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.ife on Earth depends on oxygenic photosynthesis, which enables plants, algae, and cyanobacteria to convert light into chemical energy. Sunlight powers the transfer of electrons from water to NADP + by the activity of two photosystems (PS), PSII and PSI, thus generating NADPH and ATP to sustain cell metabolism. Natural environmental conditions are highly variable, and sudden changes in irradiation can drastically affect the flow of excitation energy and electrons. At the same time, the ATP and NADPH consumption rate is also highly dynamic because of a continuous metabolic regulation (1-3). Photosynthetic organisms evolved several mechanisms to modulate the flow of excitation energy and electrons according to metabolic constraints, diverting/ feeding electrons from/to the linear transport chain (3). These pathways modulate the ATP/NADPH ratio, as in the cyclic electron transport (CET) around PSI, where electrons are redirected from PSI to plastoquinone (PQ) or Cytb 6 f (4), contributing to proton translocation and ATP synthesis, but not to NADPH formation (5-9).In cyanobacteria, the Flavodiiron proteins (known as FLV) have been identified as an additional component of electron transport chain (10-12). FLV proteins are constituted by three distinct domains: a N-terminal β-lactamase-like domain, a flavodoxin-like domain, and a C-terminal NAD(P)H-flavin reductase-like domain. The former two domains are also found in FLV proteins from archaea and anaerobic bacteria, where they are involved in O 2 or NO reduction, whereas the latter is typical only of FLVs from oxygenic photosynthetic organisms (11-13). Recent studies showed that in cyanobacteria, the FLV1/FLV3 heterodimer catalyzes the light-dependent reduction of O 2 to water, using NADPH as electron donor (10, 11), protecting PSI from light stress (10). Another FLVs heterodimer, FLV2/FLV4, instead, has been shown to be active in photo-protection of PSII (14-16). FLVs also were found expre...
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.LHCSR | nonphotochemical quenching | photosystem | plants evolution | PSBS S unlight provides energy supporting the life of photosynthetic organisms but also leads to the formation of reactive oxygen species when in excess (1, 2). During early Devonian, when plants first colonized terrestrial habitats, they underwent no competition by other organisms. However, they had to adapt to harsher physicochemical conditions than in the original water ecosystem (3) because in the atmosphere concentration of oxygen, an inhibitor of photosynthesis, is higher (4) and concentration of carbon dioxide, the final acceptor of electrons extracted from water by photosystems, is lower. Moreover, the sessile form of life acquired on land prevented escape from rapid changes in light intensity by swimming deeper, a behavior typical of algae (5). The combination of these conditions makes it more likely that light is harvested in excess with respect to the maximal rate of photochemical reactions, and a fast and efficient photoprotection response is essential for survival.The fastest response to high light stress is provided by nonphotochemical quenching (NPQ), which consists of the thermal dissipation of the chlorophyll excited singlet states ( 1 Chl*) (6-8). NPQ has two major components: energy quenching (qE), which is activated within seconds on an increase in light intensity, and inhibitory quenching, which is slower and relaxes within 1-2 h in the dark (7, 9). In vascular plants, qE activation requires PSBS, a protein homologous to light harvesting antenna subunits of photosystems (Lhc) (10), which is activated by the accumulation of protons in the chloroplast lumen and the protonation of two glutamate residues (11). Activated PSBS induces a decrease...
ORCID IDs: 0000-0003-4818-7778 (A.A.); 0000-0002-0803-7591 (T.M.); 0000-0003-4735-8811 (R.B.).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.
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.
SummaryAlthough 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.
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.
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.
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 histidinetagged 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.
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