Spliceosomal complex components are critical for adjusting the C:N balance during high-light acclimation.

Plant acclimation to an ever-changing environment is decisive for growth, reproduction, and survival. Light availability limits biomass production on both ends of the intensity spectrum. Therefore, the adjustment of plant metabolism is central to high-light (HL) acclimation, and the accumulation of photoprotective anthocyanins is commonly observed. However, mechanisms and factors regulating the HL acclimation response are less clear. Two Arabidopsis mutants of spliceosome components exhibiting a pronounced anthocyanin overaccumulation in HL were isolated from a forward genetic screen for new factors crucial for plant acclimation. Time-resolved physiological, transcriptome, and metabolome analysis revealed a vital function of the spliceosome components for rapidly adjusting gene expression and metabolism. Deficiency of INCREASED LEVEL OF POLYPLOIDY1 (ILP1), NTC-RELATED PROTEIN1 (NTR1), and PLEIOTROPIC REGULATORY LOCUS1 (PRL1) resulted in a marked overaccumulation of carbohydrates and strongly diminished amino acid biosynthesis in HL. While not generally limited in N-assimilation, ilp1, ntr1, and prl1 showed higher glutamate levels and reduced amino acid biosynthesis in HL. The comprehensive analysis reveals a function of the spliceosome components in the conditional regulation of the carbon:nitrogen balance and the accumulation of anthocyanins during HL acclimation. The importance of gene expression, metabolic regulation, and re-direction of carbon towards anthocyanin biosynthesis for HL acclimation are discussed.

Intervening dark periods negatively affect the photosynthetic performance of Chlamydomonas reinhardtii during growth under fluctuating high light.

The acclimation of the green algae Chlamydomoas reinhardtii to high light (HL) has been studied predominantly under continuous illumination of the cells. Here, we investigated the impact of fluctuating HL in alternation with either low light (LL) or darkness on photosynthetic performance and on photoprotective responses. Compared to intervening LL phases, dark phases led to (1) more pronounced reduction of the photosystem II quantum efficiency, (2) reduced degradation of the PsbS protein, (3) lower energy dissipation capacity and (4) an increased pool size of the xanthophyll cycle pigments. These characteristics indicate increased photo-oxidative stress when HL periods are interrupted by dark phases instead of LL phases. This overall trend was similar when comparing long (8 h) and short (30 min) HL phases being interrupted by long (16 h) and short (60 min) phases of dark or low light, respectively. Only the degradation of PsbS was clearly more efficient during long (16 h) LL phases when compared to short (60 min) LL phases.

Chlorophyll and Pheophytin Dephytylating Enzymes Required for Efficient Repair of PSII in Synechococcus elongatus PCC 7942.

The Chlorophyll Dephytylase1 (CLD1) and pheophytinase (PPH) proteins of Arabidopsis thaliana are homologous proteins characterized respectively as a dephytylase for chlorophylls (Chls) and pheophytin a (Phein a) and a Phein a-specific dephytylase. Three genes encoding CLD1/PPH homologs (dphA1, dphA2 and dphA3) were found in the genome of the cyanobacterium Synechococcus elongatus PCC 7942 and shown to be conserved in most cyanobacteria. His6-tagged DphA1, DphA2 and DphA3 proteins were expressed in Escherichia coli, purified to near homogeneity, and shown to exhibit significant levels of dephytylase activity for Chl a and Phein a. Each DphA protein showed similar dephytylase activities for Chl a and Phein a, but the three proteins were distinct in their kinetic properties, with DphA3 showing the highest and lowest Vmax and Km values, respectively, among the three. Transcription of dphA1 and dphA3 was enhanced under high-light conditions, whereas that of dphA2 was not affected by the light conditions. None of the dphA single mutants of S. elongatus showed profound growth defects under low (50 µmol photons m-2 s-1) or high (400 µmol photons m-2 s-1) light conditions. The triple dphA mutant did not show obvious growth defects under these conditions, either, but under illumination of 1,000 µmol photons m-2 s-1, the mutant showed more profound growth retardation compared with wild type (WT). The repair of photodamaged photosystem II (PSII) was much slower in the triple mutant than in WT. These results revealed that dephytylation of Chl a or Phein a or of both is required for efficient repair of photodamaged PSII.

The plastid-nucleus localized DNA-binding protein WHIRLY1 is required for acclimation of barley leaves to high light.

