PSI Photoinhibition and Changing CO2 Levels Initiate Retrograde Signals to Modify Nuclear Gene Expression.

Photosystem I (PSI) is a critical component of the photosynthetic machinery in plants. Under conditions of environmental stress, PSI becomes photoinhibited, leading to a redox imbalance in the chloroplast. PSI photoinhibition is caused by an increase in electron pressure within PSI, which damages the iron-sulfur clusters. In this study, we investigated the susceptibility of PSI to photoinhibition in plants at different concentrations of CO2, followed by global gene expression analyses of the differentially treated plants. PSI photoinhibition was induced using a specific illumination protocol that inhibited PSI with minimal effects on PSII. Unexpectedly, the varying CO2 levels combined with the PSI-PI treatment neither increased nor decreased the likelihood of PSI photodamage. All PSI photoinhibition treatments, independent of CO2 levels, upregulated genes generally involved in plant responses to excess iron and downregulated genes involved in iron deficiency. PSI photoinhibition also induced genes encoding photosynthetic proteins that act as electron acceptors from PSI. We propose that PSI photoinhibition causes a release of iron from damaged iron-sulfur clusters, which initiates a retrograde signal from the chloroplast to the nucleus to modify gene expression. In addition, the deprivation of CO2 from the air initiated a signal that induced flavonoid biosynthesis genes, probably via jasmonate production.

Photosynthetic and transcriptome responses to fluctuating light in Arabidopsis thylakoid ion transport triple mutant.

Fluctuating light intensity challenges fluent photosynthetic electron transport in plants, inducing photoprotection while diminishing carbon assimilation and growth, and also influencing photosynthetic signaling for regulation of gene expression. Here, we employed in vivo chlorophyll-a fluorescence and P700 difference absorption measurements to demonstrate the enhancement of photoprotective energy dissipation of both photosystems in wild-type Arabidopsis thaliana after 6 h exposure to fluctuating light as compared with constant light conditions. This acclimation response to fluctuating light was hampered in a triple mutant lacking the thylakoid ion transport proteins KEA3, VCCN1, and CLCe, leading to photoinhibition of photosystem I. Transcriptome analysis revealed upregulation of genes involved in biotic stress and defense responses in both genotypes after exposure to fluctuating as compared with constant light, yet these responses were demonstrated to be largely upregulated in triple mutant already under constant light conditions compared with wild type. The current study illustrates the rapid acclimation of plants to fluctuating light, including photosynthetic, transcriptomic, and metabolic adjustments, and highlights the connection among thylakoid ion transport, photosynthetic energy balance, and cell signaling.

The Arabidopsis thylakoid chloride channel ClCe regulates ATP availability for light-harvesting complex II protein phosphorylation.

Coping with changes in light intensity is challenging for plants, but well-designed mechanisms allow them to acclimate to most unpredicted situations. The thylakoid K+/H+ antiporter KEA3 and the voltage-dependent Cl- channel VCCN1 play important roles in light acclimation by fine-tuning electron transport and photoprotection. Good evidence exists that the thylakoid Cl- channel ClCe is involved in the regulation of photosynthesis and state transitions in conditions of low light. However, a detailed mechanistic understanding of this effect is lacking. Here we report that the ClCe loss-of-function in Arabidopsis thaliana results in lower levels of phosphorylated light-harvesting complex II (LHCII) proteins as well as lower levels of the photosystem I-LHCII complexes relative to wild type (WT) in low light conditions. The phosphorylation of the photosystem II core D1/D2 proteins was less affected either in low or high light conditions. In low light conditions, the steady-state levels of ATP synthase conductivity and of the total proton flux available for ATP synthesis were lower in ClCe loss-of-function mutants, but comparable to WT at standard and high light intensity. As a long-term acclimation strategy, expression of the ClCe gene was upregulated in WT plants grown in light-limiting conditions, but not in WT plants grown in standard light even when exposed for up to 8 h to low light. Taken together, these results suggest a role of ClCe in the regulation of the ATP synthase activity which under low light conditions impacts LHCII protein phosphorylation and state transitions.

