Role of serine/threonine protein kinase STN7 in the formation of two distinct photosystem I supercomplexes in Physcomitrium patens.

Reversible thylakoid protein phosphorylation provides most flowering plants with dynamic acclimation to short-term changes in environmental light conditions. Here, through generating Serine/Threonine protein kinase 7 (STN7)-depleted mutants in the moss Physcomitrella (Physcomitrium patens), we identified phosphorylation targets of STN7 kinase and their roles in short- and long-term acclimation of the moss to changing light conditions. Biochemical and mass spectrometry analyses revealed STN7-dependent phosphorylation of N-terminal Thr in specific Light-Harvesting Complex II (LHCII) trimer subunits (LHCBM2 and LHCBM4/8) and provided evidence that phospho-LHCBM accumulation is responsible for the assembly of two distinct Photosystem I (PSI) supercomplexes (SCs), both of which are largely absent in STN7-depleted mutants. Besides the canonical state transition complex (PSI-LHCI-LHCII), we isolated the larger moss-specific PSI-Large (PSI-LHCI-LHCB9-LHCII) from stroma-exposed thylakoids. Unlike PSI-LHCI-LHCII, PSI-Large did not demonstrate short-term dynamics for balancing the distribution of excitation energy between PSII and PSI. Instead, PSI-Large contributed to a more stable increase in PSI antenna size in Physcomitrella, except under prolonged high irradiance. Additionally, the STN7-depleted mutants revealed altered light-dependent phosphorylation of a monomeric antenna protein, LHCB6, whose phosphorylation displayed a complex regulation by multiple kinases. Collectively, the unique phosphorylation plasticity and dynamics of Physcomitrella monomeric LHCB6 and trimeric LHCBM isoforms, together with the presence of PSI SCs with different antenna sizes and responsiveness to light changes, reflect the evolutionary position of mosses between green algae and vascular plants, yet with clear moss-specific features emphasizing their adaptation to terrestrial low-light environments.

Thylakoid Protein Phosphorylation Dynamics in a Moss Mutant Lacking SERINE/THREONINE PROTEIN KINASE STN8.

In all eukaryotes, protein phosphorylation is a key regulatory mechanism in several cellular processes, including the acclimation of photosynthesis to environmental cues. Despite being a well-conserved regulatory mechanism in the chloroplasts of land plants, distinct differences in thylakoid protein phosphorylation patterns have emerged from studies on species of different phylogenetic groups. Here, we analyzed thylakoid protein phosphorylation in the moss Physcomitrella patens, assessing the thylakoid phospho-protein profile and dynamics in response to changes in white light intensity. Compared with Arabidopsis (Arabidopsis thaliana), parallel characterization of wild-type P patens and the knockout mutant stn8 (depleted in SER/THR PROTEIN KINASE8 [STN8]) disclosed a moss-specific pattern of thylakoid protein phosphorylation, both with respect to specific targets and to their dynamic phosphorylation in response to environmental cues. Unlike vascular plants, (1) phosphorylation of the PSII protein D1 in P patens was negligible under all light conditions, (2) phosphorylation of the PSII core subunits CP43 and D2 showed only minor changes upon fluctuations in light intensity, and (3) absence of STN8 completely abolished all PSII core protein phosphorylation. Further, we detected light-induced phosphorylation in the minor light harvesting complex (LHC) antenna protein LHCB6, which was dependent on STN8 kinase activity, and found specific phosphorylations on LHCB3. Data presented here provide further insights into the appearance and physiological role of thylakoid protein phosphorylation during evolution of oxygenic photosynthetic organisms and their colonization of land.

Role of cyclic and pseudo-cyclic electron transport in response to dynamic light changes in Physcomitrella patens.

Photosynthetic organisms support cell metabolism by harvesting sunlight and driving the electron transport chain at the level of thylakoid membranes. Excitation energy and electron flow in the photosynthetic apparatus is continuously modulated in response to dynamic environmental conditions. Alternative electron flow around photosystem I plays a seminal role in this regulation contributing to photoprotection by mitigating overreduction of the electron carriers. Different pathways of alternative electron flow coexist in the moss Physcomitrella patens, including cyclic electron flow mediated by the PGRL1/PGR5 complex and pseudo-cyclic electron flow mediated by the flavodiiron proteins FLV. In this work, we generated P. patens plants carrying both pgrl1 and flva knock-out mutations. A comparative analysis of the WT, pgrl1, flva, and pgrl1 flva lines suggests that cyclic and pseudo-cyclic processes have a synergic role in the regulation of photosynthetic electron transport. However, although both contribute to photosystem I protection from overreduction by modulating electron flow following changes in environmental conditions, FLV activity is particularly relevant in the first seconds after a light change whereas PGRL1 has a major role upon sustained strong illumination.

