Abstract Changes in intracellular calcium ion (Ca²⁺) concentrations generate characteristic signatures that are decoded by specialized Ca²⁺-binding proteins (CaBP). Although substantial progress has been made in understanding cytosolic calcium signaling pathways, calcium signaling within organelles, particularly chloroplasts, remains poorly understood, partly because only a few EF-hand CaBP have been identified in organelles. Here, we describe a novel EF-hand protein of 18 kDa, that was found to be associated with the chloroplast envelope and peroxisomal membrane and was therefore named OEF18 (ORGANELLAR EF-HAND PROTEIN OF 18 kDa). OEF18 has a very unusual structure, containing an N-terminal myristoylation site, followed by one EF-hand in the N-terminus facing to the cytosol, and a transmembrane domain in the C-terminus. OEF18 membrane-targeting was found to be mediated by ANKYRIN REPEAT-CONTAINING PROTEIN 2A (AKR2A) via the C-terminal transmembrane domain of OEF18. Furthermore, the EF-hand in OEF18 bound Ca²⁺ at a physiological concentration that led to a large protein conformational change, inducing oligomerization of the N-terminal part. We found that oef18 mutants accumulated less jasmonic acid (JA) and its bioactive conjugate JA-Ile, likely causing a defect in the insect herbivore response. Wild-type OEF18 complemented the herbivory phenotype of oef18 mutants, whereas an EF-hand point mutant lacking Ca²⁺-binding capacity failed to restore the wild-type response. Furthermore, OEF18 was required for resistance to salt stress in combination with dark-induced senescence. Together, these results establish OEF18 as a previously unrecognized organellar Ca²⁺ sensor that couples Ca²⁺ perception to JA-mediated defense and abiotic stress responses in plants.

Cultivar Désirée is an important model for potato functional genomics studies to assist breeding strategies. Here, we present a haplotype-resolved genome assembly of Désirée, achieved by assembling PacBio HiFi reads and Hi-C scaffolding, resulting in a high-contiguity chromosome-level assembly. We implemented a comprehensive annotation pipeline incorporating gene models and functional annotations from the Solanum tuberosum Phureja DM reference genome alongside RNA-seq reads to provide high-quality gene and transcript annotations. Additionally, we provide a genome-wide DNA methylation profile using Oxford Nanopore reads, enabling insights into potato epigenetics. The assembled genome, annotations, methylation and expression data are visualised in a publicly accessible genome browser, providing a valuable resource for the potato research community.

Plants rely on water and light for photosynthesis, but water droplets on leaves can focus light into high-intensity spots, risking photodamage. Excessive light can impair growth or induce cell death, making it essential for plants to detect and respond to light fluctuations. While Ca2+ signaling has been linked to high light (HL) acclimation, the subcellular dynamics remain unclear. Here, we investigate Ca2+ responses to HL exposure in Arabidopsis thaliana. Using a glass bead to simulate light-focusing by water droplets, a biphasic increase of Ca2+ concentration was detected in the chloroplast stroma by the genetically encoded calcium indicator YC3.6 and confirmed using a newly established stroma-localized R-GECO1 (NTRC-R-GECO1). The stromal response was largely independent of light wavelength and unaffected in phot1 phot2 and cry1 cry2 mutants. Chemical inhibition of photosynthetic electron transport, microscopy-based Fv/Fm experiments, and measurement of the reactive oxygen species (ROS)-redox balance with roGFP-based reporters and Singlet Oxygen Sensor Green (SOSG) chemical dye suggested that photodamage and singlet oxygen contribute to the stromal Ca2+ response. While blue and white light also triggered a Ca2+ response in the cytosol and nucleus, pharmacological inhibition with cyclopiazonic acid (CPA) and loss-of-function mutants of the Ca2+ transporters BIVALENT CATION TRANSPORTER 2 (BICAT2) and endoplasmic reticulum (ER)-type Ca2+-ATPase (ECA) suggested that the HL response depends on a Ca2+ exchange between the ER and chloroplast stroma. The response was primarily light dependent but accelerated by increasing external temperature. This study implicates a novel Ca2+-mediated acclimation mechanism to HL stress, a process of growing relevance in the context of climate change.

