of the short distances between chromophores in Lhc proteins, the possibility that other pigments are involved in the red-shifted spectral forms could not be ruled out. In this study, we have analyzed the pigment-pigment interactions between nearest neighboring chromophores in Lhca4. This was done by deleting individual chlorophyll binding sites by mutagenesis, and analyzing the changes in the spectroscopic properties of recombinant proteins refolded in vitro. The red-shifted (733 nm) fluorescence peak, the major target of this analysis, was lost upon mutations affecting sites A4, A5, and B5 and was modified by mutating site B6. In agreement with the shorter distance between chlorophylls A5 and B5 (7.9 A) versus A4 and A5 (12.2 A) in Lhca4 (Ben-Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630-635), we conclude that the low energy spectral form originates from an interaction involving pigments in sites A5 and B5. Mutation at site B6, although inducing a 15-nm blue-shift of the emission peak, maintains the red-shifted emission. This implies that chromophores responsible for the interaction are conserved and suggests a modification in the pigment organization. Besides the A5-B5 pair, evidence for additional pigment-pigment interactions between chlorophylls in sites B3-A3 and B6-A6 was obtained. However, these features do not affect the red-most spectral form responsible for the 733-nm fluorescence emission band.