Biology subjects

Pogson, B. J.

Publications and source records attributed to Pogson, B. J..

15 recordsLinked to original sources

bioRxiv2026

Specialisation of photosynthetic biochemistry in C4 chloroplasts potentiates distinct cell-specific functionality of different chloroplast-to-nucleus retrograde signalling pathways.

Chloroplasts are key environmental sensors for plant cells. Upon exposure to abiotic stresses such as excess sunlight and heat, reactive oxygen species (ROS) in chloroplasts are sensed to trigger chloroplast-to-nucleus signalling pathways for acclimatory nuclear gene expression. There is extensive biochemical- and cell type-specialisation of chloroplasts, such as those in heat-resilient plants performing C4 photosynthesis, but how these specialisations intersect with chloroplast-to-nucleus signalling remains unclear. Here we show that specific chloroplast-to-nucleus signalling networks are functional in specialised leaf cell types of the C4 model species, Setaria viridis. RNA-sequencing, electron paramagnetic resonance (EPR) spectroscopy and metabolomics on cell type-enriched fractions, complemented with mutant analyses provided evidence for cell-specific enrichment of chloroplast-to-nucleus retrograde signalling pathways correlating with the photosynthetic specialisation in these cell types. Singlet oxygen (1O2)-responsive beta-cyclocitral ({beta}-CC) /dihydroactinidiolide (DHA) signalling and hydrogen peroxide (H2O2)-responsive 3'-phosphoadenosine 5'-phosphate (PAP) signalling pathways were specific to mesophyll cells, in line with enrichment of photosystem II and linear electron flow to photosystem I, respectively in this cell type. In contrast, bundle sheath cells were enriched for the rapid, ROS-independent retrograde signalling pathway mediated by the Calvin cycle product, dihydroxyacetone phosphate (DHAP), consistent with the exclusive localisation of the Calvin cycle to C4 bundle sheath cells. Taken together, our results provide insights into how chloroplast- and cell type-specialisation intersect with stress signalling in plant cells and identify photosynthetic biochemistry as a key driver of cell-specific cellular signalling.

plant biology↗

bioRxiv2025

Arabidopsis DXO1 is an evolutionarily diverged homolog that impacts ribosome loading and mRNA surveillance

The Rai1/Dxo1/DXO protein family are involved in eukaryotic 5'-end RNA quality control by hydrolyzing non-canonical mRNA cap structures, such as the NAD+ 5'-cap. The plant DXO1 ortholog shows distinct biochemical properties, compared to its fungal and mammalian counterparts, and only minor phenotypic traits can be attributed to its NAD+-decapping (deNADding) activity. On the other hand, the chloroplast and growth defects observed in dxo1 null mutants appear linked to the presence of a serine-rich domain at its N-terminus. Here, we report that plant DXO1 is a distinct ortholog that likely diverged earlier than its fungal and mammalian counterparts. We attribute this to the presence of a serine-rich N-terminal domain, which is found almost exclusively in plants and likely arose during the emergence of angiosperms. Degradome profiling revealed that Arabidopsis DXO1 contributes to mRNA surveillance, via examination of exon junction complex and ribosome footprints, and this activity is reliant on the presence of the serine-rich N-terminal domain in Arabidopsis. Furthermore, polysome profiling revealed that plants lacking DXO1 have severe ribosome loading defects, which may explain the loss of mRNA surveillance and alterations in pre-rRNA processing. Taken together, our data leads us to propose a model whereby Arabidopsis DXO1 is involved in 5'-end RNA quality control, which is critical for ribosome loading that impacts translational fidelity and translation-dependent mRNA surveillance.

plant biology↗

bioRxiv2025

High-throughput screening and structure-guided design of small molecules enable modulation of SAL-PAP stress signaling

