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.     

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.     

Non-photochemical quenching-dependent acclimation and thylakoid organization of Chlamydomonas reinhardtii to high light stress

Light is essential for all photosynthetic organisms while an excess of it can lead to damage mainly the photosystems of the thylakoid membrane. In this study, we have grown Chlamydomonas reinhardtii cells in different intensities of high light to understand the photosynthetic process with reference to thylakoid membrane organization during its acclimation process. We observed, the cells acclimatized to long-term response to high light intensities of 500 and 1000 µmol m^?2 s^?1 with faster growth and more biomass production when compared to cells at 50 µmol m^?2 s^?1 light intensity. The ratio of Chl a/b was marginally decreased from the mid-log phase of growth at the high light intensity. Increased level of zeaxanthin and LHCSR3 expression was also found which is known to play a key role in non-photochemical quenching (NPQ) mechanism for photoprotection. Changes in photosynthetic parameters were observed such as increased levels of NPQ, marginal change in electron transport rate, and many other changes which demonstrate that cells were acclimatized to high light which is an adaptive mechanism. Surprisingly, PSII core protein contents have marginally reduced when compared to peripherally arranged LHCII in high light-grown cells. Further, we also observed alterations in stromal subunits of PSI and low levels of PsaG, probably due to disruption of PSI assembly and also its association with LHCI. During the process of acclimation, changes in thylakoid organization occurred in high light intensities with reduction of PSII supercomplex formation. This change may be attributed to alteration of protein–pigment complexes which are in agreement with circular dichoism spectra of high light-acclimatized cells, where decrease in the magnitude of psi-type bands indicates changes in ordered arrays of PSII–LHCII supercomplexes. These results specify that acclimation to high light stress through NPQ mechanism by expression of LHCSR3 and also observed changes in thylakoid protein profile/supercomplex formation lead to low photochemical yield and more biomass production in high light condition.

     

In vitro and in vivo investigation of chlorophyll binding sites involved in non‐photochemical quenching in Chlamydomonas reinhardtii

Non‐photochemical quenching (NPQ) of the light energy absorbed is one of the main photoprotective mechanisms evolved by oxygenic photosynthetic organisms to avoid photodamage, at a cost of reduced photosynthetic efficiency. Tuning of NPQ has been reported as a promising biotechnological strategy to increase productivity in both higher plants and unicellular microalgae. Engineering of NPQ induction requires the comprehension of its molecular mechanism(s), strongly debated in the last three decades with several different models proposed. In this work, the molecular details of NPQ induction was investigated at intramolecular level by in vitro and in vitro site‐specific mutagenesis on chlorophyll binding sites of the Light‐Harvesting Complex Stress‐Related 3 (LHCSR3) protein, the pigment binding complexes identified as the quencher during NPQ induction in the model organism for green algae Chlamydomonas reinhardtii. The results obtained demonstrate a correlation between the quenching activity of LHCSR3 variants in vitro and the NPQ phenotypes observed in vivo. In particular, multiple quenching sites in LHCSR3 cooperatively dissipating the excitation energy were revealed with a peculiar role of Chl 613, a chromophore located a close distance to carotenoid binding site L1.     

Light-Harvesting Complex Stress-Related Proteins Catalyze Excess Energy Dissipation in Both Photosystems of Physcomitrella patens

Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR.     

Acclimation and endogenous abscisic acid in the moss Physcomitrella patens during acquisition of desiccation tolerance

The moss Physcomitrella patens has been used as a model organism to study the induction of desiccation tolerance (DT), but links between dehydration rate, the accumulation of endogenous abscisic acid (ABA) and DT remain unclear. In this study, we show that prolonged acclimation of P. patens at 89% relative humidity (RH) [?16 MPa] can induce tolerance of desiccation at 33% RH (?153 MPa) in both protonema and gametophore stages. During acclimation, significant endogenous ABA accumulation occurred after 1 day in gametophores and after 2 days in protonemata. Physcomitrella patens expressing the ABA‐inducible EARLY METHIONINE promoter fused to a cyan fluorescent protein (CFP) reporter gene revealed a mostly uniform distribution of the CFP increasing throughout the tissues during acclimation. DT was measured by day 6 of acclimation in gametophores, but not until 9 days of acclimation for protonemata. These results suggest that endogenous ABA accumulating when moss cells experience moderate water loss requires sufficient time to induce the changes that permit cells to survive more severe desiccation. These results provide insight for ongoing studies of how acclimation induces metabolic changes to enable DT in P. patens.     

