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Environmental changes trigger stress responses in living organisms. Although the underlying mechanisms are only partly understood, they involve intricate signaling pathways and transcription factors (TFs). Mediator is a conserved co-regulator complex required for transcriptional regulation of all eukaryotic protein-encoding genes. However, its function in abiotic stress responses is elusive. Here, we describe global gene expression changes induced by salt stress in Arabidopsis thaliana . To investigate the involvement of Mediator, we analyzed med9, med16, med18 , and cdk8 mutants, each representing one of the four Mediator modules. Our results demonstrate that promoters of differentially expressed genes (DEGs) for each mutant are enriched for binding sites of specific TFs. Phenotypic analyses further support the transcriptomic data: med16 and med18 , and to a lesser extent cdk8 , exhibit defects typical to mutations that affect abscisic acid and anthocyanin metabolism and we identify dysregulated signaling molecules, TFs, and target genes in these pathways. Our results reveal how signals from different stress response pathways are dependent on, and integrated by, Mediator subunits to coordinate a functional response to salt stress.

We have established a method for chromatin immunoprecipitation coupled to mass spectrometry (ChIP-MS) in Arabidopsis thaliana. We demonstrate its utility by investigating proteins associated with histone H3 lysine 27 acetylation (H3K27ac), a key epigenetic mark regulating photosynthesis-associated nuclear genes (PhANGs) during chloroplast development and establishment of photosynthesis. Purification of chromatin-associated proteins from light-grown Arabidopsis cell cultures identified 66 proteins associated with H3K27ac that met the selection criteria in the two replicate experiments: (i) 2-fold change in relation to IgG, (ii) at least two unique peptides, and (iii) relevant biological annotations. The identified proteins included chromatin remodelers, chromatin regulators and transcription factors with potential roles in H3K27ac deposition. To evaluate the physiological role of the candidates associated with the H3K27ac mark, we developed a rapid and reproducible phenotyping method based on controlled light scanning to determine chlorophyll accumulation in mutant seedlings. We complemented with pigment quantification and analysis of photosynthesis-associated nuclear genes (PhANGs) expression. Several mutants displayed altered greening, pigment accumulation, or affected photosynthetic gene expression consistent with a role during chloroplast development. Notably, chr11, chr17, and atpds5a mutants showed impaired pigment accumulation and reduced expression of PhANGs, whereas hmgb4 and mbd10 mutants exhibited increased greening and induction of PhANGs. Together, these findings establish ChIP-MS as a robust approach to identify histone mark-associated proteins in plants and provide a first set of candidate regulators of H3K27ac during chloroplast biogenesis. This technical advance opens new possibilities to discover chromatin-based regulation of plant development and environmental responses.

Alternative splicing (AS) enables cells to produce multiple protein isoforms from single genes, fine-tuning protein function across numerous cellular processes. However, despite its biological importance, researchers lack effective tools to compare the domain composition of AS-derived protein isoforms because such comparisons require both structural data and specialized methods. Recent advances in AI-driven protein structure prediction, particularly AlphaFold2, now make accurate structural determination of splicing isoforms accessible, enabling functional AS analysis at the protein structure level. Here, we present IsoformMapper, a web resource that analyzes AS through network community analysis of protein structures. This approach captures 3D physical interactions between protein regions often missed by traditional domain analysis, enabling structural comparisons of isoforms across any biological system. We illustrate our tool by analyzing validated human Bcl-X protein isoforms, revealing how AS creates distinct community structures with antagonistic functional roles. As a proof of concept, we apply our tool to investigate how GENOMES UNCOUPLED1 (GUN1)-dependent retrograde signaling regulates plant de-etiolation through alternative splicing in Arabidopsis. In response to light, gun1 shows alterations in spliceosome component expression, suggesting that GUN1 contributes to AS regulation of genes essential for photosynthetic establishment. The gun1 mutant displays altered splice variant ratios for PNSL2, CHAOS, and SIG5. Our tool reveals that these isoforms form distinct protein community structures, demonstrating how AS impacts protein function and validating IsoformMapper's practical value.