Chloroplasts sense environmental stress and activate chloroplast-to-nucleus retrograde signalling, reprogramming nuclear gene expression to drive plant acclimation. One such pathway is regulated by the chloroplastic phosphatase, SAL, which hydrolyses the nucleotide signal 3-phosphoadenosine 5-phosphate (PAP). In Arabidopsis thaliana, genetic loss of AtSAL1 elevates PAP and enhances stress tolerance but causes pleiotropic growth defects, complicating the interpretation of the role of PAP in cellular signalling. To uncouple stress signalling from genetic pleiotropy, we conducted a high-throughput in vitro screen of 13,000 small molecules and identified V20, a competitive inhibitor of AtSAL1 with three-fold greater potency than the known Li inhibitor. Structural analoguing of V20 and biochemical assays defined key pharmacophore features required for inhibition, including an ortho-halogenated aromatic ring, hydrogen-bond donor capacity at the triazole carboxamide, and electron-withdrawing substitutions that enhance {pi}-{pi} stacking with aromatic residues. Accelerated molecular dynamics simulations using a new high-resolution crystal structure revealed two previously uncharacterised V20 binding pockets adjacent to the catalytic site. V20 binding induces conformational changes which restrict substrate access to the catalytic site. Exogenous application of V20 to Arabidopsis led to increased PAP accumulation, activated PAP-responsive gene expression and enhanced oxidative tolerance, demonstrating cellular uptake and in vivo efficacy in the energy organelles, chloroplasts and mitochondria, where AtSAL1 is localised. Collectively, our findings reveal new insights into the regulatory domains of SAL enzymatic activity for control of PAP-mediated signalling and establish a proof-of-concept for targeted chemical modulation of SAL activity, which offers novel strategies to selectively manipulate chloroplast-to-nucleus retrograde signalling in plants. Significance StatementWe applied drug discovery strategies to test 13,000 small molecules for inhibition of AtSAL1, a key regulator of plant stress signalling from chloroplasts. Using biochemical assays, chemical analogues and molecular dynamic simulations, we defined the structural-activity relationship of a lead compound, V20, and elucidated its inhibitory mechanism. Application of V20 to plant leaves elevated the levels of AtSAL1s natural substrate, PAP (a chloroplast stress signalling molecule), activated stress-responsive genes and improved oxidative stress tolerance in Arabidopsis. This on-demand activation of SAL-PAP chloroplast signalling avoids pleotropic effects observed in loss-of-function mutants. Our approach provides a new strategy to study chloroplast-mediated cellular signaling pathways that are crucial for plant acclimation to stressful environmental conditions such as excessive sunlight.

plant biology↗

bioRxiv2025

Priming of retrograde signaling in wheat across multiple natural environments reveal how responses to dynamic stimuli can be integrated to alter yield, yield stability and water productivity

O_LIChloroplast-to-nucleus retrograde signaling enables rapid stress responses in plants, but whether these signals accumulate to affect crop performance across entire growing seasons under field conditions remains unknown. C_LIO_LIWe generated wheat mutants with targeted deletions in specific SAL gene copies from two distinct homeologous groups (TaSAL1 and TaSAL2), creating lines with enhanced stress signal responsiveness. We tested these lines across 15 field trials spanning diverse Australian environments with varying temperatures, rainfall, and irrigation regimes, measuring physiological responses, yield, biomass, and water productivity. C_LIO_LILines with TaSAL2 gene deletions showed 4-8% yield improvements with enhanced water productivity, while TaSAL1 deletions reduced yields. The TaSAL2 mutants maintained superior photosynthetic function under drought stress, showed improved relative water content, and demonstrated enhanced yield stability across environments. Canopy temperature measurements revealed dynamic stomatal regulation, with increased closure during midday stress periods but normal aperture under benign conditions. Significantly, specific SAL modifications enhanced photosynthetic efficiency and stress resilience without traditional yield penalties. C_LIO_LITargeted modification of specific SAL homeologous groups can simultaneously improve both yield and stress tolerance in wheat. This demonstrates that retrograde signaling integrates environmental information across the plant lifecycle, and highlights the importance of locus-specific targeting and multi-environment field validation for crop modifications. C_LI

plant biology↗

bioRxiv2024

PGR5 promotes energy-dependent non-photochemical quenching to enable efficient C4 photosynthesis under fluctuating light