Flavodiiron proteins act as safety valve for electrons in Physcomitrella patens.

Photosynthetic organisms support cell metabolism by harvesting sunlight to fuel the photosynthetic electron transport. The flow of excitation energy and electrons in the photosynthetic apparatus needs to be continuously modulated to respond to dynamics of environmental conditions, and Flavodiiron (FLV) proteins are seminal components of this regulatory machinery in cyanobacteria. FLVs were lost during evolution by flowering plants, but are still present in nonvascular plants such as Physcomitrella patens We generated P. patens mutants depleted in FLV proteins, showing their function as an electron sink downstream of photosystem I for the first seconds after a change in light intensity. flv knock-out plants showed impaired growth and photosystem I photoinhibition when exposed to fluctuating light, demonstrating FLV's biological role as a safety valve from excess electrons on illumination changes. The lack of FLVs was partially compensated for by an increased cyclic electron transport, suggesting that in flowering plants, the FLV's role was taken by other alternative electron routes.

Conservation of core complex subunits shaped the structure and function of photosystem I in the secondary endosymbiont alga Nannochloropsis gaditana.

Photosystem I (PSI) is a pigment protein complex catalyzing the light-driven electron transport from plastocyanin to ferredoxin in oxygenic photosynthetic organisms. Several PSI subunits are highly conserved in cyanobacteria, algae and plants, whereas others are distributed differentially in the various organisms. Here we characterized the structural and functional properties of PSI purified from the heterokont alga Nannochloropsis gaditana, showing that it is organized as a supercomplex including a core complex and an outer antenna, as in plants and other eukaryotic algae. Differently from all known organisms, the N. gaditana PSI supercomplex contains five peripheral antenna proteins, identified by proteome analysis as type-R light-harvesting complexes (LHCr4-8). Two antenna subunits are bound in a conserved position, as in PSI in plants, whereas three additional antennae are associated with the core on the other side. This peculiar antenna association correlates with the presence of PsaF/J and the absence of PsaH, G and K in the N. gaditana genome and proteome. Excitation energy transfer in the supercomplex is highly efficient, leading to a very high trapping efficiency as observed in all other PSI eukaryotes, showing that although the supramolecular organization of PSI changed during evolution, fundamental functional properties such as trapping efficiency were maintained.

In Vivo Identification of Photosystem II Light Harvesting Complexes Interacting with PHOTOSYSTEM II SUBUNIT S.

Light is the primary energy source for photosynthetic organisms, but in excess, it can generate reactive oxygen species and lead to cell damage. Plants evolved multiple mechanisms to modulate light use efficiency depending on illumination intensity to thrive in a highly dynamic natural environment. One of the main mechanisms for protection from intense illumination is the dissipation of excess excitation energy as heat, a process called nonphotochemical quenching. In plants, nonphotochemical quenching induction depends on the generation of a pH gradient across thylakoid membranes and on the presence of a protein called PHOTOSYSTEM II SUBUNIT S (PSBS). Here, we generated Physcomitrella patens lines expressing histidine-tagged PSBS that were exploited to purify the native protein by affinity chromatography. The mild conditions used in the purification allowed copurifying PSBS with its interactors, which were identified by mass spectrometry analysis to be mainly photosystem II antenna proteins, such as LIGHT-HARVESTING COMPLEX B (LHCB). PSBS interaction with other proteins appears to be promiscuous and not exclusive, although the major proteins copurified with PSBS were components of the LHCII trimers (LHCB3 and LHCBM). These results provide evidence of a physical interaction between specific photosystem II light-harvesting complexes and PSBS in the thylakoids, suggesting that these subunits are major players in heat dissipation of excess energy.

Photoacclimation of photosynthesis in the Eustigmatophycean Nannochloropsis gaditana.

Nannochloropsis is an eukaryotic alga of the phylum Heterokonta, originating from a secondary endosymbiotic event. In this work, we investigated how the photosynthetic apparatus responds to growth in different light regimes in Nannochloropsis gaditana. We found that intense illumination induces the decrease of both photosystem I and II contents and their respective antenna sizes. Cells grown in high light showed a larger capacity for electron transport, with enhanced cyclic electron transport around photosystem I, contributing to photoprotection from excess illumination. Even when exposed to excess light intensities for several days, N. gaditana cells did not activate constitutive responses such as nonphotochemical quenching and the xanthophyll cycle. These photoprotection mechanisms in N. gaditana thus play a role in acclimation to fast changes in illumination within a time range of minutes, while regulation of the electron flow capacity represents a long-term response to prolonged exposure to excess light.