Identification of pH-sensing Sites in the Light Harvesting Complex Stress-related 3 Protein Essential for Triggering Non-photochemical Quenching in Chlamydomonas reinhardtii

Light harvesting complex stress-related 3 (LHCSR3) is the protein essential for photoprotective excess energy dissipation (non-photochemical quenching, NPQ) in the model green alga Chlamydomonas reinhardtii. Activation of NPQ requires low pH in the thylakoid lumen, which is induced in excess light conditions and sensed by lumen-exposed acidic residues. In this work we have used site-specific mutagenesis in vivo and in vitro for identification of the residues in LHCSR3 that are responsible for sensing lumen pH. Lumen-exposed protonatable residues, aspartate and glutamate, were mutated to asparagine and glutamine, respectively. By expression in a mutant lacking all LHCSR isoforms, residues Asp¹¹⁷, Glu²²¹, and Glu²²⁴ were shown to be essential for LHCSR3-dependent NPQ induction in C. reinhardtii. Analysis of recombinant proteins carrying the same mutations refolded in vitro with pigments showed that the capacity of responding to low pH by decreasing the fluorescence lifetime, present in the wild-type protein, was lost. Consistent with a role in pH sensing, the mutations led to a substantial reduction in binding the NPQ inhibitor dicyclohexylcarbodiimide.     

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.

Phosphorylation-dependent formation of photosystem I supercomplexes provides both short- and long-term acclimation of moss photosynthetic apparatus to changing environmental cues.     

Slow zeaxanthin accumulation and the enhancement of CP26 collectively contribute to an atypical non-photochemical quenching in macroalga Ulva prolifera under high light

Non-photochemical quenching (NPQ) is an important photoprotective mechanism in plants, which dissipates excess energy and further protects the photosynthetic apparatus under high light stress. NPQ can be dissected into a number of components: qE, qZ, and qI. In general, NPQ is catalyzed by two independent mechanisms, with the faster-activated quenching catalyzed by the monomeric light-harvesting complex (LHCII) proteins and the slowly activated quenching catalyzed by LHCII trimers, both processes depending on zeaxanthin but to different extent. Here, we studied the NPQ of the intertidal green macroalga, Ulva prolifera, and found that the NPQ of U. prolifera lack the faster-activated quenching, and showed much greater sensitivity to dithiothreitol (DTT) than to dicyclohexylcarbodiimide (DCCD). Further results suggested that the monomeric LHC proteins in U. prolifera included only CP29 and CP26, but lacked CP24, unlike Arabidopsis thaliana and the moss Physcomitrella patens. Moreover, the expression levels of CP26 increased significantly following exposure to high light, but the concentrations of the two important photoprotective proteins (PsbS and light-harvesting complex stress-related [LhcSR]) did not change upon the same conditions. Analysis of the xanthophyll cycle pigments showed that, upon exposure to high light, zeaxanthin synthesis in U. prolifera was gradual and much slower than that in P. patens, and could effectively be inhibited by DTT. Based on these results, we speculate the enhancement of CP26 and slow zeaxanthin accumulation provide an atypical NPQ, making this green macroalga well adapted to the intertidal environments.     

Photosynthetic Acclimation of Erythrophleum guineense and Dalbergia odorifera to Winter Low Temperature in a Marginal Tropical Area

In marginal tropical areas, air temperature in winter usually decreases by 10℃ compared with summer at night/day. Although tropical plants are sensitive to low temperature, the mechanism underlying photosynthetic acclimation of tropical trees to winter low temperature is unclear. To address this question, the photosystem I (PSI) and photosystem II (PSII) activities, and energy distribution in PSI and PSII were examined in summer and winter in two tropical high quality timber tree species Erythrophleum guineense and Dalbergia odorifera grown in a marginal tropical area (21°54′N, 101°46′E). Our results indicated that the photosynthetic apparatus of Eguineense and Dodorifera was maintained stable in winter. The effective quantum yield of PSII decreased significantly in winter, but non photochemical quenching (NPQ) significantly increased. In winter, cyclic electron flow (CEF) was significantly stimulated in both species, which was significantly and positively correlated with NPQ. Meanwhile, the stimulation of CEF led to an increase in P700 oxidation ratio and the over reduction of PSI acceptor side was prevented. Antimycin A (a specific inhibitor of PGR5 dependent CEF) significantly aggravated PSII photoinhibition under high light in both species. These results suggested that stimulation of CEF is an important mechanism for photosynthetic acclimation to winter low temperature in a marginal tropical area in the two tropical tree species.     