PROTON GRADIENT REGULATION 5 (PGR5) is essential for generating proton motive force across thylakoid membranes in C3 plants and supporting photoprotection under fluctuating light conditions. It is proposed that this function is achieved by regulating cyclic electron flow around Photosystem I. During the evolutionary transition from C3 to C4 photosynthesis, the leaf abundance of PGR5 has increased, coinciding with a rise in the cyclic electron flow rate. To investigate the contribution of PGR5 to photoprotection in C4 photosynthesis, we generated model C4 monocot Setaria viridis with null pgr5 alleles. We show that plants lacking PGR5 struggle to establish proton motive force and energy-dependent non-photochemical quenching (qE) at higher irradiances during instantaneous measurements. This leads to a progressive decline in maximum Photosystem I activity when leaves are exposed to repeated cycles of high irradiance. Additionally, plants without PGR5 exhibit severely reduced growth and photosynthesis compared to wild type plants when grown under fluctuating daylight but not under constant daylight. In the absence of PGR5, a slower-relaxing, zeaxanthin-dependent form of non-photochemical quenching supports growth under fluctuating light, albeit at the cost of reduced photochemical efficiency and assimilation rate. Our findings underscore the role of PGR5 in enabling efficient C4 photosynthesis under fluctuating light by establishing proton motive force for the rapid upregulation of qE and preventing photodamage to the electron transport machinery. This study highlights the importance of various non-photochemical quenching mechanisms for C4 photosynthesis and emphasises the role of PGR5 in the evolution of C4 plants.

plant biology↗

bioRxiv2024

The LYCOPENE EPSILON CYCLASE untranslated mRNA leader modulates carotenoid feedback and post-transcriptional regulation

Metabolic feedback is proposed to modulate nuclear gene expression and carotenoid biosynthesis in plastids, however few mechanisms have been identified so far in plants. Utilising mutants, overexpression lines, and chemical inhibitors, we demonstrate that Arabidopsis LYCOPENE EPSILON CYCLASE ({varepsilon}LCY) mRNA levels correlate with changes in {beta}-carotenoid accumulation. Transgenic seedlings harbouring the{varepsilon} LCY 5 leader sequence fused to FIREFLY LUCIFERASE (FiLUC) showed reporter responsiveness to metabolic feedback triggered by norflurazon or loss-of-function in the CAROTENOID ISOMERASE (CRTISO). The{varepsilon} LCY 5UTR harboured three alternative transcription start sites (TSS). The most abundant -133bp sequence generated in dark and light grown seedlings harboured a 5 conserved domain (CD) with other Brassicaceae species and a viral internal ribosome entry site (IRES) proximal to the start codon. In silico modelling predicted the 5UTR formed two energetically separated RNA structural probabilities having a minimal free energy consistent with metabolite-binding RNA riboswitches that was distinguished by hairpin structures within the CD. Site-specific mutations were used to stabilize the 5UTR into a single RNA shape definition having negligible separation between the mountain plot structure prediction curves and a distal terminator-like hairpin structure. Stabilizing the 5UTR shape triggered the posttranscriptional repression of FiLUC activity enabled by the CaMV35S promoter in tobacco transient assays and stable transgenic Arabidopsis lines. The stabilised shape fragment became responsive to metabolic feedback induced by norflurazon and in crtiso mutant etiolated and de-etiolated seedlings. The{varepsilon} LCY 5UTR resembles a conformational RNA regulatory switch harbouring a posttranscriptional expression platform and aptamer domain responsive to carotenoid-mediated feedback signalling.

