Amino acid and protein specificity of protein fatty acylation in C. elegans.

Protein lipidation plays critical roles in regulating protein function and localization. However, the chemical diversity and specificity of fatty acyl group utilization have not been investigated using untargeted approaches, and it is unclear to what extent structures and biosynthetic origins of S-acyl moieties differ from N- and O-fatty acylation. Here, we show that fatty acylation patterns in Caenorhabditis elegans differ markedly between different amino acid residues. Hydroxylamine capture revealed predominant cysteine S-acylation with 15-methylhexadecanoic acid (isoC17:0), a monomethyl branched-chain fatty acid (mmBCFA) derived from endogenous leucine catabolism. In contrast, enzymatic protein hydrolysis showed that N-terminal glycine was acylated almost exclusively with straight-chain myristic acid, whereas lysine was acylated preferentially with two different mmBCFAs and serine was acylated promiscuously with a broad range of fatty acids, including eicosapentaenoic acid. Global profiling of fatty acylated proteins using a set of click chemistry-capable alkyne probes for branched- and straight-chain fatty acids uncovered 1,013 S-acylated proteins and 510 hydroxylamine-resistant N- or O-acylated proteins. Subsets of S-acylated proteins were labeled almost exclusively by either a branched-chain or a straight-chain probe, demonstrating acylation specificity at the protein level. Acylation specificity was confirmed for selected examples, including the S-acyltransferase DHHC-10. Last, homology searches for the identified acylated proteins revealed a high degree of conservation of acylation site patterns across metazoa. Our results show that protein fatty acylation patterns integrate distinct branches of lipid metabolism in a residue- and protein-specific manner, providing a basis for mechanistic studies at both the amino acid and protein levels.

Composition and function of stress granules and P-bodies in plants.

Stress Granules (SGs) and Processing-bodies (P-bodies) are biomolecular condensates formed in the cell with the highly conserved purpose of maintaining balance between storage, translation, and degradation of mRNA. This balance is particularly important when cells are exposed to different environmental conditions and adjustments have to be made in order for plants to respond to and tolerate stressful conditions. While P-bodies are constitutively present in the cell, SG formation is a stress-induced event. Typically thought of as protein-RNA aggregates, SGs and P-bodies are formed by a process called liquid-liquid phase separation (LLPS), and both their function and composition are very dynamic. Both foci are known to contain proteins involved in translation, protein folding, and ATPase activity, alluding to their roles in regulating mRNA and protein expression levels. From an RNA perspective, SGs and P-bodies primarily consist of mRNAs, though long non-coding RNAs (lncRNAs) have also been observed, and more focus is now being placed on the specific RNAs associated with these aggregates. Recently, metabolites such as nucleotides and amino acids have been reported in purified plant SGs with implications for the energetic dynamics of these condensates. Thus, even though the field of plant SGs and P-bodies is relatively nascent, significant progress has been made in understanding their composition and biological role in stress responses. In this review, we discuss the most recent discoveries centered around SG and P-body function and composition in plants.

Stress-related biomolecular condensates in plants.

Biomolecular condensates are membraneless organelle-like structures that can concentrate molecules and often form through liquid-liquid phase separation. Biomolecular condensate assembly is tightly regulated by developmental and environmental cues. Although research on biomolecular condensates has intensified in the past 10 years, our current understanding of the molecular mechanisms and components underlying their formation remains in its infancy, especially in plants. However, recent studies have shown that the formation of biomolecular condensates may be central to plant acclimation to stress conditions. Here, we describe the mechanism, regulation, and properties of stress-related condensates in plants, focusing on stress granules and processing bodies, 2 of the most well-characterized biomolecular condensates. In this regard, we showcase the proteomes of stress granules and processing bodies in an attempt to suggest methods for elucidating the composition and function of biomolecular condensates. Finally, we discuss how biomolecular condensates modulate stress responses and how they might be used as targets for biotechnological efforts to improve stress tolerance.

Hello darkness, my old friend: 3-KETOACYL-COENZYME A SYNTHASE4 is a branch point in the regulation of triacylglycerol synthesis in Arabidopsis thaliana.