Gene expression and organization of thylakoid protein complexes in the PSII-less mutant of Synechocystis sp. PCC 6803.

Photosystems I and II (PSI and PSII) are the integral components of the photosynthetic electron transport chain that utilize light to provide chemical energy for CO2 fixation. In this study, we investigated how the deficiency of PSII affects the gene expression, accumulation, and organization of thylakoid protein complexes as well as physiological characteristics of Synechocystis sp. PCC 6803 by combining biochemical, biophysical, and transcriptomic approaches. RNA-seq analysis showed upregulated expression of genes encoding the PSII core proteins, and downregulation of genes associated with interaction between light-harvesting phycobilisomes and PSI. Two-dimensional separation of thylakoid protein complexes confirmed the lack of PSII complexes, yet unassembled PSII subunits were detected. The content of PsaB representing PSI was lower, while the content of cytochrome b6f complexes was higher in the PSII-less strain as compared with control (CS). Application of oxygraph measurements revealed higher rates of dark respiration and lower PSI activity in the mutant. The latter likely resulted from the detected decrease in the accumulation of PSI, PSI monomerization, increased proportion of energetically decoupled phycobilisomes in PSII-less cultures, and low abundance of phycocyanin. Merging the functional consequences of PSII depletion with differential protein and transcript accumulation in the mutant, in comparison to CS, identified signal transduction from the photosynthetic apparatus to the genome level.

Patterning of the Autotrophic, Mixotrophic, and Heterotrophic Proteomes of Oxygen-Evolving Cyanobacterium Synechocystis sp. PCC 6803.

Proteomes of an oxygenic photosynthetic cyanobacterium, Synechocystis sp. PCC 6803, were analyzed under photoautotrophic (low and high CO2, assigned as ATLC and ATHC), photomixotrophic (MT), and light-activated heterotrophic (LAH) conditions. Allocation of proteome mass fraction to seven sub-proteomes and differential expression of individual proteins were analyzed, paying particular attention to photosynthesis and carbon metabolism-centered sub-proteomes affected by the quality and quantity of the carbon source and light regime upon growth. A distinct common feature of the ATHC, MT, and LAH cultures was low abundance of inducible carbon-concentrating mechanisms and photorespiration-related enzymes, independent of the inorganic or organic carbon source. On the other hand, these cells accumulated a respiratory NAD(P)H dehydrogenase I (NDH-11) complex in the thylakoid membrane (TM). Additionally, in glucose-supplemented cultures, a distinct NDH-2 protein, NdbA, accumulated in the TM, while the plasma membrane-localized NdbC and terminal oxidase decreased in abundance in comparison to both AT conditions. Photosynthetic complexes were uniquely depleted under the LAH condition but accumulated under the ATHC condition. The MT proteome displayed several heterotrophic features typical of the LAH proteome, particularly including the high abundance of ribosome as well as amino acid and protein biosynthesis machinery-related components. It is also noteworthy that the two equally light-exposed ATHC and MT cultures allocated similar mass fractions of the total proteome to the seven distinct sub-proteomes. Unique trophic condition-specific expression patterns were likewise observed among individual proteins, including the accumulation of phosphate transporters and polyphosphate polymers storing energy surplus in highly energetic bonds under the MT condition and accumulation under the LAH condition of an enzyme catalyzing cyanophycin biosynthesis. It is concluded that the rigor of cell growth in the MT condition results, to a great extent, by combining photosynthetic activity with high intracellular inorganic carbon conditions created upon glucose breakdown and release of CO2, besides the direct utilization of glucose-derived carbon skeletons for growth. This combination provides the MT cultures with excellent conditions for growth that often exceeds that of mere ATHC.

Photosystem I Inhibition, Protection and Signalling: Knowns and Unknowns.