plant biology↗

bioRxiv2024

Genome-wide association study elucidates the genetic architecture of manganese tolerance in Brassica napus

Brassica napus (canola) is a significant contributor to the worlds oil production and is cultivated across continents, yet acidic soils with Al3+ and Mn2+ toxicities limit its production. The genetic determinants underlying acidic soil tolerance in canola are unknown and require to be uncovered for canola breeding and production. Here, through comprehensive phenotyping, whole genome resequencing, and genome-wide association analysis, we identified three QTLs for tolerance to Mn2+ toxicity on chromosomes A09, C03, and C09. Allelism tests between four tolerance sources confirmed that at least one locus on A09 controls Mn2+ tolerance in B. napus. Integrated analysis of genomic and expression QTL and Mn2+ tolerance data reveals that BnMTP8.A09, in conjunction with BnMATE.C03, BnMTP8.C04 and BnMTP8.C08, play a central role in conferring Mn2+ tolerance in B. napus. Gene expression analysis revealed a high correlation (R2 = 0.74) between Mn2+ tolerance and the BnMTP8.A09 expression. Yeast complementation assays show that BnMTP8.A09 can complement manganese-hypersensitive yeast mutant strain PMR1{Delta} and restore Mn2+ tolerance to wild-type levels. Inductively coupled plasma mass spectrometry revealed that Mn2+ tolerant accessions accumulate less Mn in the shoots compared to Mn2+ sensitives, suggesting that the BnMTP8.A09 transporter likely sequesters Mn2+ into the tonoplast. Taken together, our research unveils the genetic architecture of Mn2+ tolerance and identifies BnMTP8.A09 as a major gene imparting tolerance to Mn2+ toxicity in B. napus.

plant biology↗

bioRxiv2023

Cell-specialized chloroplast signaling orchestrates photosynthetic and extracellular reactive oxygen species for stress responses

Cellular responses to abiotic stress involve multiple signals including secondary messengers such as reactive oxygen species (ROS) and Ca2+, phytohormones such as abscisic acid (ABA) and chloroplast-to-nucleus retrograde signals such as 3-phosphoadenosine 5-phosphate (PAP). Mechanism(s) by which these messengers, produced in different subcellular compartments, intersect for cell regulation remain enigmatic. Previously we showed that the chloroplast retrograde signal PAP, similar to ABA, induces an increase in ROS levels in guard cells (Pornsiriwong et al, 2017). Here we demonstrate a mechanistic link enabling ABA and PAP to coordinate both chloroplast and plasma membrane ROS production. In whole leaves, PAP alters various ROS-related processes including plasmodesmal permeability as well as responses to ozone and the bacterial elicitor flg22, but mainly initiates processes that quench ROS during oxidative stress. Conversely, we show in guard cells, both PAP and ABA induce an increase in ROS levels in both chloroplasts via photosynthetic electron transport, and the apoplast via the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH). Both subcellular ROS sources were necessary for ABA- and PAP-mediated stomatal closure. However, PAP signaling diverges from ABA by activating RBOHD, instead of RBOHF, for apoplastic ROS mediated stomatal closure. We identified three calcium-dependent protein kinases (CPKs) as the post-translational activators of RBOHD-mediated ROS production. CPK13, CPK32, and CPK34 were transcriptionally induced by PAP and concurrently activate RBOHD and the slow anion channel SLAC1 by phosphorylating two Serine (S) residues, including S120 which is also targeted by the core ABA signaling kinase OPEN STOMATA 1 (OST1). Consequently, overexpression of the PAP-induced CPKs rescues stomatal closure in ost1. Our data identify stomatal chloroplasts, to be nodes in the multifaceted cellular stress response networks as they are both sources and mediators of ROS and retrograde signals such PAP. Thus, chloroplasts are not just mediators of photosynthesis in response to, for example, excess light, but can serve as critical nodes in the multifaceted cellular stress response networks in specialized cells via retrograde signals, providing support to the concept of sensory plastids. Significance StatementThe chloroplast is an environmental sensor for stresses such as excess light and drought via the activation of photosynthetic-mediated retrograde signals. However, how does it function in specialized cells for which carbon fixation is secondary? Here we show the chloroplast is an important node to coordinate multiple plant signaling pathways in response to stresses such as drought. The chloroplast retrograde signal 3-phosphoadenosine 5-phosphate (PAP) plays multiple roles in reactive oxygen species (ROS) signaling and homeostasis. While PAP suppresses ROS in photosynthetic tissue, PAP instead induces guard cell ROS in chloroplasts and extracellular space to induce stomatal closure. We decipher how PAP-induced proteins activate both extracellular ROS production and anion channels for stomatal closure, thus providing a mechanism by which chloroplasts provide a strategic complement to canonical hormonal pathways in regulating plant physiological responses in specialized cells.