Plant lipids are important as alternative sources of carbon and energy when sugars or starch are limited. Here, we applied combined heat and darkness or extended darkness to a panel of ∼300 Arabidopsis (Arabidopsis thaliana) accessions to study lipid remodeling under carbon starvation. Natural allelic variation at 3-KETOACYL-COENZYME A SYNTHASE4 (KCS4), a gene encoding an enzyme involved in very long chain fatty acid (VLCFA) synthesis, underlies the differential accumulation of polyunsaturated triacylglycerols (puTAGs) under stress. Ectopic expression of KCS4 in yeast and plants proved that KCS4 is a functional enzyme localized in the endoplasmic reticulum with specificity for C22 and C24 saturated acyl-CoA. Allelic mutants and transient overexpression in planta revealed the differential role of KCS4 alleles in VLCFA synthesis and leaf wax coverage, puTAG accumulation, and biomass. Moreover, the region harboring KCS4 is under high selective pressure and allelic variation at KCS4 correlates with environmental parameters from the locales of Arabidopsis accessions. Our results provide evidence that KCS4 plays a decisive role in the subsequent fate of fatty acids released from chloroplast membrane lipids under carbon starvation. This work sheds light on both plant response mechanisms and the evolutionary events shaping the lipidome under carbon starvation.

Tyr-Asp inhibition of glyceraldehyde 3-phosphate dehydrogenase affects plant redox metabolism.

How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr-Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr-Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr-Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched-chain amino acid-containing dipeptides, but not by Tyr-Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small-molecule regulators at the nexus of stress, protein degradation, and metabolism.

Identification and functional annotation of long intergenic non-coding RNAs in Brassicaceae.

Long intergenic noncoding RNAs (lincRNAs) are a large yet enigmatic class of eukaryotic transcripts that can have critical biological functions. The wealth of RNA-sequencing (RNA-seq) data available for plants provides the opportunity to implement a harmonized identification and annotation effort for lincRNAs that enables cross-species functional and genomic comparisons as well as prioritization of functional candidates. In this study, we processed >24 Tera base pairs of RNA-seq data from >16,000 experiments to identify ∼130,000 lincRNAs in four Brassicaceae: Arabidopsis thaliana, Camelina sativa, Brassica rapa, and Eutrema salsugineum. We used nanopore RNA-seq, transcriptome-wide structural information, peptide data, and epigenomic data to characterize these lincRNAs and identify conserved motifs. We then used comparative genomic and transcriptomic approaches to highlight lincRNAs in our data set with sequence or transcriptional conservation. Finally, we used guilt-by-association analyses to assign putative functions to lincRNAs within our data set. We tested this approach on a subset of lincRNAs associated with germination and seed development, observing germination defects for Arabidopsis lines harboring T-DNA insertions at these loci. LincRNAs with Brassicaceae-conserved putative miRNA binding motifs, small open reading frames, or abiotic-stress modulated expression are a few of the annotations that will guide functional analyses into this cryptic portion of the transcriptome.

Don't let go: co-fractionation mass spectrometry for untargeted mapping of protein-metabolite interactomes.

The chemical complexity of metabolomes goes hand in hand with their functional diversity. Small molecules have many essential roles, many of which are executed by binding and modulating the function of a protein partner. The complex and dynamic protein-metabolite interaction (PMI) network underlies most if not all biological processes, but remains under-characterized. Herein, we highlight how co-fractionation mass spectrometry (CF-MS), a well-established approach to map protein assemblies, can be used for proteome and metabolome identification of the PMIs. We will review recent CF-MS studies, discuss the main advantages and limitations, summarize the available CF-MS guidelines, and outline future challenges and opportunities.

Protein interactome of 3',5'-cAMP reveals its role in regulating the actin cytoskeleton.