MAIN CONCLUSION: In accordance with a key role of WHIRLY1 in light-acclimation mechanisms, typical features of acclimation to high light, including photosynthesis and leaf morphology, are compromised in WHIRLY1 deficient plants. Acclimation to the environment requires efficient communication between chloroplasts and the nucleus. Previous studies indicated that the plastid-nucleus located WHIRLY1 protein is required for the communication between plastids and the nucleus in situations of high light exposure. To investigate the consequences of WHIRLY1 deficiency on the light acclimation of photosynthesis and leaf anatomy, transgenic barley plants with an RNAi-mediated knockdown of HvWHIRLY1 were compared to wild-type plants when growing at low and high irradiance. While wild-type plants showed the typical light acclimation responses, i.e. higher photosynthetic capacity and thicker leaves, the WHIRLY1 deficient plants were not able to respond to differences in irradiance. The results revealed a systemic role of WHIRLY1 in light acclimation by coordinating responses at the level of the chloroplast and the level of leaf morphology.

High Light Acclimation Mechanisms Deficient in a PsbS-Knockout Arabidopsis Mutant.

The photosystem II PsbS protein of thylakoid membranes is responsible for regulating the energy-dependent, non-photochemical quenching of excess chlorophyll excited states as a short-term mechanism for protection against high light (HL) stress. However, the role of PsbS protein in long-term HL acclimation processes remains poorly understood. Here we investigate the role of PsbS protein during long-term HL acclimation processes in wild-type (WT) and npq4-1 mutants of Arabidopsis which lack the PsbS protein. During long-term HL illumination, photosystem II photochemical efficiency initially dropped, followed by a recovery of electron transport and photochemical quenching (qL) in WT, but not in npq4-1 mutants. In addition, we observed a reduction in light-harvesting antenna size during HL treatment that ceased after HL treatment in WT, but not in npq4-1 mutants. When plants were adapted to HL, more reactive oxygen species (ROS) were accumulated in npq4-1 mutants compared to WT. Gene expression studies indicated that npq4-1 mutants failed to express genes involved in plastoquinone biosynthesis. These results suggest that the PsbS protein regulates recovery processes such as electron transport and qL during long-term HL acclimation by maintaining plastoquinone biosynthetic gene expression and enhancing ROS homeostasis.

High light acclimation of Chromera velia points to photoprotective NPQ.

It has previously been shown that the long-term treatment of Arabidopsis thaliana with the chloroplast inhibitor lincomycin leads to photosynthetic membranes enriched in antennas, strongly reduced in photosystem II reaction centers (PSII) and with enhanced nonphotochemical quenching (NPQ) (Belgio et al. Biophys J 102:2761-2771, 2012). Here, a similar physiological response was found in the microalga Chromera velia grown under high light (HL). In comparison to cells acclimated to low light, HL cells displayed a severe re-organization of the photosynthetic membrane characterized by (1) a reduction of PSII but similar antenna content; (2) partial uncoupling of antennas from PSII; (3) enhanced NPQ. The decrease in the number of PSII represents a rather unusual acclimation response compared to other phototrophs, where a smaller PSII antenna size is more commonly found under high light. Despite the diminished PSII content, no net damage could be detected on the basis of the Photosynthesis versus irradiance curve and electron transport rates pointing at the excess capacity of PSII. We therefore concluded that the photoinhibition is minimized under high light by a lower PSII content and that cells are protected by NPQ in the antennas.

Transcriptional and posttranscriptional regulation of cyanobacterial photosynthesis.

Cyanobacteria are well established model organisms for the study of oxygenic photosynthesis, nitrogen metabolism, toxin biosynthesis, and salt acclimation. However, in comparison to other model bacteria little is known about regulatory networks, which allow cyanobacteria to acclimate to changing environmental conditions. The current work has begun to illuminate how transcription factors modulate expression of different photosynthetic regulons. During the past few years, the research on other regulatory principles like RNA-based regulation showed the importance of non-protein regulators for bacterial lifestyle. Investigations on modulation of photosynthetic components should elucidate the contributions of all factors within the context of a larger regulatory network. Here, we focus on regulation of photosynthetic processes including transcriptional and posttranscriptional mechanisms, citing examples from a limited number of cyanobacterial species. Though, the general idea holds true for most species, important differences exist between various organisms, illustrating diversity of acclimation strategies in the very heterogeneous cyanobacterial clade. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Prof Conrad Mullineaux.