Potato (Solanum tuberosum) is highly water and space efficient but susceptible to abiotic stresses such as heat, drought, and flooding, which are severely exacerbated by climate change. Our understanding of crop acclimation to abiotic stress, however, remains limited. Here, we present a comprehensive molecular and physiological high-throughput profiling of potato (Solanum tuberosum, cv. Désirée) under heat, drought, and waterlogging applied as single stresses or in combinations designed to mimic realistic future scenarios. Stress responses were monitored via daily phenotyping and multi-omics analyses of leaf samples comprising proteomics, targeted transcriptomics, metabolomics, and hormonomics at several timepoints during and after stress treatments. Additionally, critical metabolites of tuber samples were analyzed at the end of the stress period. We performed integrative multi-omics data analysis using a bioinformatic pipeline that we established based on machine learning and knowledge networks. Waterlogging produced the most immediate and dramatic effects on potato plants, interestingly activating ABA responses similar to drought stress. In addition, we observed distinct stress signatures at multiple molecular levels in response to heat or drought and to a combination of both. In response to all treatments, we found a downregulation of photosynthesis at different molecular levels, an accumulation of minor amino acids, and diverse stress-induced hormones. Our integrative multi-omics analysis provides global insights into plant stress responses, facilitating improved breeding strategies toward climate-adapted potato varieties.

Our work identified Ca2+-dependent and -independent changes in protein contents upon oxidative stress, showing that Ca2+ signaling shapes the early oxidative stress response and identifying potential targets for stress resilience research. Calcium (Ca2+) and reactive oxygen species (ROS) are key secondary messengers in plant stress signaling, yet their interplay in regulating proteome-wide responses remains poorly understood. We employed label-free quantitative (LFQ) proteomics to investigate Ca2+-dependent and -independent proteome changes in Arabidopsis thaliana leaves upon oxidative stress induced by hydrogen peroxide (H2O2). To dissect the role of Ca2+ signaling, we inhibited H2O2-induced Ca2+ transients by pre-treatment with the Ca2+ influx blocker LaCl3. Throughout all four treatment samples - control, H2O2-treated, LaCl3-treated, H2O2- and LaCl3-treated - we identified a total of 3724 and 3757 proteins after 10 and 30 min, respectively. Of these, 581 proteins showed significant changes in abundance between the 10 min and 909 proteins between the 30 min sample groups. The combined LaCl3 and H2O2 treatment resulted in the highest number of differentially abundant proteins (DAPs), indicating a strong attenuating effect of Ca2+ signaling on the oxidative stress response. By contrast, only 37 and 57 proteins responded to H2O2 alone with distinct subsets of strictly Ca2+-dependent, partially Ca2+-dependent, and Ca2+-independent proteins. Ca2+-independent H2O2-responsive proteins predominantly showed increased abundance, while strictly Ca2+-dependent proteins exhibited decreased abundance, suggesting a role for Ca2+ signaling in protein degradation. Furthermore, three proteins—WLIM1, CYP97C1, and AGAP1—underwent shifts in Ca2+-dependency between the two time points, pointing to a dynamic Ca2+-regulation. This study provides insight into short-term Ca2+-dependent and independent regulation of the Arabidopsis leaf proteome in response to oxidative stress, thereby identifying potential new targets for research on plant stress resilience mechanisms.

Calcium (Ca 2+ ) and reactive oxygen species (ROS) are key secondary messengers in plant stress signaling, yet their interplay in regulating proteome-wide responses remains poorly understood. In this study, we employed label-free quantitative (LFQ) proteomics to investigate Ca 2+ -dependent and independent changes in the proteome of Arabidopsis thaliana leaves upon oxidative stress induced by hydrogen peroxide (H 2 O 2 ). To dissect the role of Ca 2+ signaling, we inhibited H 2 O 2 -induced Ca 2+ transients by pretreatment with LaCl 3 , a plasma membrane Ca 2+ channel blocker. We then analysed the proteome of plants treated with H 2 0 2 or ddH 2 O after 10 and 30 min of treatment and detected 3724 and 3757 proteins, respectively. From these, 581 proteins showed significant changes in abundance after 10 min and 909 proteins after 30 min. Remarkably, the combined LaCl 3 and H 2 O 2 treatment resulted in the highest number of differentially abundant proteins (DAPs), indicating a strong attenuating effect of Ca 2+ signaling on the oxidative stress response. Specifically responsive to only H 2 O 2 were 37 and 57 proteins with distinct subsets of strictly Ca 2+ -dependent, partially Ca 2+ - dependent, and Ca 2+ -independent proteins. Notably, Ca 2+ -independent H 2 O 2 -responsive proteins predominantly showed increased abundance, while strictly Ca 2+ -dependent proteins exhibited decreased abundance, suggesting a role for Ca 2+ signaling in protein degradation. Furthermore, three proteins—WLIM1, CYP97C1, and AGAP1—underwent Ca 2+ -dependent shifts between the two time points, pointing to a dynamic nature of Ca 2+ -regulated proteomic changes. This study provides novel insights into short-term Ca 2+ -dependent and independent regulation of the Arabidopsis leaf proteome in response to oxidative stress, identifying key stress-responsive proteins and potential new targets for further research on plant stress resilience mechanisms.