plant biology↗

bioRxiv2023

Reducing PSY activity fine tunes threshold levels of a cis-carotene-derived signal that regulates the PIF3/HY5 module and plastid biogenesis

PHYTOENE SYNTHASE (PSY) is a rate-limiting enzyme catalysing the first committed step of carotenoid biosynthesis, and changes in PSY gene expression and/or protein activity alter carotenoid composition and plastid differentiation in plants. Here we identified four genetic variants of PSY (psy-4, psy-90, psy-130 and psy-145) using a forward genetics approach that rescued leaf virescence phenotypes displayed by the Arabidopsis CAROTENOID ISOMERASE (CRTISO) mutant ccr2 (carotenoid and chloroplast regulation 2) when grown under a shorter photoperiod. The four non-lethal mutations affected alternative splicing, enzyme-substrate interactions, and PSY:ORANGE multi-enzyme complex binding, constituting the dynamic posttranscriptional fine-tuning of PSY levels and activity without changing localization to the stroma and protothylakoid membranes. psy genetic variants did not alter overall total xanthophyll or cis-carotene accumulation in ccr2 yet reduced specific acyclic linear cis-carotenes linked to the biosynthesis of a yet-to-be-identified apocarotenoid signal. ccr2 psy variants modulated the ratio of PHYTOCHROME-INTERACTING FACTOR 3/ELONGATED HYPOCOTYL 5 (PIF3/HY5), displayed a normal PLB formation in etioplasts, and chlorophyll accumulation during seedling photomorphogenesis. Thus, suppressing PSY activity and impairing PSY:ORANGE protein interactions reveals how threshold specific cis-carotene levels can be fine-tuned through holoenzyme-metabolon interactions to control plastid development. HighlightsManipulation of the PHYTOENE SYNTHASE catalytic activity in concert with its regulatory protein, ORANGE, reduces threshold levels of acyclic linear cis-carotenes that signal control over plastid biogenesis in dark and light grown Arabidopsis seedlings

plant biology↗

bioRxiv2022

Dynamics of mRNA fate during light stress and recovery: from transcription to stability and translation

Transcript stability is an important determinant of its abundance and, consequently, translation. However, it is unclear the extent to which it is modulated between environmental conditions. We previously hypothesised that recovery-induced transcript destabilisation facilitated a phenomenon of rapid recovery gene down-regulation (RRGD) in Arabidopsis thaliana following stress, based on mathematical calculations to account for ongoing transcription. Here, we test this hypothesis, and investigate processes regulating transcript abundance and fate, by quantifying changes in transcription, stability, and translation before, during, and after light stress. We adapt syringe infiltration to apply a transcriptional inhibitor to soil-grown plants in combination with stress. Compared to measurements in juvenile plants and cell culture, we find reduced stability in a range of transcripts. We also observe transcript destabilisation during light stress, followed by stabilisation upon recovery. Alongside fast transcriptional shut-off in recovery, this destabilisation appears to facilitate RRGD. Translation was dynamic over the course of light stress and recovery, with substantial transcript-specific increases in ribosome-association, independent of changes in total transcript abundance, observed after 30 minutes of light stress. Taken together, we provide evidence for the combinatorial regulation of transcription, stability, and translation that occurs to facilitate responses to light stress and recovery.