Zeaxanthin binds to light-harvesting complex stress-related protein to enhance nonphotochemical quenching in Physcomitrella patens.

Nonphotochemical quenching (NPQ) dissipates excess energy to protect the photosynthetic apparatus from excess light. The moss Physcomitrella patens exhibits strong NPQ by both algal-type light-harvesting complex stress-related (LHCSR)-dependent and plant-type S subunit of Photosystem II (PSBS)-dependent mechanisms. In this work, we studied the dependence of NPQ reactions on zeaxanthin, which is synthesized under light stress by violaxanthin deepoxidase (VDE) from preexisting violaxanthin. We produced vde knockout (KO) plants and showed they underwent a dramatic reduction in thermal dissipation ability and enhanced photoinhibition in excess light conditions. Multiple mutants (vde lhcsr KO and vde psbs KO) showed that zeaxanthin had a major influence on LHCSR-dependent NPQ, in contrast with previous reports in Chlamydomonas reinhardtii. The PSBS-dependent component of quenching was less dependent on zeaxanthin, despite the near-complete violaxanthin to zeaxanthin exchange in LHC proteins. Consistent with this, we provide biochemical evidence that native LHCSR protein binds zeaxanthin upon excess light stress. These findings suggest that zeaxanthin played an important role in the adaptation of modern plants to the enhanced levels of oxygen and excess light intensity of land environments.

Characterization of the photosynthetic apparatus of the Eustigmatophycean Nannochloropsis gaditana: evidence of convergent evolution in the supramolecular organization of photosystem I.

Nannochloropsis gaditana belongs to Eustigmatophyceae, a class of eukaryotic algae resulting from a secondary endosymbiotic event. Species of this class have been poorly characterized thus far but are now raising increasing interest in the scientific community because of their possible application in biofuel production. Nannochloropsis species have a peculiar photosynthetic apparatus characterized by the presence of only chlorophyll a, with violaxanthin and vaucheriaxanthin esters as the most abundant carotenoids. In this study, the photosynthetic apparatus of this species was analyzed by purifying the thylakoids and isolating the different pigment-binding complexes upon mild solubilization. The results from the biochemical and spectroscopic characterization showed that the photosystem II antenna is loosely bound to the reaction center, whereas the association is stronger in photosystem I, with the antenna-reaction center super-complexes surviving purification. Such a supramolecular organization was found to be conserved in photosystem I from several other photosynthetic eukaryotes, even though these taxa are evolutionarily distant. A hypothesis on the possible selective advantage of different associations of the antenna complexes of photosystems I and II is discussed.

Photoprotective sites in the violaxanthin-chlorophyll a binding Protein (VCP) from Nannochloropsis gaditana.

Violaxanthin-chlorophyll a binding protein (VCP) is the major light harvesting complex (LHC) of the Heterokonta Nannochloropsis gaditana. It binds chlorophyll a, violaxanthin and vaucheriaxanthin, the last in the form of 19' deca/octanoate esters. Photosynthetic apparatus of algae belonging to this group have been poorly characterized in the past, but they are now receiving an increasing interest also because of their possible biotechnological application in biofuel production. In this work, isolated VCP proteins have been studied by means of advanced EPR techniques in order to prove the presence of the photoprotective mechanism based on the triplet-triplet energy transfer (TTET), occurring between chlorophyll and carotenoid molecules. This process has been observed before in several light harvesting complexes belonging to various photosynthetic organisms. We used Optically Detected Magnetic Resonance (ODMR) to identify the triplet states populated by photo-excitation, and describe the optical properties of the chromophores carrying the triplet states. In parallel, time-resolved EPR (TR-EPR) and pulse EPR have been employed to get insight into the TTET mechanism and reveal the structural features of the pigment sites involved in photoprotection. The analysis of the spectroscopic data shows a strong similarity among VCP, FCP from diatoms and LHC-II from higher plants. Although these antenna proteins have differentiated sequences and bind different pigments, results suggest that in all members of the LHC superfamily there is a protein core with a conserved structural organization, represented by two central carotenoids surrounded by five chlorophyll a molecules, which plays a fundamental photoprotective role in Chl triplet quenching through carotenoid triplet formation.