PsbS contributes to photoprotection in Chlamydomonas reinhardtii independently of energy dissipation

Photosynthetic organisms are frequently exposed to excess light conditions and hence to photo-oxidative stress. To counteract photo-oxidative damage, land plants and most algae make use of non- photochemical quenching (NPQ) of excess light energy, in particular the rapidly inducible and relaxing qE-mechanism. In vascular plants, the constitutively active PsbS protein is the key regulator of qE. In the green algae C. reinhardtii, however, qE activation is only possible after initial high-light (HL) acclimation for several hours and requires the synthesis of LHCSR proteins which act as qE regulators. The precise function of PsbS, which is transiently expressed during HL acclimation in C. reinhardtii, is still unclear. Here, we investigated the impact of different PsbS amounts on HL acclimation characteristics of C. reinhardtii cells. We demonstrate that lower PsbS amounts negatively affect HL acclimation at different levels, including NPQ capacity, electron transport characteristics, antenna organization and morphological changes, resulting in an overall increased HL sensitivity and lower vitality of cells. Contrarily, higher PsbS amounts do not result in a higher NPQ capacity, but nevertheless provide higher fitness and tolerance towards HL stress. Strikingly, constitutively expressed PsbS protein was found to be degraded during HL acclimation. We propose that PsbS is transiently required during HL acclimation for the reorganization of thylakoid membranes and/or antenna proteins along with the activation of NPQ and adjustment of electron transfer characteristics, and that degradation of PsbS is essential in the fully HL acclimated state.     

Effect of the energy supply on filamentous growth and development in Physcomitrella patens

The filamentous gametophyte of the moss Physcomitrella patens consists of two filament types called chloronemata and caulonemata. Chloronemal cells are photosynthetically active with numerous chloroplasts, while caulonemata help to spread the colony by radial growth. The balance between the two filament types is affected by external factors such as light and plant hormones. In the present study, caulonema formation and chloronemal branching have been monitored during high and low light conditions and in the presence of glucose, auxin, or cytokinin. These experiments were performed both in a wild-type strain and in a hxk1 knockout mutant which lacks the major hexokinase of Physcomitrella. It was found that caulonema formation is induced by high energy conditions such as high light and external glucose, while chloronemal branching is stimulated by low energy conditions such as reduced light, and in the hxk1 mutant. The hxk1 mutation also causes buds to appear on chloronemal filaments, which is rarely seen in the wild type, and shows increased sensitivity to cytokinin and abscisic acid. Based on these findings a model is proposed in which the energy supply of the moss colony regulates the balance between chloronemal and caulonemal growth.     

几内亚格木和降香黄檀对热带北缘地区冬季低温的光合适应

在热带北缘地区,冬季气温较夏季下降10℃左右,虽然热带植物对零上低温敏感,但是大部分热带树木能够适应热带北缘地区的冬季气温,其光合生理机制并不清楚。我们通过测定种植在热带北缘地区（21°54′N,101°46′E）的两种热带树木（几内亚格木和降香黄檀）的光系统Ⅰ和Ⅱ活性以及光系统Ⅰ和Ⅱ的能量分配的季节变化,发现这两个树种的光系统Ⅰ和Ⅱ活性在冬季并没有下降。两个树种的光系统Ⅱ的有效量子产额在冬季明显下降,同时伴随着热耗散激发。在冬季,环式电子传递的激发与热耗散的激发呈现显著的正相关。环式电子传递的激发使得氧化态P700比例的上升,从而避免了光系统Ⅰ受体端的过度还原。化学试剂抗霉素A（PGR5途径环式电子传递的一种特异性抑制剂）处理过的叶片较对照组表现出更强光损伤程度。这些结果表明环式电子传递的激发是热带树木适应热带北缘地区冬季低温的一个重要的光合生理机制。     

In silico and biochemical analysis of Physcomitrella patens photosynthetic antenna: identification of subunits which evolved upon land adaptation

Background

In eukaryotes the photosynthetic antenna system is composed of subunits encoded by the light harvesting complex (Lhc) multigene family. These proteins play a key role in photosynthesis and are involved in both light harvesting and photoprotection. The moss Physcomitrella patens is a member of a lineage that diverged from seed plants early after land colonization and therefore by studying this organism, we may gain insight into adaptations to the aerial environment.

Principal Findings

In this study, we characterized the antenna protein multigene family in Physcomitrella patens, by sequence analysis as well as biochemical and functional investigations. Sequence identification and analysis showed that some antenna polypeptides, such as Lhcb3 and Lhcb6, are present only in land organisms, suggesting they play a role in adaptation to the sub-aerial environment. Our functional analysis which showed that photo-protective mechanisms in Physcomitrella patens are very similar to those in seed plants fits with this hypothesis. In particular, Physcomitrella patens also activates Non Photochemical Quenching upon illumination, consistent with the detection of an ortholog of the PsbS protein. As a further adaptation to terrestrial conditions, the content of Photosystem I low energy absorbing chlorophylls also increased, as demonstrated by differences in Lhca3 and Lhca4 polypeptide sequences, in vitro reconstitution experiments and low temperature fluorescence spectra.

Conclusions

This study highlights the role of Lhc family members in environmental adaptation and allowed proteins associated with mechanisms of stress resistance to be identified within this large family.     