Photosynthesis is the process that harnesses, converts and stores light energy in the form of chemical energy in bonds of organic compounds. Oxygenic photosynthetic organisms (i.e., plants, algae and cyanobacteria) employ an efficient apparatus to split water and transport electrons to high-energy electron acceptors. The photosynthetic system must be finely balanced between energy harvesting and energy utilisation, in order to limit generation of dangerous compounds that can damage the integrity of cells. Insight into how the photosynthetic components are protected, regulated, damaged, and repaired during changing environmental conditions is crucial for improving photosynthetic efficiency in crop species. Photosystem I (PSI) is an integral component of the photosynthetic system located at the juncture between energy-harnessing and energy consumption through metabolism. Although the main site of photoinhibition is the photosystem II (PSII), PSI is also known to be inactivated by photosynthetic energy imbalance, with slower reactivation compared to PSII; however, several outstanding questions remain about the mechanisms of damage and repair, and about the impact of PSI photoinhibition on signalling and metabolism. In this review, we address the knowns and unknowns about PSI activity, inhibition, protection, and repair in plants. We also discuss the role of PSI in retrograde signalling pathways and highlight putative signals triggered by the functional status of the PSI pool.

Characterization of the Free and Membrane-Associated Fractions of the Thylakoid Lumen Proteome in Arabidopsis thaliana.

The thylakoid lumen houses proteins that are vital for photosynthetic electron transport, including water-splitting at photosystem (PS) II and shuttling of electrons from cytochrome b6f to PSI. Other lumen proteins maintain photosynthetic activity through biogenesis and turnover of PSII complexes. Although all lumen proteins are soluble, these known details have highlighted interactions of some lumen proteins with thylakoid membranes or thylakoid-intrinsic proteins. Meanwhile, the functional details of most lumen proteins, as well as their distribution between the soluble and membrane-associated lumen fractions, remain unknown. The current study isolated the soluble free lumen (FL) and membrane-associated lumen (MAL) fractions from Arabidopsis thaliana, and used gel- and mass spectrometry-based proteomics methods to analyze the contents of each proteome. These results identified 60 lumenal proteins, and clearly distinguished the difference between the FL and MAL proteomes. The most abundant proteins in the FL fraction were involved in PSII assembly and repair, while the MAL proteome was enriched in proteins that support the oxygen-evolving complex (OEC). Novel proteins, including a new PsbP domain-containing isoform, as well as several novel post-translational modifications and N-termini, are reported, and bi-dimensional separation of the lumen proteome identified several protein oligomers in the thylakoid lumen.

Rapid Transcriptional Reprogramming Triggered by Alteration of the Carbon/Nitrogen Balance Has an Impact on Energy Metabolism in Nostoc sp. PCC 7120.

Nostoc (Anabaena) sp. PCC 7120 is a filamentous cyanobacterial species that fixes N2 to nitrogenous compounds using specialised heterocyst cells. Changes in the intracellular ratio of carbon to nitrogen (C/N balance) is known to trigger major transcriptional reprogramming of the cell, including initiating the differentiation of vegetative cells to heterocysts. Substantial transcriptional analysis has been performed on Nostoc sp. PCC 7120 during N stepdown (low to high C/N), but not during C stepdown (high to low C/N). In the current study, we shifted the metabolic balance of Nostoc sp. PCC 7120 cultures grown at 3% CO2 by introducing them to atmospheric conditions containing 0.04% CO2 for 1 h, after which the changes in gene expression were measured using RNAseq transcriptomics. This analysis revealed strong upregulation of carbon uptake, while nitrogen uptake and metabolism and early stages of heterocyst development were downregulated in response to the shift to low CO2. Furthermore, gene expression changes revealed a decrease in photosynthetic electron transport and increased photoprotection and reactive oxygen metabolism, as well a decrease in iron uptake and metabolism. Differential gene expression was largely attributed to change in the abundances of the metabolites 2-phosphoglycolate and 2-oxoglutarate, which signal a rapid shift from fluent photoassimilation to glycolytic metabolism of carbon after transition to low CO2. This work shows that the C/N balance in Nostoc sp. PCC 7120 rapidly adjusts the metabolic strategy through transcriptional reprogramming, enabling survival in the fluctuating environment.