Physiological Responses to Salt Stress at the Seedling Stage in Wild (Oryza rufipogon Griff.) and Cultivated (Oryza sativa L.) Rice.

Domesticated rice Oryza sativa L. is a major staple food worldwide, and the cereal most sensitive to salinity. It originated from the wild ancestor Oryza rufipogon Griff., which was reported to possess superior salinity tolerance. Here, we examined the morpho-physiological responses to salinity stress (80 mM NaCl for 7 days) in seedlings of an O. rufipogon accession and two Italian O. sativa genotypes, Baldo (mildly tolerant) and Vialone Nano (sensitive). Under salt treatment, O. rufipogon showed the highest percentage of plants with no to moderate stress symptoms, displaying an unchanged shoot/root biomass ratio, the highest Na+ accumulation in roots, the lowest root and leaf Na+/K+ ratio, and highest leaf relative water content, leading to a better preservation of the plant architecture, ion homeostasis, and water status. Moreover, O. rufipogon preserved the overall leaf carbon to nitrogen balance and photosynthetic apparatus integrity. Conversely, Vialone Nano showed the lowest percentage of plants surviving after treatment, and displayed a higher reduction in the growth of shoots rather than roots, with leaves compromised in water and ionic balance, negatively affecting the photosynthetic performance (lowest performance index by JIP-test) and apparatus integrity. Baldo showed intermediate salt tolerance. Being O. rufipogon interfertile with O. sativa, it resulted a good candidate for pre-breeding towards salt-tolerant lines.

NPQ activation reduces chlorophyll triplet state formation in the moss Physcomitrella patens.

Plants live in variable environments in which light intensity can rapidly change, from limiting to excess conditions. Non-photochemical quenching (NPQ) is a regulatory mechanism which protects plants from oxidative stress by dissipating excess Chl singlet excitation. In this work, the physiological role of NPQ was assessed by monitoring its influence on the population of the direct source of light excess damage, i.e., Chl triplets ((3)Chl*). (3)Chl* formation was evaluated in vivo, with the moss Physcomitrella patens, by exploiting the high sensitivity of fluorescence-detected magnetic resonance (FDMR). A dark adapted sample was compared with a pre-illuminated sample in which NPQ was activated, the latter showing a strong reduction in (3)Chl* yield. In line with this result, mutants unable to activate NPQ showed only a minor effect in (3)Chl* yield upon pre-illumination.The decrease in (3)Chl* yield is equally experienced by all the Chl pools associated with PSII, suggesting that NPQ is effective in protecting both the core and the peripheral antenna complexes. Moreover, the FDMR results show that the structural reorganization in the photosynthetic apparatus, required by NPQ, does not lead to the formation of new (3)Chl* traps in the LHCs. This work demonstrates that NPQ activation leads to effective photoprotection, promoting a photosystem II state characterized by a reduced probability of (3)Chl* formation, due to a decreased singlet excited state population, while maintaining an efficient quenching of the (3)Chl* eventually formed by carotenoids.

Evolution of photoprotection mechanisms upon land colonization: evidence of PSBS-dependent NPQ in late Streptophyte algae.

Light is the energy source for photosynthetic organisms but, if absorbed in excess, it can drive to the formation of reactive oxygen species and photoinhibition. One major mechanism to avoid oxidative damage in plants and algae is the dissipation of excess excitation energy as heat, called non-photochemical quenching (NPQ). Eukaryotic algae and plants, however, rely on two different proteins for NPQ activation, the former mainly depending on LHCSR (Lhc-like protein Stress Related; previously called Li818, Light Induced protein 818), whereas in the latter the major role is played by a distinct protein, PSBS (photosystem II subunit S). In the moss Physcomitrella patens, which diverged from vascular plants early after land colonization, both these proteins were found to be present and active in inducing NPQ, suggesting that during plants evolution both mechanisms co-existed. In order to investigate in more detail NPQ adaptation toward land colonization, we analyzed Streptophyte algae, the latest organisms to diverge from the land plants ancestors. Among them we found evidence of a PSBS-dependent NPQ in species belonging to Charales, Coleochaetales and Zygnematales, the latest groups to diverge from land plants ancestors. On the contrary earlier diverging algae, as Mesostigmatales and Klebsormidiales, likely rely on LHCSR for their NPQ activation. Presented evidence thus suggests that PSBS-dependent NPQ, although possibly present in some Chlorophyta, was stably acquired in the Cambrian period about 500 million years ago, before late Streptophyte algae diverged from plants ancestors.