Plants align their physiology with daily environmental cycles through the circadian clock, which integrates light and metabolic signals to optimize growth and stress responses. While light entrainment has been extensively studied, emerging evidence highlights the central role of metabolism—particularly from chloroplasts and mitochondria—in tuning circadian rhythms. In this review, we explore the bidirectional relationship between organelle metabolism and the circadian clock, focusing on how metabolic signals such as sugars, ROS, and organic acids function as entrainment cues. We discuss how the clock regulates organelle function at multiple levels, including transcriptional, translational, and post-translational mechanisms, and how organelle-derived signals feedback to modulate core clock components through retrograde pathways. Special attention is given to the integration of chloroplast and mitochondrial signals, emphasizing their synergistic roles in maintaining cellular homeostasis. Drawing on the “three-body problem” analogy, we illustrate the dynamic and reciprocal interactions among light, clock, and metabolism. This perspective underscores the need to reframe the circadian system, not merely as light-driven but also as a central integrator of energy status and environmental cues. Understanding this integrated network is essential to improve plant performance and resilience under fluctuating environmental conditions.

Retrograde signalling networks originating in the organelles dictate nuclear gene expression and are essential for control and regulation of cellular energy metabolism. We investigate whether such plastid retrograde signals control nuclear gene expression by altering the chromatin state during the establishment of photosynthetic function in response to light. An Arabidopsis thaliana cell culture provides the required temporal resolution to map four histone modifications during the greening process. We uncover sequential and distinct epigenetic reprogramming events where an epigenetic switch from a histone methylation to an acetylation at photosynthesis-associated loci is dependent on a plastid retrograde signal. The transcription factors VIVIPAROUS1/ABI3-LIKE (VAL1), RELATIVE OF EARLY FLOWERING 6 (REF6) and GOLDEN2-LIKE FACTOR1/2 (GLKs) are linked to the H3K27ac deposition at photosynthesis associated loci that precedes full activation of the photosynthesis genes. Our work demonstrates that retrograde signals play a role in the epigenetic reprogramming essential to the establishment of photosynthesis in plant cells.

The components of the mediator kinase module are highly conserved across all eukaryotic lineages, and cyclin-dependent kinase 8 (CDK8) is essential for correct cell proliferation and differentiation in diverse eukaryotic systems. We show that CDK8 couples leaf development with the establishment of correct stomata patterning for prevailing CO₂ conditions. In Arabidopsis, the basic helix-loop-helix (bHLH) transcription factor SPEECHLESS (SPCH) controls cellular entry into the stomatal cell lineage, and CDK8 interacts with and phosphorylates SPCH, controlling SPCH protein levels and thereby also expression of the SPCH target genes encoding key regulators of cell fate and asymmetric cell divisions. The lack of the CDK8-mediated control of SPCH results in an increased number of meristemoid and guard mother cells, and increased stomata index in the cdk8 mutants. Increasing atmospheric CO₂ concentrations trigger a developmental programme controlling cell entry into stomatal lineage by limiting the asymmetric divisions. In cdk8, the number of meristemoids and guard mother cells remains the same under ambient and high CO₂ concentrations, as the accumulated levels of SPCH caused by the lack of CDK8 appear to override the negative regulation of increased CO₂. Thus, our work provides novel mechanistic understanding of how plants alter critical leaf properties in response to increasing atmospheric CO₂.

Rapid metabolic responses to pathogens are essential for plant survival and depend on numerous transcription factors. Mediator is the major transcriptional co-regulator for integration and transmission of signals from transcriptional regulators to RNA polymerase II. Using four Arabidopsis Mediator mutants, med16, med18, med25 and cdk8, we studied how differences in regulation of their transcript and metabolite levels correlate to their responses to Pseudomonas syringae infection. We found that med16 and cdk8 were susceptible, while med25 showed increased resistance. Glucosinolate, phytoalexin and carbohydrate levels were reduced already before infection in med16 and cdk8, but increased in med25, which also displayed increased benzenoids levels. Early after infection, wild type plants showed reduced glucosinolate and nucleoside levels, but increases in amino acids, benzenoids, oxylipins and the phytoalexin camalexin. The Mediator mutants showed altered levels of these metabolites and in regulation of genes encoding key enzymes for their metabolism. At later stage, mutants displayed defective levels of specific amino acids, carbohydrates, lipids and jasmonates which correlated to their infection response phenotypes. Our results reveal that MED16, MED25 and CDK8 are required for a proper, coordinated transcriptional response of genes which encode enzymes involved in important metabolic pathways for Arabidopsis responses to Pseudomonas syringae infections.