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions over time. By using multiple imaging sensors, many traits of interest can be assessed, including plant biomass, photosynthetic efficiency, canopy temperature, and leaf reflectance indices. Plants are frequently exposed to multiple stresses under field conditions where severe heat waves, flooding, and drought events seriously threaten crop productivity. When stresses coincide, resulting effects on plants can be distinct due to synergistic or antagonistic interactions. To elucidate how potato plants respond to single and combined stresses that resemble naturally occurring stress scenarios, five different treatments were imposed on a selected potato cultivar ( Solanum tuberosum L., cv. Lady Rosetta) at the onset of tuberization, i.e. control, drought, heat, waterlogging, and combinations of heat, drought, and waterlogging stresses. Our analysis shows that waterlogging stress had the most detrimental effect on plant performance, leading to fast and drastic physiological responses related to stomatal closure, including a reduction in the quantum yield and efficiency of photosystem II and an increase in canopy temperature and water index. Under heat and combined stress treatments, the relative growth rate was reduced in the early phase of stress. Under drought and combined stresses, plant volume and photosynthetic performance dropped with an increased temperature and stomata closure in the late phase of stress. The combination of optimized stress treatment under defined environmental conditions together with selected phenotyping protocols allowed to reveal the dynamics of morphological and physiological responses to single and combined stresses. Here, a useful tool is presented for plant researchers looking to identify plant traits indicative of resilience to several climate change-related stresses.

Calcium is a universal messenger in different kingdoms of living organisms and regulates most physiological processes, including defense against pathogens. The threat of viral infections in humans has become very clear in recent years, and this has triggered detailed research into all aspects of host-virus interactions, including the suppression of calcium signaling in infected cells. At the same time, however, the threat of plant viral infections is underestimated in society, and research in the field of calcium signaling during plant viral infections is scarce. Here we highlight an emerging role of calcium signaling for antiviral protection in plants, in parallel with the known evidence from studies of animal cells. Obtaining more knowledge in this domain might open up new perspectives for future crop protection and the improvement of food security.

Stress Knowledge Map (SKM; https://skm.nib.si) is a publicly available resource containing two complementary knowledge graphs that describe the current knowledge of biochemical, signaling, and regulatory molecular interactions in plants: a highly curated model of plant stress signaling (PSS; 543 reactions) and a large comprehensive knowledge network (488 390 interactions). Both were constructed by domain experts through systematic curation of diverse literature and database resources. SKM provides a single entry point for investigations of plant stress response and related growth trade-offs, as well as interactive explorations of current knowledge. PSS is also formulated as a qualitative and quantitative model for systems biology and thus represents a starting point for a plant digital twin. Here, we describe the features of SKM and show, through two case studies, how it can be used for complex analyses, including systematic hypothesis generation and design of validation experiments, or to gain new insights into experimental observations in plant biology.