A Feed-Forward Loop Consisting of the Response Regulator RpaB and the Small RNA PsrR1 Controls Light Acclimation of Photosystem I Gene Expression in the Cyanobacterium Synechocystis sp. PCC 6803.

Since cyanobacteria need to decrease PSI content to avoid absorption of excess light energy, down-regulation of PSI gene expression is one of the key characteristics of the high-light (HL) acclimation response. The transcriptional regulator RpaB and the small RNA PsrR1 (photosynthesis regulatory RNA1) have been suggested to be the two most critical factors for this response in Synechocystis sp. PCC 6803. In this study, we found that the HLR1 DNA-binding motif, the recognition sequence for RpaB, is highly conserved in the core promoter region of the psrR1 gene among cyanobacterial species. Gel mobility shift assay revealed that RpaB binds to the HLR1 sequence of psrR1 in vitro. RNA gel blot analysis together with chromatin affinity purification (ChAP) analysis suggested that PSI genes are activated and the psrR1 gene is repressed by the binding of RpaB under low-light (LL) conditions. A decrease in DNA binding affinity of RpaB occurs within 5 min after the shift from LL to HL conditions, leading to the prompt decrease in PSI promoter activity together with derepression of psrR1 gene expression. Accumulating PsrR1 molecules then prevent translation from pre-existing PSI transcripts. By this dual repression at transcriptional and post-transcriptional levels, rapid and strict down-regulation of PSI expression under HL is secured. Our findings suggest that RpaB and PsrR1 constitute a feed-forward loop for the regulation of PSI gene expression to achieve a rapid acclimation response to the damaging HL conditions.

The chloroplast NADPH thioredoxin reductase C, NTRC, controls non-photochemical quenching of light energy and photosynthetic electron transport in Arabidopsis.

High irradiances may lead to photooxidative stress in plants, and non-photochemical quenching (NPQ) contributes to protection against excess excitation. One of the NPQ mechanisms, qE, involves thermal dissipation of the light energy captured. Importantly, plants need to tune down qE under light-limiting conditions for efficient utilization of the available quanta. Considering the possible redox control of responses to excess light implying enzymes, such as thioredoxins, we have studied the role of the NADPH thioredoxin reductase C (NTRC). Whereas Arabidopsis thaliana plants lacking NTRC tolerate high light intensities, these plants display drastically elevated qE, have larger trans-thylakoid ΔpH and have 10-fold higher zeaxanthin levels under low and medium light intensities, leading to extremely low linear electron transport rates. To test the impact of the high qE on plant growth, we generated an ntrc-psbs double-knockout mutant, which is devoid of qE. This double mutant grows faster than the ntrc mutant and has a higher chlorophyll content. The photosystem II activity is partially restored in the ntrc-psbs mutant, and linear electron transport rates under low and medium light intensities are twice as high as compared with plants lacking ntrc alone. These data uncover a new role for NTRC in the control of photosynthetic yield.

Essential Role of Acyl-ACP Synthetase in Acclimation of the Cyanobacterium Synechococcus elongatus Strain PCC 7942 to High-Light Conditions.

Most organisms capable of oxygenic photosynthesis have an aas gene encoding an acyl-acyl carrier protein synthetase (Aas), which activates free fatty acids (FFAs) via esterification to acyl carrier protein. Cyanobacterial aas mutants are often used for studies aimed at photosynthetic production of biofuels because the mutation leads to intracellular accumulation of FFAs and their secretion into the external medium, but the physiological significance of the production of FFAs and their recycling involving Aas has remained unclear. Using an aas-deficient mutant of Synechococcus elongatus strain PCC 7942, we show here that remodeling of membrane lipids is activated by high-intensity light and that the recycling of FFAs is essential for acclimation to high-light conditions. Unlike wild-type cells, the mutant cells could not increase their growth rate as the light intensity was increased from 50 to 400 µmol photons m(-2) s(-1), and the high-light-grown mutant cells accumulated FFAs and the lysolipids derived from all the four major classes of membrane lipids, revealing high-light-induced lipid deacylation. The high-light-grown mutant cells showed much lower PSII activity and Chl contents as compared with the wild-type cells or low-light-grown mutant cells. The loss of Aas accelerated photodamage of PSII but did not affect the repair process of PSII, indicating that PSII is destabilized in the mutant. Thus, Aas is essential for acclimation of the cyanobacterium to high-light conditions. The relevance of the present finding s to biofuel production using cyanobacteria is discussed.