The development of a seedling into a photosynthetically active plant is a crucial process. Despite its importance, we do not fully understand the regulatory mechanisms behind the establishment of functional chloroplasts. We herein provide new insight into the early light response by identifying the function of three basic region/leucine zipper (bZIP) transcription factors: bZIP16, bZIP68, and GBF1. These proteins are involved in the regulation of key components required for the establishment of photosynthetically active chloroplasts. The activity of these bZIPs is dependent on the redox status of a conserved cysteine residue, which provides a mechanism to finetune light-responsive gene expression. The blue light cryptochrome (CRY) photoreceptors provide one of the major light-signaling pathways, and bZIP target genes overlap with one-third of CRY-regulated genes with an enrichment for photosynthesis/chloroplast-associated genes. bZIP16, bZIP68, and GBF1 were demonstrated as novel interaction partners of CRY1. The interaction between CRY1 and bZIP16 was stimulated by blue light. Furthermore, we demonstrate a genetic link between the bZIP proteins and cryptochromes as the cry1cry2 mutant is epistatic to the cry1cry2bzip16bzip68gbf1 mutant. bZIP16, bZIP68, and GBF1 regulate a subset of photosynthesis associated genes in response to blue light critical for a proper greening process in Arabidopsis.

Increasing plants' photosynthetic efficiency is a major challenge that must be addressed in order to cover the food demands of the growing population in the changing climate. Photosynthesis is greatly limited at the initial carboxylation reaction, where CO₂ is converted to the organic acid 3-PGA, catalyzed by the RuBisCO enzyme. RuBisCO has poor affinity for CO₂, but also the CO₂ concentration at the RuBisCO site is limited by the diffusion of atmospheric CO₂ through the various leaf compartments to the reaction site. Beyond genetic engineering, nanotechnology can offer a materials-based approach for enhancing photosynthesis, and yet, it has mostly been explored for the light-dependent reactions. In this work, we developed polyethyleneimine-based nanoparticles for enhancing the carboxylation reaction. We demonstrate that the nanoparticles can capture CO₂ in the form of bicarbonate and increase the CO₂ that reacts with the RuBisCO enzyme, enhancing the 3-PGA production in in vitro assays by 20%. The nanoparticles can be introduced to the plant via leaf infiltration and, because of the functionalization with chitosan oligomers, they do not induce any toxic effect to the plant. In the leaves, the nanoparticles localize in the apoplastic space but also spontaneously reach the chloroplasts where photosynthetic activity takes place. Their CO₂ loading-dependent fluorescence verifies that, in vivo, they maintain their ability to capture CO₂ and can be therefore reloaded with atmospheric CO₂ while in planta. Our results contribute to the development of a nanomaterials-based CO₂-concentrating mechanism in plants that can potentially increase photosynthetic efficiency and overall plants' CO₂ storage.

• One of the most dramatic challenges in the life of a plant occurs when the seedling emerges from the soil and exposure to light triggers expression of genes required for establishment of photosynthesis.
• This process needs to be tightly regulated, as premature accumulation of light-harvesting proteins and photoreactive Chl precursors causes oxidative damage when the seedling is first exposed to light. Photosynthesis genes are encoded by both nuclear and plastid genomes, and to establish the required level of control, plastid-to-nucleus (retrograde) signalling is necessary to ensure correct gene expression.
• We herein show that a negative GENOMES UNCOUPLED1 (GUN1)-mediated retrograde signal restricts chloroplast development in darkness and during early light response by regulating the transcription of several critical transcription factors linked to light response, photomorphogenesis, and chloroplast development, and consequently their downstream target genes in Arabidopsis.
• Thus, the plastids play an essential role during skotomorphogenesis and the early light response, and GUN1 acts as a safeguard during the critical step of seedling emergence from darkness.