Photosynthetic signalling during high light stress and recovery: targets and dynamics.

The photosynthetic apparatus is one of the major primary sensors of the plant's external environment. Changes in environmental conditions affect the balance between harvested light energy and the capacity to deal with excited electrons in the stroma, which alters the redox homeostasis of the photosynthetic electron transport chain. Disturbances to redox balance activate photosynthetic regulation mechanisms and trigger signalling cascades that can modify the transcription of nuclear genes. H2O2 and oxylipins have been identified as especially prominent regulators of gene expression in response to excess light stress. This paper explores the hypothesis that photosynthetic imbalance triggers specific signals that target discrete gene profiles and biological processes. Analysis of the major retrograde signalling pathways engaged during high light stress and recovery demonstrates both specificity and overlap in gene targets. This work reveals distinct, time-resolved profiles of gene expression that suggest a regulatory interaction between rapidly activated abiotic stress response and induction of secondary metabolism and detoxification processes during recovery. The findings of this study show that photosynthetic electron transport provides a finely tuned sensor for detecting and responding to the environment through chloroplast retrograde signalling. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

The small Ca2+-binding protein CSE links Ca2+ signalling with nitrogen metabolism and filament integrity in Anabaena sp. PCC 7120.

BACKGROUND: Filamentous cyanobacteria represent model organisms for investigating multicellularity. For many species, nitrogen-fixing heterocysts are formed from photosynthetic vegetative cells under nitrogen limitation. Intracellular Ca2+ has been implicated in the highly regulated process of heterocyst differentiation but its role remains unclear. Ca2+ is known to operate more broadly in metabolic signalling in cyanobacteria, although the signalling mechanisms are virtually unknown. A Ca2+-binding protein called the Ca2+ Sensor EF-hand (CSE) is found almost exclusively in filamentous cyanobacteria. Expression of asr1131 encoding the CSE protein in Anabaena sp. PCC 7120 was strongly induced by low CO2 conditions, and rapidly downregulated during nitrogen step-down. A previous study suggests a role for CSE and Ca2+ in regulation of photosynthetic activity in response to changes in carbon and nitrogen availability. RESULTS: In the current study, a mutant Anabaena sp. PCC 7120 strain lacking asr1131 (Δcse) was highly prone to filament fragmentation, leading to a striking phenotype of very short filaments and poor growth under nitrogen-depleted conditions. Transcriptomics analysis under nitrogen-replete conditions revealed that genes involved in heterocyst differentiation and function were downregulated in Δcse, while heterocyst inhibitors were upregulated, compared to the wild-type. CONCLUSIONS: These results indicate that CSE is required for filament integrity and for proper differentiation and function of heterocysts upon changes in the cellular carbon/nitrogen balance. A role for CSE in transmitting Ca2+ signals during the first response to changes in metabolic homeostasis is discussed.

PROTEIN PHOSPHATASE 2A-B'γ Controls Botrytis cinerea Resistance and Developmental Leaf Senescence.

Plants optimize their growth and survival through highly integrated regulatory networks that coordinate defensive measures and developmental transitions in response to environmental cues. Protein phosphatase 2A (PP2A) is a key signaling component that controls stress reactions and growth at different stages of plant development, and the PP2A regulatory subunit PP2A-B'γ is required for negative regulation of pathogenesis responses and for maintenance of cell homeostasis in short-day conditions. Here, we report molecular mechanisms by which PP2A-B'γ regulates Botrytis cinerea resistance and leaf senescence in Arabidopsis (Arabidopsis thaliana). We extend the molecular functionality of PP2A-B'γ to a protein kinase-phosphatase interaction with the defense-associated calcium-dependent protein kinase CPK1 and present indications this interaction may function to control CPK1 activity. In presenescent leaf tissues, PP2A-B'γ is also required to negatively control the expression of salicylic acid-related defense genes, which have recently proven vital in plant resistance to necrotrophic fungal pathogens. In addition, we find the premature leaf yellowing of pp2a-b'γ depends on salicylic acid biosynthesis via SALICYLIC ACID INDUCTION DEFICIENT2 and bears the hallmarks of developmental leaf senescence. We propose PP2A-B'γ age-dependently controls salicylic acid-related signaling in plant immunity and developmental leaf senescence.