A deficiency in chloroplastic ferredoxin 2 facilitates effective photosynthetic capacity during long-term high light acclimation in Arabidopsis thaliana.

Photosynthetic electron transport is the major energy source for cellular metabolism in plants, and also has the potential to generate excess reactive oxygen species that cause irreversible damage to photosynthetic apparatus under adverse conditions. Ferredoxins (Fds), as the electron-distributing hub in the chloroplast, contribute to redox regulation and antioxidant defense. However, the steady-state levels of photosynthetic Fd decrease in plants when they are exposed to environmental stress conditions. To understand the effect of Fd down-regulation on plant growth, we characterized Arabidopsis thaliana plants lacking Fd2 (Fd2-KO) under long-term high light (HL) conditions. Unexpectedly, Fd2-KO plants exhibited efficient photosynthetic capacity and stable thylakoid protein complexes. At the transcriptional level, photoprotection-related genes were up-regulated more in the mutant plants, suggesting that knockout Fd2 lines possess a relatively effective photo-acclimatory responses involving enhanced plastid redox signaling. In contrast to the physiological characterization of Fd2-KO under short-term HL, the plastoquinone pool returned to a relatively balanced redox state via elevated PGR5-dependent cyclic electron flow during extended HL. fd2 pgr5 double mutant plants displayed severely impaired photosynthetic capacity under HL treatment, further supporting a role for PGR5 in adaptation to HL in the Fd2-KO plants. These results suggest potential benefits of reducing Fd levels in plants grown under long-term HL conditions.

Identification of Small RNAs During High Light Acclimation in Arabidopsis thaliana.

The biological significance of non-coding RNAs (ncRNAs) has been firmly established to be important for the regulation of genes involved in stress acclimation. Light plays an important role for the growth of plants providing the energy for photosynthesis; however, excessive light conditions can also cause substantial defects. Small RNAs (sRNAs) are a class of non-coding RNAs that regulate transcript levels of protein-coding genes and mediate epigenetic silencing. Next generation sequencing facilitates the identification of small non-coding RNA classes such as miRNAs (microRNAs) and small-interfering RNAs (siRNAs), and long non-coding RNAs (lncRNAs), but changes in the ncRNA transcriptome in response to high light are poorly understood. We subjected Arabidopsis plants to high light conditions and performed a temporal in-depth study of the transcriptome data after 3 h, 6 h, and 2 days of high light treatment. We identified a large number of high light responsive miRNAs and sRNAs derived from NAT gene pairs, lncRNAs and TAS transcripts. We performed target predictions for differentially expressed miRNAs and correlated their expression levels through mRNA sequencing data. GO analysis of the targets revealed an overrepresentation of genes involved in transcriptional regulation. In A. thaliana, sRNA-mediated regulation of gene expression in response to high light treatment is mainly carried out by miRNAs and sRNAs derived from NAT gene pairs, and from lncRNAs. This study provides a deeper understanding of sRNA-dependent regulatory networks in high light acclimation.

Slr0320 Is Crucial for Optimal Function of Photosystem II during High Light Acclimation in Synechocystis sp. PCC 6803.

Upon exposure of photosynthetic organisms to high light (HL), several HL acclimation responses are triggered. Herein, we identified a novel gene, slr0320, critical for HL acclimation in Synechocystis sp. PCC 6803. The growth rate of the Δslr0320 mutant was similar to wild type (WT) under normal light (NL) but severely declined under HL. Net photosynthesis of the mutant was lower under HL, but maximum photosystem II (PSII) activity was higher under NL and HL. Immunodetection revealed the accumulation and assembly of PSII were similar between WT and the mutant. Chlorophyll fluorescence traces showed the stable fluorescence of the mutant under light was much higher. Kinetics of single flash-induced chlorophyll fluorescence increase and decay revealed the slower electron transfer from QA to QB in the mutant. These data indicate that, in the Δslr0320 mutant, the number of functional PSIIs was comparable to WT even under HL but the electron transfer between QA and QB was inefficient. Quantitative proteomics and real-time PCR revealed that expression profiles of psbL, psbH and psbI were significantly altered in the Δslr0320 mutant. Thus, Slr0320 protein plays critical roles in optimizing PSII activity during HL acclimation and is essential for PSII electron transfer from QA to QB.