Identification of protein interactors is ideally suited for the functional characterization of small molecules. 3',5'-cAMP is an evolutionary ancient signaling metabolite largely uncharacterized in plants. To tap into the physiological roles of 3',5'-cAMP, we used a chemo-proteomics approach, thermal proteome profiling (TPP), for the unbiased identification of 3',5'-cAMP protein targets. TPP measures shifts in the protein thermal stability upon ligand binding. Comprehensive proteomics analysis yielded a list of 51 proteins significantly altered in their thermal stability upon incubation with 3',5'-cAMP. The list contained metabolic enzymes, ribosomal subunits, translation initiation factors, and proteins associated with the regulation of plant growth such as CELL DIVISION CYCLE 48. To functionally validate obtained results, we focused on the role of 3',5'-cAMP in regulating the actin cytoskeleton suggested by the presence of actin among the 51 identified proteins. 3',5'-cAMP supplementation affected actin organization by inducing actin-bundling. Consistent with these results, the increase in 3',5'-cAMP levels, obtained either by feeding or by chemical modulation of 3',5'-cAMP metabolism, was sufficient to partially rescue the short hypocotyl phenotype of the actin2 actin7 mutant, severely compromised in actin level. The observed rescue was specific to 3',5'-cAMP, as demonstrated using a positional isomer 2',3'-cAMP, and true for the nanomolar 3',5'-cAMP concentrations reported for plant cells. In vitro characterization of the 3',5'-cAMP-actin pairing argues against a direct interaction between actin and 3',5'-cAMP. Alternative mechanisms by which 3',5'-cAMP would affect actin dynamics, such as by interfering with calcium signaling, are discussed. In summary, our work provides a specific resource, 3',5'-cAMP interactome, as well as functional insight into 3',5'-cAMP-mediated regulation in plants.

Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices.

Mass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics-derived data.

Regulation of a single inositol 1-phosphate synthase homeologue by HSFA6B contributes to fibre yield maintenance under drought conditions in upland cotton.

Drought stress substantially impacts crop physiology resulting in alteration of growth and productivity. Understanding the genetic and molecular crosstalk between stress responses and agronomically important traits such as fibre yield is particularly complicated in the allopolyploid species, upland cotton (Gossypium hirsutum), due to reduced sequence variability between A and D subgenomes. To better understand how drought stress impacts yield, the transcriptomes of 22 genetically and phenotypically diverse upland cotton accessions grown under well-watered and water-limited conditions in the Arizona low desert were sequenced. Gene co-expression analyses were performed, uncovering a group of stress response genes, in particular transcription factors GhDREB2A-A and GhHSFA6B-D, associated with improved yield under water-limited conditions in an ABA-independent manner. DNA affinity purification sequencing (DAP-seq), as well as public cistrome data from Arabidopsis, were used to identify targets of these two TFs. Among these targets were two lint yield-associated genes previously identified through genome-wide association studies (GWAS)-based approaches, GhABP-D and GhIPS1-A. Biochemical and phylogenetic approaches were used to determine that GhIPS1-A is positively regulated by GhHSFA6B-D, and that this regulatory mechanism is specific to Gossypium spp. containing the A (old world) genome. Finally, an SNP was identified within the GhHSFA6B-D binding site in GhIPS1-A that is positively associated with yield under water-limiting conditions. These data lay out a regulatory connection between abiotic stress and fibre yield in cotton that appears conserved in other systems such as Arabidopsis.

Arabidopsis PROTODERMAL FACTOR2 binds lysophosphatidylcholines and transcriptionally regulates phospholipid metabolism.

Plant homeodomain leucine zipper IV (HD-Zip IV) transcription factors (TFs) contain an evolutionarily conserved steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. While the START domain is required for TF activity, its presumed role as a lipid sensor is not clear. Here we used tandem affinity purification from Arabidopsis cell cultures to demonstrate that PROTODERMAL FACTOR2 (PDF2), a representative member that controls epidermal differentiation, recruits lysophosphatidylcholines (LysoPCs) in a START-dependent manner. Microscale thermophoresis assays confirmed that a missense mutation in a predicted ligand contact site reduces lysophospholipid binding. We additionally found that PDF2 acts as a transcriptional regulator of phospholipid- and phosphate (Pi) starvation-related genes and binds to a palindromic octamer with consensus to a Pi response element. Phospholipid homeostasis and elongation growth were altered in pdf2 mutants according to Pi availability. Cycloheximide chase experiments revealed a role for START in maintaining protein levels, and Pi starvation resulted in enhanced protein destabilization, suggesting a mechanism by which lipid binding controls TF activity. We propose that the START domain serves as a molecular sensor for membrane phospholipid status in the epidermis. Our data provide insights toward understanding how the lipid metabolome integrates Pi availability with gene expression.