A Genome-Wide Association Study of Non-Photochemical Quenching in response to local seasonal climates in Arabidopsis thaliana.

Field-grown plants have variable exposure to sunlight as a result of shifting cloud-cover, seasonal changes, canopy shading, and other environmental factors. As a result, they need to have developed a method for dissipating excess energy obtained from periodic excessive sunlight exposure. Non-photochemical quenching (NPQ) dissipates excess energy as heat, however, the physical and molecular genetic mechanics of NPQ variation are not understood. In this study, we investigated the genetic loci involved in NPQ by first growing different Arabidopsis thaliana accessions in local and seasonal climate conditions, then measured their NPQ kinetics through development by chlorophyll fluorescence. We used genome-wide association studies (GWAS) to identify 15 significant quantitative trait loci (QTL) for a range of photosynthetic traits, including a QTL co-located with known NPQ gene PSBS (AT1G44575). We found there were large alternative regulatory segments between the PSBS promoter regions of the functional haplotypes and a significant difference in PsbS protein concentration. These findings parallel studies in rice showing recurrent regulatory evolution of this gene. The variation in the PSBS promoter and the changes underlying other QTLs could give insight to allow manipulations of NPQ in crops to improve their photosynthetic efficiency and yield.

Photoinhibition of Photosystem I Provides Oxidative Protection During Imbalanced Photosynthetic Electron Transport in Arabidopsis thaliana.

Photosynthesis involves the conversion of sunlight energy into stored chemical energy, which is achieved through electron transport along a series of redox reactions. Excess photosynthetic electron transport might be dangerous due to the risk of molecular oxygen reduction, generating reactive oxygen species (ROS) over-accumulation. Avoiding excess ROS production requires the rate of electron transport to be coordinated with the capacity of electron acceptors in the chloroplast stroma. Imbalance between the donor and acceptor sides of photosystem I (PSI) can lead to inactivation, which is called PSI photoinhibition. We used a light-inducible PSI photoinhibition system in Arabidopsis thaliana to resolve the time dynamics of inhibition and to investigate its impact on ROS production and turnover. The oxidation state of the PSI reaction center and rates of CO2 fixation both indicated strong and rapid PSI photoinhibition upon donor side/acceptor side imbalance, while the rate of inhibition eased during prolonged imbalance. PSI photoinhibition was not associated with any major changes in ROS accumulation or antioxidant activity; however, a lower level of lipid oxidation correlated with lower abundance of chloroplast lipoxygenase in PSI-inhibited leaves. The results of this study suggest that rapid activation of PSI photoinhibition under severe photosynthetic imbalance protects the chloroplast from over-reduction and excess ROS formation.

The unique photosynthetic apparatus of Pinaceae: analysis of photosynthetic complexes in Picea abies.