The xanthophyll cycle in diatom Phaeodactylum tricornutum in response to light stress.

Chosen aspects of the functioning of diadinoxanthin cycle in a model diatom Phaeodactylum tricornutum grown under low light conditions (LL) and under high light conditions (HL), which cause activation of violaxanthin cycle, were examined. Heterogeneity of the kinetics of diadinoxanthin ↔ diatoxanthin conversions regulated by de-epoxidase/epoxidase enzymes was detected. Three different rates of diadinoxanthin de-epoxidation (τ > 20 min, 5 min > τ > 1.5 min and τ ≤ 1 min) were observed. Appearance and contribution of these phases depended on the light conditions and xanthophylls subpopulations in membranes. Moreover, diadinoxanthin de-epoxidation was postulated to occur in darkness and its rate was estimated to be almost two times faster (τ ≈ 14 min) than diatoxanthin-epoxidation in LL- and HL-grown diatoms collected after the dark phase of the photoperiod and exposed to very high light and subsequent darkness. The level of lipid hydroperoxides and the expression of genes encoding xanthophyll cycle enzymes was measured. Our observations suggest that isoforms of these enzymes may participate in carotenoid synthesis or be exclusively involved in xanthophyll conversions. Violaxanthin cycle pigments present in HL-acclimated diatoms change thermodynamic properties of thylakoid membranes. Zeaxanthin is known to localize preferentially in the inner part of the lipid bilayer and diatoxanthin in its outer part. The different localization of these pigments probably decide about their complementary action in protection of the membranes against reactive oxygen 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.

Mutation of the Atypical Kinase ABC1K3 Partially Rescues the PROTON GRADIENT REGULATION 6 Phenotype in Arabidopsis thaliana.

Photosynthesis is an essential pathway providing the chemical energy and reducing equivalents that sustain higher plant metabolism. It relies on sunlight, which is an inconstant source of energy that fluctuates in both intensity and spectrum. The fine and rapid tuning of the photosynthetic apparatus is essential to cope with changing light conditions and increase plant fitness. Recently PROTON GRADIENT REGULATION 6 (PGR6-ABC1K1), an atypical plastoglobule-associated kinase, was shown to regulate a new mechanism of light response by controlling the homeostasis of photoactive plastoquinone (PQ). PQ is a crucial electron carrier existing as a free neutral lipid in the photosynthetic thylakoid membrane. Perturbed homeostasis of PQ impairs photosynthesis and plant acclimation to high light. Here we show that a homologous kinase, ABC1K3, which like PGR6-ABC1K1 is associated with plastoglobules, also contributes to the homeostasis of the photoactive PQ pool. Contrary to PGR6-ABC1K1, ABC1K3 disfavors PQ availability for photosynthetic electron transport. In fact, in the abc1k1/abc1k3 double mutant the pgr6(abc1k1) the photosynthetic defect seen in the abc1k1 mutant is mitigated. However, the PQ concentration in the photoactive pool of the double mutant is comparable to that of abc1k1 mutant. An increase of the PQ mobility, inferred from the kinetics of its oxidation in dark, contributes to the mitigation of the pgr6(abc1k1) photosynthetic defect. Our results also demonstrate that ABC1K3 contributes to the regulation of other mechanisms involved in the adaptation of the photosynthetic apparatus to changes in light quality and intensity such as the induction of thermal dissipation and state transitions. Overall, we suggests that, besides the absolute concentration of PQ, its mobility and exchange between storage and active pools are critical for light acclimation in plants.

Requirement of ONE-HELIX PROTEIN 1 (OHP1) in early Arabidopsis seedling development and under high light intensity.

Plant ONE-HELIX PROTEINS (OHPs) are part of the light-harvesting complex superfamily whose members are involved in various processes related to sensing and capturing light as well as light protection. We recently showed the requirement of a functional OHP1-OHP2 heterodimer for efficient D1 synthesis. Interestingly, while the ohp1 knockout mutant showed a strong defect in accumulation of the photosystem II and is hardly viable, virus-induced gene silencing of OHP1 had no detectable impact on plant growth and performance under standard growth conditions. However, in vivo labeling assays with 35S-methionine indicate a reduced D1 synthesis rate. Here, we show that VIGS-OHP1 plants are more susceptible towards elevated light intensities than control plants. This underlines an obligatory function of OHP1 for light acclimation.