The genetic and physiological basis of Arabidopsis thaliana tolerance to Pseudomonas viridiflava.

The opportunistic pathogen Pseudomonas viridiflava colonizes > 50 agricultural crop species and is the most common Pseudomonas in the phyllosphere of European Arabidopsis thaliana populations. Belonging to the P. syringae complex, it is genetically and phenotypically distinct from well-characterized P. syringae sensu stricto. Despite its prevalence, we lack knowledge of how A. thaliana responds to its native isolates at the molecular level. Here, we characterize the host response in an A. thaliana - P. viridiflava pathosystem. We measured host and pathogen growth in axenic infections and used immune mutants, transcriptomics, and metabolomics to determine defense pathways influencing susceptibility to P. viridiflava infection. Infection with P. viridiflava increased jasmonic acid (JA) levels and the expression of ethylene defense pathway marker genes. The immune response in a susceptible host accession was delayed compared with a tolerant one. Mechanical injury rescued susceptibility, consistent with an involvement of JA. The JA/ethylene pathway is important for suppression of P. viridiflava, yet suppression capacity varies between accessions. Our results shed light on how A. thaliana can suppress the ever-present P. viridiflava, but further studies are needed to understand how P. viridiflava evades this suppression to spread broadly across A. thaliana populations.

Past accomplishments and future challenges of the multi-omics characterization of leaf growth.

The advent of omics technologies has revolutionized biology and advanced our understanding of all biological processes, including major developmental transitions in plants and animals. Here, we review the vast knowledge accumulated concerning leaf growth in terms of transcriptional regulation before turning our attention to the historically less well-characterized alterations at the protein and metabolite level. We will then discuss how the advent of biochemical methods coupled with metabolomics and proteomics can provide insight into the protein-protein and protein-metabolite interactome of the growing leaves. We finally highlight the substantial challenges in detection, spatial resolution, integration, and functional validation of the omics results, focusing on metabolomics as a prerequisite for a comprehensive understanding of small-molecule regulation of plant growth.

2',3'-cAMP treatment mimics the stress molecular response in Arabidopsis thaliana.

The role of the RNA degradation product 2',3'-cyclic adenosine monophosphate (2',3'-cAMP) is poorly understood. Recent studies have identified 2',3'-cAMP in plant material and determined its role in stress signaling. The level of 2',3'-cAMP increases upon wounding, in the dark, and under heat, and 2',3'-cAMP binding to an RNA-binding protein, Rbp47b, promotes stress granule (SG) assembly. To gain further mechanistic insights into the function of 2',3'-cAMP, we used a multi-omics approach by combining transcriptomics, metabolomics, and proteomics to dissect the response of Arabidopsis (Arabidopsis thaliana) to 2',3'-cAMP treatment. We demonstrated that 2',3'-cAMP is metabolized into adenosine, suggesting that the well-known cyclic nucleotide-adenosine pathway of human cells might also exist in plants. Transcriptomics analysis revealed only minor overlap between 2',3'-cAMP- and adenosine-treated plants, suggesting that these molecules act through independent mechanisms. Treatment with 2',3'-cAMP changed the levels of hundreds of transcripts, proteins, and metabolites, many previously associated with plant stress responses, including protein and RNA degradation products, glucosinolates, chaperones, and SG components. Finally, we demonstrated that 2',3'-cAMP treatment influences the movement of processing bodies, confirming the role of 2',3'-cAMP in the formation and motility of membraneless organelles.

Metabolism of crown tissue is crucial for drought tolerance and recovery after stress cessation in Lolium/Festuca forage grasses.