Pinaceae are the predominant photosynthetic species in boreal forests, but so far no detailed description of the protein components of the photosynthetic apparatus of these gymnosperms has been available. In this study we report a detailed characterization of the thylakoid photosynthetic machinery of Norway spruce (Picea abies (L.) Karst). We first customized a spruce thylakoid protein database from translated transcript sequences combined with existing protein sequences derived from gene models, which enabled reliable tandem mass spectrometry identification of P. abies thylakoid proteins from two-dimensional large pore blue-native/SDS-PAGE. This allowed a direct comparison of the two-dimensional protein map of thylakoid protein complexes from P. abies with the model angiosperm Arabidopsis thaliana. Although the subunit composition of P. abies core PSI and PSII complexes is largely similar to that of Arabidopsis, there was a high abundance of a smaller PSI subcomplex, closely resembling the assembly intermediate PSI* complex. In addition, the evolutionary distribution of light-harvesting complex (LHC) family members of Pinaceae was compared in silico with other land plants, revealing that P. abies and other Pinaceae (also Gnetaceae and Welwitschiaceae) have lost LHCB4, but retained LHCB8 (formerly called LHCB4.3). The findings reported here show the composition of the photosynthetic apparatus of P. abies and other Pinaceae members to be unique among land plants.

A novel Ca2+-binding protein influences photosynthetic electron transport in Anabaena sp. PCC 7120.

Ca2+ is a potent signalling molecule that regulates many cellular processes. In cyanobacteria, Ca2+ has been linked to cell growth, stress response and photosynthesis, and to the development of specialist heterocyst cells in certain nitrogen-fixing species. Despite this, the pathways of Ca2+ signal transduction in cyanobacteria are poorly understood, and very few protein components are known. The current study describes a previously unreported Ca2+-binding protein which was called the Ca2+ Sensor EF-hand (CSE), which is conserved in filamentous, nitrogen-fixing cyanobacteria. CSE is shown to bind Ca2+, which induces a conformational change in the protein structure. Poor growth of a strain of Anabaena sp. PCC 7120 overexpressing CSE was attributed to diminished photosynthetic performance. Transcriptomics, biophysics and proteomics analyses revealed modifications in the light-harvesting phycobilisome and photosynthetic reaction centre protein complexes.

Consequences of photosystem-I damage and repair on photosynthesis and carbon use in Arabidopsis thaliana.

Natural growth environments commonly include fluctuating conditions that can disrupt the photosynthetic energy balance and induce photoinhibition through inactivation of the photosynthetic apparatus. Photosystem II (PSII) photoinhibition is efficiently reversed by the PSII repair cycle, whereas photoinhibited photosystem I (PSI) recovers much more slowly. In the current study, treatment of the Arabidopsis thaliana mutant proton gradient regulation 5 (pgr5) with excess light was used to compromise PSI functionality in order to investigate the impact of photoinhibition and subsequent recovery on photosynthesis and carbon metabolism. The negative impact of PSI photoinhibition on CO2 fixation was especially deleterious under low irradiance. Impaired starch accumulation after PSI photoinhibition was reflected in reduced respiration in the dark, but this was not attributed to impaired sugar synthesis. Normal chloroplast and mitochondrial metabolisms were shown to recover despite the persistence of substantial PSI photoinhibition for several days. The results of this study indicate that the recovery of PSI function involves the reorganization of the light-harvesting antennae, and suggest a pool of surplus PSI that can be recruited to support photosynthesis under demanding conditions.

Interaction between photosynthetic electron transport and chloroplast sinks triggers protection and signalling important for plant productivity.

The photosynthetic light reactions provide energy that is consumed and stored in electron sinks, the products of photosynthesis. A balance between light reactions and electron consumption in the chloroplast is vital for plants, and is protected by several photosynthetic regulation mechanisms. Photosystem I (PSI) is particularly susceptible to photoinhibition when these factors become unbalanced, which can occur in low temperatures or in high light. In this study we used the pgr5 Arabidopsis mutant that lacks ΔpH-dependent regulation of photosynthetic electron transport as a model to study the consequences of PSI photoinhibition under high light. We found that PSI damage severely inhibits carbon fixation and starch accumulation, and attenuates enzymatic oxylipin synthesis and chloroplast regulation of nuclear gene expression after high light stress. This work shows that modifications to regulation of photosynthetic light reactions, which may be designed to improve yield in crop plants, can negatively impact metabolism and signalling, and thereby threaten plant growth and stress tolerance.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'.

Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch.

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.