plant biology↗

bioRxiv2021

Enzymes degraded under high light maintain proteostasis by transcriptional regulation in Arabidopsis

Photo-inhibitory high light stress in Arabidopsis leads to increases in markers of protein degradation and transcriptional upregulation of proteases and proteolytic machinery, but proteostasis is largely maintained. We find significant increases in the in vivo degradation rate for specific molecular chaperones, nitrate reductase, glyceraldehyde-3 phosphate dehydrogenase, and phosphoglycerate kinase and other plastid, mitochondrial, peroxisomal, and cytosolic enzymes involved in redox shuttles. Coupled analysis of protein degradation rates, mRNA levels, and protein abundance reveal that 57% of the nuclear-encoded enzymes with higher degradation rates also had high light-induced transcriptional responses to maintain proteostasis. In contrast, plastid-encoded proteins with enhanced degradation rates showed decreased transcript abundances and must maintain protein abundance by other processes. This analysis reveals a light-induced transcriptional program for nuclear-encoded genes, beyond the regulation of PSII D1 subunit and the function of PSII, to replace key protein degradation targets in plants and ensure proteostasis under high light stress.

plant biology↗

bioRxiv2021

Autophagy promotes photomorphogenesis during seedling development in Arabidopsis in carbon limiting conditions

Autophagy is a conserved catabolic process that plays an essential role under nutrient starvation condition and influences different developmental processes. We observed that seedlings of autophagy mutants (atg2, atg5, atg7, and atg9) germinated in the dark showed delayed chloroplast development following illumination. The delayed chloroplast development was characterized by a decrease in photosynthetic and chlorophyll biosynthetic proteins, lower chlorophyll content, reduced chloroplast size, and increased levels of proteins involved in lipid biosynthesis. Confirming the biological impact of these differences, photosynthetic performance was impaired in autophagy mutants 12h post illumination. We investigated if the delayed chloroplast development could be explained by lower lipid import to the chloroplast or lower triglyceride (TAG) turnover. We observed that the limitations in the chloroplast lipid import imposed by trigalactosyldiacylglycerol1 are unlikely to explain the delay in photomorphogenesis. However, we found that lower TAG mobility in the triacylglycerol lipase mutant sugardependent1 significantly affected photomorphogenesis. Moreover, we showed that lower levels of carbon resources exacerbated the delay in photomorphogenesis whereas higher levels of carbon resources had an opposite effect. This work provides evidence that autophagic process operate during de-etiolation in a manner that contributes to photomorphogenesis through increasing lipid turnover to physically or energetically sustain photomorphogenesis.

plant biology↗

bioRxiv2021

Retrograde Control of Cytosolic Translation Targets Synthesis of Plastid Localized Proteins and Nuclear Responses for Efficient Light Acclimation.

Canonical retrograde signalling comprises information transmission from organelles to the nucleus and in particular controls gene expression for organellar proteins. The need to re-assess this paradigm was suggested by discrepancies between de novo protein synthesis and transcript abundance in response to excess light. Here we uncover major components of a translation-dependent retrograde signalling pathway that first impacts translation and then transcription. The response realization depends on the kinases Mitogen-activated protein kinase 6 (MPK6) and Sucrose non-fermenting 1-related kinase (SnRK1) subunit, AKIN10. Global ribosome foot-printing revealed differential ribosome association of 951 transcripts within 10 min after transfer from low to high light. Despite predominant translational repression, 15 % of transcripts were increased in translation and enriched for chloroplast-localized photosynthetic proteins. About one third of these transcripts, including Stress associated proteins (SAP) 2 and 3, share regulatory motifs in their 5'-UTR that act as binding sites for glyceraldehyde-3-phosphate dehydrogenase (GAPC) and light responsive RNA binding proteins (RBPs). SAP2 and 3 are both translationally regulated and interact with the calcium sensor Calmodulin-like 49 (CML49), which promotes relocation to the nucleus inducing a translation-dependent nuclear stress response. Thus, translation-dependent retrograde signalling bifurcates to directly regulate a translational circuit of chloroplast proteins and simultaneously initiate a nuclear circuit synchronizing retrograde and anterograde response pathways, serving as a rapid mechanism for functional acclimation of the chloroplast.