A process of plant recovery after drought cessation is a complex trait which has not been fully recognized. The most important organ associated with this phenomenon in monocots, including forage grasses, is the crown tissue located between shoots and roots. The crown tissue is a meristematic crossroads for metabolites and other compounds between these two plant organs. Here, for the first time, we present a metabolomic and lipidomic study focused on the crown tissue under drought and recovery in forage grasses, important for agriculture in European temperate regions. The plant materials involve high (HDT) and low drought-tolerant (LDT) genotypes of Festuca arundinacea, and Lolium multiflorum/F. arundinacea introgression forms. The obtained results clearly demonstrated that remodeling patterns of the primary metabolome and lipidome in the crown under drought and recovery were different between HDT and LDT plants. Furthermore, HDT plants accumulated higher contents of primary metabolites under drought in the crown tissue, especially carbohydrates which could function as osmoprotectants and storage materials. On the other hand, LDT plants characterized by higher membranes damage under drought, simultaneously accumulated membrane phospholipids in the crown and possessed the capacity to recover their metabolic functions after stress cessation to the levels observed in HDT plants.

Rewiring of the protein-protein-metabolite interactome during the diauxic shift in yeast.

In budding yeast Saccharomyces cerevisiae, the switch from aerobic fermentation to respiratory growth is separated by a period of growth arrest, known as the diauxic shift, accompanied by a significant metabolic rewiring, including the derepression of gluconeogenesis and the establishment of mitochondrial respiration. Previous studies reported hundreds of proteins and tens of metabolites accumulating differentially across the diauxic shift transition. To assess the differences in the protein-protein (PPIs) and protein-metabolite interactions (PMIs) yeast samples harvested in the glucose-utilizing, fermentative phase, ethanol-utilizing and early stationary respiratory phases were analysed using isothermal shift assay (iTSA) and a co-fractionation mass spectrometry approach, PROMIS. Whereas iTSA monitors changes in protein stability and is informative towards protein interaction status, PROMIS uses co-elution to delineate putative PPIs and PMIs. The resulting dataset comprises 1627 proteins and 247 metabolites, hundreds of proteins and tens of metabolites characterized by differential thermal stability and/or fractionation profile, constituting a novel resource to be mined for the regulatory PPIs and PMIs. The examples discussed here include (i) dissociation of the core and regulatory particle of the proteasome in the early stationary phase, (ii) the differential binding of a co-factor pyridoxal phosphate to the enzymes of amino acid metabolism and (iii) the putative, phase-specific interactions between proline-containing dipeptides and enzymes of central carbon metabolism.

The Role of Triacylglycerol in the Protection of Cells against Lipotoxicity under Drought in Lolium multiflorum/Festucaarundinacea Introgression Forms.

Triacylglycerol is a key lipid compound involved in maintaining homeostasis of both membrane lipids and free fatty acids (FFA) in plant cells under adverse environmental conditions. However, its role in the process of lipid remodeling has not been fully recognized, especially in monocots, including grass species. For our study, two closely related introgression forms of Lolium multiflorum (Italian ryegrass) and Festuca arundinacea (tall fescue), distinct in their level of drought tolerance, were selected as plant models to study rearrangements in plant lipidome under water deficit and further re-watering. The low drought tolerant (LDT) form revealed an elevated level of cellular membrane damage accompanied by an increased content of polyunsaturated FFA and triacylglycerol under water deficit, compared with the high drought tolerant (HDT) form. However, the LDT introgression form demonstrated also the ability to regenerate its membranes after stress cessation. The obtained results clearly indicated that accumulation of triacylglycerol under advanced drought in the LDT form could serve as a cellular protective mechanism against overaccumulation of toxic polyunsaturated FFA and other lipid intermediates. Furthermore, accumulation of triacylglycerol under drought conditions could serve also as storage of substrates required for further regeneration of membranes after stress cessation. The rearrangements in triacylglycerol metabolism were supported by the upregulation of several genes, involved in a biosynthesis of triacylglycerol. With respect to this process, diacylglycerol O-acyltransferase DGAT2 seems to play the most important role in the analyzed grasses.