Physcomitrella patens mutants affected on heat dissipation clarify the evolution of photoprotection mechanisms upon land colonization.

Light is the source of energy for photosynthetic organisms; when in excess, however, it also drives the formation of reactive oxygen species and, consequently, photoinhibition. Plants and algae have evolved mechanisms to regulate light harvesting efficiency in response to variable light intensity so as to avoid oxidative damage. Nonphotochemical quenching (NPQ) consists of the rapid dissipation of excess excitation energy as heat. Although widespread among oxygenic photosynthetic organisms, NPQ shows important differences in its machinery. In land plants, such as Arabidopsis thaliana, NPQ depends on the presence of PSBS, whereas in the green alga Chlamydomonas reinhardtii it requires a different protein called LHCSR. In this work, we show that both proteins are present in the moss Physcomitrella patens. By generating KO mutants lacking PSBS and/or LHCSR, we also demonstrate that both gene products are active in NPQ. Plants lacking both proteins are more susceptible to high light stress than WT, implying that they are active in photoprotection. These results suggest that NPQ is a fundamental mechanism for survival in excess light and that upon land colonization, photosynthetic organisms evolved a unique mechanism for excess energy dissipation before losing the ancestral one found in algae.

A red-shifted antenna protein associated with photosystem II in Physcomitrella patens.

Antenna systems of plants and green algae are made up of pigment-protein complexes belonging to the light-harvesting complex (LHC) multigene family. LHCs increase the light-harvesting cross-section of photosystems I and II and catalyze photoprotective reactions that prevent light-induced damage in an oxygenic environment. The genome of the moss Physcomitrella patens contains two genes encoding LHCb9, a new antenna protein that bears an overall sequence similarity to photosystem II antenna proteins but carries a specific motif typical of photosystem I antenna proteins. This consists of the presence of an asparagine residue as a ligand for Chl 603 (A5) chromophore rather than a histidine, the common ligand in all other LHCbs. Asparagine as a Chl 603 (A5) ligand generates red-shifted spectral forms associated with photosystem I rather than with photosystem II, suggesting that in P. patens, the energy landscape of photosystem II might be different with respect to that of most green algae and plants. In this work, we show that the in vitro refolded LHCb9-pigment complexes carry a red-shifted fluorescence emission peak, different from all other known photosystem II antenna proteins. By using a specific antibody, we localized LHCb9 within PSII supercomplexes in the thylakoid membranes. This is the first report of red-shifted spectral forms in a PSII antenna system, suggesting that this biophysical feature might have a special role either in optimization of light use efficiency or in photoprotection in the specific environmental conditions experienced by this moss.

Coexistence of plant and algal energy dissipation mechanisms in the moss Physcomitrella patens.

Although light is the source of energy for photosynthetic organisms, it causes oxidative stress when in excess. Plants and algae prevent reactive oxygen species (ROS) formation by activation of nonphotochemical quenching (NPQ), which dissipates excess excitation energy as heat. Although NPQ is found in both algae and plants, these organisms rely on two different proteins for its activation, Light harvesting complex stress-related (LHCSR) and Photosystem II subunit S (PSBS). In the moss Physcomitrella patens, both proteins are present and active. Several P. patens lines depleted in or over-expressing PSBS and/or LHCSR at various levels were generated by exploiting the ability of Physcomitrella to undergo homologous recombination. The analysis of the transgenic lines showed that either protein is sufficient, alone, for NPQ activation independently of the other, supporting the idea that they rely on different activation mechanisms. Modulation of PSBS and/or LHCSR contents was found to be correlated with NPQ amplitude, indicating that plants and algae can directly modulate their ability to dissipate energy simply by altering the accumulation level of one or both of these proteins. The availability of a large range of P. patens genotypes differing in PSBS and LHCSR content allowed comparison of their activation mechanisms and discussion of implications for the evolution of photoprotection during land colonization.

Role of PSBS and LHCSR in Physcomitrella patens acclimation to high light and low temperature.

Photosynthetic organisms respond to strong illumination by activating several photoprotection mechanisms. One of them, non-photochemical quenching (NPQ), consists in the thermal dissipation of energy absorbed in excess. In vascular plants NPQ relies on the activity of PSBS, whereas in the green algae Chlamydomonas reinhardtii it requires a different protein, LHCSR. The moss Physcomitrella patens is the only known organism in which both proteins are present and active in triggering NPQ, making this organism particularly interesting for the characterization of this protection mechanism. We analysed the acclimation of Physcomitrella to high light and low temperature, finding that these conditions induce an increase in NPQ correlated to overexpression of both PSBS and LHCSR. Mutants depleted of PSBS and/or LHCSR showed that modulation of their accumulation indeed determines NPQ amplitude. All mutants with impaired NPQ also showed enhanced photosensitivity when exposed to high light or low temperature, indicating that in this moss the fast-responding NPQ mechanism is also involved in long-term acclimation.