plant biology↗

bioRxiv2019

A cis-carotene derived apocarotenoid regulates etioplast and chloroplast development

Carotenoids are core plastid components, yet a regulatory function during plastid biogenesis remains enigmatic. A unique carotenoid biosynthesis mutant, carotenoid chloroplast regulation 2 (ccr2), that has no prolamellar body (PLB) and normal PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR) levels, was used to demonstrate a regulatory function for carotenoids under varied dark-light regimes. A forward genetics approach revealed how an epistatic interaction between a (-carotene isomerase mutant (ziso-155) and ccr2 blocked the biosynthesis of specific cis-carotenes and restored PLB formation in etioplasts. We attributed this to a novel apocarotenoid signal, as chemical inhibition of carotenoid cleavage dioxygenase activity restored PLB formation in ccr2 etioplasts during skotomorphogenesis. The apocarotenoid acted in parallel to the transcriptional repressor of photomorphogenesis, DEETIOLATED1 (DET1), to post-transcriptionally regulate PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR), PHYTOCHROME INTERACTING FACTOR3 (PIF3) and ELONGATED HYPOCOTYL5 (HY5) protein levels. The apocarotenoid signal and det1 complemented each other to restore POR levels and PLB formation, thereby controlling plastid development. One-sentence summaryCarotenoids are not just required as core components for plastid biogenesis, they can be cleaved into an apocarotenoid signal that regulates etioplast and chloroplast development during extended periods of darkness.

plant biology↗

bioRxiv2018

Excess light priming in Arabidopsis thaliana with altered DNA methylomes

Plants must continuously react to the ever-fluctuating nature of their environment. Repeated exposure to stressful conditions can lead to priming, whereby prior encounters heighten a plants ability to respond to future events. A clear example of priming is provided by the model plant Arabidopsis thaliana (Arabidopsis), in which photosynthetic and photoprotective responses are enhanced following recurring light stress. While there are various post-translational mechanisms underpinning photoprotection, an unresolved question is the relative importance of transcriptional changes towards stress priming and, consequently, the potential contribution from DNA methylation - a heritable chemical modification of DNA capable of influencing gene expression. Here, we systematically investigate the potential molecular underpinnings of physiological priming against recurring excess-light (EL), specifically DNA methylation and transcriptional regulation: the latter having not been examined with respect to EL priming. The capacity for physiological priming of photosynthetic and photoprotective parameters following a recurring EL treatment was not impaired in Arabidopsis mutants with perturbed establishment, maintenance, or removal of DNA methylation. Importantly, no differences in development or basal photoprotective capacity were identified in the mutants that may confound the above result. Little evidence for a causal transcriptional component of physiological priming was identified; in fact, most alterations in primed plants presented as a transcriptional dampening in response to an additional EL exposure, likely a consequence of physiological priming. However, a set of transcripts uniquely regulated in primed plants provide preliminary evidence for a novel transcriptional component of recurring EL priming, independent of physiological changes. Thus, we propose that physiological priming of recurring EL in Arabidopsis occurs independently of DNA methylation; and that the majority of the associated transcriptional alterations are a consequence, not cause, of this physiological priming.\n\nOne sentence summaryPhotoprotection and priming against recurring excess light is functional despite impaired maintenance of the DNA methylome.

plant biology↗