A manipulation of carotenoid metabolism influence biomass partitioning and fitness in tomato.

Improving yield, nutritional value and tolerance to abiotic stress are major targets of current breeding and biotechnological approaches that aim at increasing crop production and ensuring food security. Metabolic engineering of carotenoids, the precursor of vitamin-A and plant hormones that regulate plant growth and response to adverse growth conditions, has been mainly focusing on provitamin A biofortification or the production of high-value carotenoids. Here, we show that the introduction of a single gene of the carotenoid biosynthetic pathway in different tomato cultivars induced profound metabolic alterations in carotenoid, apocarotenoid and phytohormones pathways. Alterations in isoprenoid- (abscisic acid, gibberellins, cytokinins) and non-isoprenoid (auxin and jasmonic acid) derived hormones together with enhanced xanthophyll content influenced biomass partitioning and abiotic stress tolerance (high light, salt, and drought), and it caused an up to 77% fruit yield increase and enhanced fruit's provitamin A content. In addition, metabolic and hormonal changes led to accumulation of key primary metabolites (e.g. osmoprotectants and antiaging agents) contributing with enhanced abiotic stress tolerance and fruit shelf life. Our findings pave the way for developing a new generation of crops that combine high productivity and increased nutritional value with the capability to cope with climate change-related environmental challenges.

A reactive oxygen species Ca2+ signalling pathway identified from a chemical screen for modifiers of sugar-activated circadian gene expression.

Sugars are essential metabolites for energy and anabolism that can also act as signals to regulate plant physiology and development. Experimental tools to disrupt major sugar signalling pathways are limited. We performed a chemical screen for modifiers of activation of circadian gene expression by sugars to discover pharmacological tools to investigate and manipulate plant sugar signalling. Using a library of commercially available bioactive compounds, we identified 75 confident hits that modified the response of a circadian luciferase reporter to sucrose in dark-adapted Arabidopsis thaliana seedlings. We validated the transcriptional effect on a subset of the hits and measured their effects on a range of sugar-dependent phenotypes for 13 of these chemicals. Chemicals were identified that appear to influence known and unknown sugar signalling pathways. Pentamidine isethionate was identified as a modifier of a sugar-activated Ca2+ signal that acts as a calmodulin inhibitor downstream of superoxide in a metabolic signalling pathway affecting circadian rhythms, primary metabolism and plant growth. Our data provide a resource of new experimental tools to manipulate plant sugar signalling and identify novel components of these pathways.

Functional characterization of proton antiport regulation in the thylakoid membrane.

During photosynthesis, energy is transiently stored as an electrochemical proton gradient across the thylakoid membrane. The resulting proton motive force (pmf) is composed of a membrane potential (ΔΨ) and a proton concentration gradient (ΔpH) and powers the synthesis of ATP. Light energy availability for photosynthesis can change very rapidly and frequently in nature. Thylakoid ion transport proteins buffer the effects that light fluctuations have on photosynthesis by adjusting pmf and its composition. Ion channel activities dissipate ΔΨ, thereby reducing charge recombinations within photosystem II. The dissipation of ΔΨ allows for increased accumulation of protons in the thylakoid lumen, generating the signal that activates feedback downregulation of photosynthesis. Proton export from the lumen via the thylakoid K+ exchange antiporter 3 (KEA3), instead, decreases the ΔpH fraction of the pmf and thereby reduces the regulatory feedback signal. Here, we reveal that the Arabidopsis (Arabidopsis thaliana) KEA3 protein homo-dimerizes via its C-terminal domain. This C-terminus has a regulatory function, which responds to light intensity transients. Plants carrying a C-terminus-less KEA3 variant show reduced feed-back downregulation of photosynthesis and suffer from increased photosystem damage under long-term high light stress. However, during photosynthetic induction in high light, KEA3 deregulation leads to an increase in carbon fixation rates. Together, the data reveal a trade-off between long-term photoprotection and a short-term boost in carbon fixation rates, which is under the control of the KEA3 C-terminus.