Programmed cell death and adaptation: two different types of abiotic stress response in a unicellular chlorophyte.

Eukaryotic microalgae are highly suitable biological indicators of environmental changes because they are exposed to extreme seasonal fluctuations. The biochemical and molecular targets and regulators of key proteins involved in the stress response in microalgae have yet to be elucidated. This study presents morphological and biochemical evidence of programmed cell death (PCD) in a low temperature strain of Chlorella saccharophila induced by exposure to NaCl stress. Morphological characteristics of PCD, including cell shrinkage, detachment of the plasma membrane from the cell wall, nuclear condensation and DNA fragmentation, were observed. Additionally, a significant production of H(2)O(2) and increase in caspase 3-like activity were detected. We demonstrated that singly applied environmental stresses such as warming or salt stress trigger a pathway of PCD. Intriguingly, the prior application of salt stress seems to reduce heat shock-induced cell death significantly, suggesting a combined effect which activates a defense mechanism in algal cells. These results suggest that C. saccharophila can undergo PCD under stress conditions, and that this PCD shares several features with metazoan PCD. Moreover, the simultaneous exposure of this unicellular chlorophyte to different abiotic stresses results in a tolerance mechanism.

PGR5 and NDH-1 systems do not function as protective electron acceptors but mitigate the consequences of PSI inhibition.

Avoidance of photoinhibition at photosystem (PS)I is based on synchronized function of PSII, PSI, Cytochrome b6f and stromal electron acceptors. Here, we used a special light regime, PSI photoinhibition treatment (PIT), in order to specifically inhibit PSI by accumulating excess electrons at the photosystem (Tikkanen and Grebe, 2018). In the analysis, Arabidopsis thaliana WT was compared to the pgr5 and ndho mutants, deficient in one of the two main cyclic electron transfer pathways described to function as protective alternative electron acceptors of PSI. The aim was to investigate whether the PGR5 (pgr5) and the type I NADH dehydrogenase (NDH-1) (ndho) systems protect PSI from excess electron stress and whether they help plants to cope with the consequences of PSI photoinhibition. First, our data reveals that neither PGR5 nor NDH-1 system protects PSI from a sudden burst of electrons. This strongly suggests that these systems in Arabidopsis thaliana do not function as direct acceptors of electrons delivered from PSII to PSI - contrasting with the flavodiiron proteins that were found to make Physcomitrella patens PSI resistant to the PIT. Second, it is demonstrated that under light-limiting conditions, the electron transfer rate at PSII is linearly dependent on the amount of functional PSI in all genotypes, while under excess light, the PGR5-dependent control of electron flow at the Cytochrome b6f complex overrides the effect of PSI inhibition. Finally, the PIT is shown to increase the amount of PGR5 and NDH-1 as well as of PTOX, suggesting that they mitigate further damage to PSI after photoinhibition rather than protect against it.

Chlorella saccharophila cytochrome f and its involvement in the heat shock response.

Cytochrome f is an essential component of the major redox complex of the thylakoid membrane. Cloning and characterization are presented here of a novel partial cDNA (ChspetA) encoding cytochrome f in the psychrophile unicellular green alga Chlorella saccharophila and its involvement in the heat shock (HS) response pathway has been analysed. Semi-quantitative reverse transcriptase PCR analysis showed that ChspetA expression is up-regulated in heat-shocked cells and the protein profile of cytochrome f highlighted a release of cytochrome f into the cytosol depending on the time lapse from the HS. Evans Blue assay, analysis of chromatin condensation, and chloroplast alterations showed the induction of cell death in cell suspensions treated with cytosolic extracts from heat-shocked cells. This study identifies cytochrome f in C. saccharophila that seems to be involved in the HS-induced programmed cell death process. The data suggest that cytochrome f fulfils its role through a modulation of its transcription and translation levels, together with its intracellular localization. This work focuses on a possible role of cytochrome f into the programmed cell death-like process in a unicellular chlorophyte and suggests the existence of chloroplast-mediated programmed cell death machinery in an organism belonging to one of the primary lineages of photosynthetic eukaryotes.