Photoprotective responses of an ice algal community in Saroma-Ko Lagoon, Hokkaido, Japan

In polar seas, ice algal communities can acclimate to extremely low light conditions. Reduced acclimation to shade in ice algal communities, as a result of shortened ice seasons at the lower latitude limits of sea ice distribution, has been suggested as advantageous for avoiding strong photoinhibition when cells are released into high light levels at the water’s surface. Thermal dissipation of excess energy by xanthophyll cycle pigments in the de-epoxidated state may occur in ice algal communities released from retreating sea ice. A light exposure experiment was conducted on ice algal communities obtained from sea ice at Saroma-Ko Lagoon in Hokkaido, Japan. Photoprotective responses to direct sunlight were examined through non-photochemical quenching (NPQ) of chlorophyll fluorescence and xanthophyll pigments. De-epoxidation of diadinoxanthin (DD) to diatoxanthin (DT) occurred rapidly, and NPQ showed a dynamic response to high light exposure. The linear relationship between the ratio of DT to chlorophyll a and NPQ followed a steeper slope than previously observed for mesophilic diatoms. The steeper slope could be explained by an apparent increase in DT for the mesophilic diatoms and induction of NPQ in response to low temperatures only in the ice algal communities. Enhanced production of DT in mesophilic diatoms could be the result of de-epoxidation of DD plus de novo synthesis, and the enhancement of NPQ might be caused by low temperature stress in the ice algae. Although the response of NPQ might be related to temperature, NPQ independent of DT synthesis should also be studied.     

Enhancing the growth of Physcomitrella patens by combination of monochromatic red and blue light - a kinetic study

In the current work we demonstrate the relevance of monochromatic light conditions in moss plant cell culture. Light intensity and illumination wavelength are important cultivation parameters due to their impact on growth and chlorophyll formation kinetics of the moss Physcomitrella patens. This moss was chosen as a model organism due to its capability to produce complex recombinant pharmaceutical proteins. Filamentous moss cells were cultivated in mineral medium in shaking flasks. The flasks were illuminated by light emitting diodes (LED) providing nearly monochromatic red and blue light as well as white light as a reference. A maximum growth rate of 0.78 day((1) was achieved under additional CO(2) aeration and no growth inhibition was observed under high light illumination. The application of dual red and blue light is the most effective way to reach high growth and chlorophyll formation rates while minimizing energy consumption of the LEDs. These observations are discussed as effects of photo sensory pigments in the moss. The combination of monochromatic red and blue light should be considered when a large scale process is set up.     

Mapping of the Physcomitrella patens proteome

The moss Physcomitrella patens is unique among land plants due to the high rate of homologous recombination in its nuclear DNA. The feasibility of gene targeting makes Physcomitrella an unrivalled model organism in the field of plant functional genomics. To further extend the potentialities of this seed-less plant we aimed at exploring the P. patens proteome. Experimental conditions had to be adopted to meet the special requirements connected to the investigations of this moss. Here we describe the identification of 306 proteins from the protonema of Physcomitrella. Proteins were separated by two dimensional electrophoresis, excised form the gel and analysed by means of mass spectrometry. This reference map will lay the basis for further profound studies in the field of Physcomitrella proteomics.     

Effect of cold acclimation on the photosynthetic performance of two ecotypes of Colobanthus quitensis (Kunth) Bartl

The effects of cold acclimation of two ecotypes (Antarctic and Andes) of Colobanthus quitensis (Kunth) Bartl. Caryophyllaceae on their photosynthetic characteristics and performance under high light (HL) were compared. Non-acclimated plants of the Antarctic ecotype exhibited a higher (34%) maximal rate of photosynthesis than the Andes ecotype. In cold-acclimated plants the light compensation point was increased. Dark respiration was significantly increased during the exposure to 4 degrees C in both ecotypes. Cold-acclimated Antarctic plants showed higher Phi(PSII) and qP compared with the Andes ecotype. In addition, the Antarctic ecotype exhibited higher heat dissipation (NPQ), especially in the cold-acclimated state, which was mainly associated with the fast relaxing component of non-photochemical quenching (NPQ(F)). By contrast, the Andes ecotype exhibited a lower NPQ(F) and a significant increase in the slowly relaxing component (NPQ(s)) at low temperature and HL, indicating higher sensitivity to low temperature-induced photoinhibition. Although the xanthophyll cycle was fully operational in both ecotypes, cold-acclimated Antarctic plants exposed to HL exhibited higher epoxidation state of the xanthophyll cycle pigments (EPS) compared with the cold-acclimated Andes ecotype. Thus, the photosynthetic apparatus of the Antarctic ecotype operates more efficiently than that of the Andes one, under a combination of low temperature and HL. The ecotype differences are discussed in relation to the different climatic conditions of the two Colobanthus.