Plants require water and light for photosynthesis, but light, when focused by water droplets on leaves, can create high light intensity spots that are harmful to plants. As excessive light intensity can reduce growth or even induce cell death, it is vital for plants to detect and react to changes in light exposure and acclimate to high light stress. Ca2+ signaling was previously implicated in high light acclimation. However, the dynamics of free Ca2+ concentration in the chloroplast, the primary site of photosynthesis, or in the nucleus and in the cytoplasm, where transcription and translation for long-term acclimation occurs, remain unknown. Here we studied the dynamics and mechanism of the Ca2+ response to high light exposure. Focusing light through a glass bead to mimic water droplets triggered an increase of the free Ca2+ concentration in the chloroplast stroma of Arabidopsis thaliana . This finding was corroborated using established and newly developed genetically encoded calcium indicators, which revealed a biphasic increase in the stromal free Ca2+ concentration when exposed to varying intensities and qualities of light. Among photosynthetic by-products, reactive oxygen and lipophilic species in particular, have been implicated in high light stress acclimation. A H2O2 signature was induced, albeit with different dynamics than the Ca2+ response, while chemical inhibition of the photosynthetic electron transport points towards singlet oxygen as a potential culprit of the high light-induced increase in stromal free Ca2+ concentration. The observed dynamics differed from those of a heat-shock induced Ca2+ signature, although temperature had a positive effect on the Ca2+ response. Based on Ca2+ inhibitor treatments and the free Ca2+ concentration dynamics, we suggest that the high light-induced stromal Ca2+ is derived from the endoplasmic reticulum rather than from the cytoplasm. In conclusion, inspired by the burning glass effect of water droplets on leaves, we uncovered a Ca2+ response that implicates a novel mechanism for plants to acclimate to high light stress—a process that will become increasingly relevant in a changing climate. ### Competing Interest Statement The authors have declared no competing interest.

Potato, the most important non-cereal crop, is highly water and space efficient but susceptible to abiotic stress such as heat, drought, or flooding. Climate change is severely increasing the likelihood of such stresses to occur individually, sequentially, or simultaneously. However, the understanding of acclimation to abiotic stress in crops in general, especially with multiple stresses, is still very limited. Here, we present a comprehensive one month-long molecular and physiological high-throughput profiling of potato ( Solanum tuberosum , cv. Désirée) under both single and multiple abiotic stresses, designed to mimic realistic future scenarios. Acclimation time-responses were monitored via daily phenomic analysis and leaf samples were processed for multi-omics spanning from transcriptomics to proteomics and hormonomics. Additionally, critical metabolites of tuber samples were analysed at the end of the period. To facilitate the multi-omics analyses, the dataset was integrated with prior knowledge, which is indispensable for development of high-throughput pipelines in agricultural research. Waterlogging had the most immediate and dramatic effects, with responses similar to drought stress. In addition, we observed distinct stress signatures at multiple molecular levels in response to heat or drought and to a combination of both. In general, there was a downregulation of photosynthesis at different molecular levels, accumulation of minor amino acids and diverse stress induced hormones. Our integrative multi-omics analysis provides global insights into plant stress responses, facilitating improved breeding strategies. One Sentence Summary Integrated multi-omics analysis of high-throughput phenotyping in potato reveals distinct molecular signatures of acclimation to single and combined abiotic stresses related to climate change. Experimentally acquired data and data required to reproduce the analysis are available from Supp. Table 5 and NIBs’ GitHub repository . Proteomics data are available via ProteomeXchange with identifier PXD052587.

Effects associated with a changing climate could severely threaten potato production in Europe. Hence, farmers need to take up adaptation measures to safeguard agricultural production. Collecting data from 553 farmers from 22 different European countries, our survey evaluates European potato farmers' perceptions regarding the influence of climate change on local potato production, and their willingness to implement adaptation strategies. An overwhelming majority of survey respondents had already experienced the effects of climatic changes on their potato production. Specifically, drought and heat were identified as the most significant threats. The planting of an adapted variety was the preferred adaptation strategy, while farmers were also willing to take up changes in agricultural management practices. Survey respondents predominantly considered yield stability as the most important characteristic of an adapted variety, closely followed by heat tolerance, disease resistance, drought tolerance, and yield potential. When choosing a variety, the personal experience of the survey respondents as well as the experience of their peers were identified as the most important sources of information. Our survey gives valuable insights into the challenges European potato farmers are facing in times of climate change. Supplying farmers with better-adapted varieties would be a well-targeted and well-accepted measure to advance climate change adaptation.

© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Special Issue Editorial

Chloroplast development is initiated by light-signals triggering the expression of nuclear encoded chloroplast genes in a first phase, followed by massive structural changes in the transition from proplastids to mature chloroplasts in the second phase. While the molecular players involved in the first phase are currently emerging, regulatory components of the second phase, demanding high plastid translational capacity and RNA processing, are still enigmatic. This is mostly due to the very limited amount of plant material at the early phases of development that makes biochemical studies such as identifying protein interaction networks very difficult. To overcome this problem, we developed a TurboID-based proximity labelling workflow that requires only very limited sample amounts to obtain mechanistic insights into protein interaction networks present in the early stages of plastid development. We used the CGL20a protein, a novel factor involved in chloroplast development, as bait for in vivo proximity labelling in developing seedlings 7 days after germination. We found that CGL20a resides in a nexus of RNA binding proteins mainly associated to ribosomal RNA (rRNA) including different ribosome-associated proteins. One-sentence summary The use of plastid-specific in vivo proximity labelling in Arabidopsis seedlings allows to identify novel components in chloroplast development in higher plants.

Phytohormones are major signaling components that contribute to nearly all aspects of plant life. They constitute an interconnected communication network to fine-tune growth and development in response to the ever-changing environment. To this end, they have to coordinate with other signaling components, such as reactive oxygen species and calcium signals. On the one hand, the two endosymbiotic organelles, plastids and mitochondria, control various aspects of phytohormone signaling and harbor important steps of hormone precursor biosynthesis. On the other hand, phytohormones have feedback actions on organellar functions. In addition, organelles and phytohormones often act in parallel in a coordinated matter to regulate cellular functions. Therefore, linking organelle functions with increasing knowledge of phytohormone biosynthesis, perception, and signaling will reveal new aspects of plant stress tolerance. In this review, we highlight recent work on organelle-phytohormone interactions focusing on the major stress-related hormones abscisic acid, jasmonates, salicylic acid, and ethylene.

Plants adjust their energy metabolism to continuous environmental fluctuations, resulting in a tremendous plasticity in their architecture. The regulatory circuits involved, however, remain largely unresolved. In Arabidopsis, moderate perturbations in photosynthetic activity, administered by short-term low light exposure or unexpected darkness, lead to increased lateral root (LR) initiation. Consistent with expression of low-energy markers, these treatments alter energy homeostasis and reduce sugar availability in roots. Here, we demonstrate that the LR response requires the metabolic stress sensor kinase Snf1-RELATED-KINASE1 (SnRK1), which phosphorylates the transcription factor BASIC LEUCINE ZIPPER63 (bZIP63) that directly binds and activates the promoter of AUXIN RESPONSE FACTOR19 (ARF19), a key regulator of LR initiation. Consistently, starvation-induced ARF19 transcription is impaired in bzip63 mutants. This study highlights a positive developmental function of SnRK1. During energy limitation, LRs are initiated and primed for outgrowth upon recovery. Hence, this study provides mechanistic insights into how energy shapes the agronomically important root system.

Comprehensive knowledge of the proteome is a crucial prerequisite to understand dynamic changes in biological systems. Particularly low-abundance proteins are of high relevance in these processes as these are often proteins involved in signal transduction and acclimation responses. Although technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS), the dynamic range in protein abundance is still the most limiting problem for the detection of low-abundance proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of high-abundance proteins. Therefore, specific enrichment strategies are still required to overcome this technical limitation of MS-based protein discovery. We have searched for novel signal transduction proteins, more specifically kinases and calcium-binding proteins, and here we describe different approaches for enrichment of these low-abundance proteins from isolated chloroplasts from pea and Arabidopsis for subsequent proteomic analysis by MS. These approaches could be extended to include other signal transduction proteins and target different organelles.

Drought is a major cause of losses in crop yield. Under field conditions, plants exposed to drought are usually also experiencing rapid changes in light intensity. Accordingly, plants need to acclimate to both, drought and light stress. Two crucial mechanisms in plant acclimation to changes in light conditions comprise thylakoid protein phosphorylation and dissipation of light energy as heat by non-photochemical quenching (NPQ). Here, we analyzed the acclimation efficacy of two different wheat varieties, by applying fluctuating light for analysis of plants, which had been subjected to a slowly developing drought stress as it usually occurs in the field. This novel approach allowed us to distinguish four drought phases, which are critical for grain yield, and to discover acclimatory responses which are independent of photodamage. In short-term, under fluctuating light, the slowdown of NPQ relaxation adjusts the photosynthetic activity to the reduced metabolic capacity. In long-term, the photosynthetic machinery acquires a drought-specific configuration by changing the PSII-LHCII phosphorylation pattern together with protein stoichiometry. Therefore, the fine-tuning of NPQ relaxation and PSII-LHCII phosphorylation pattern represent promising traits for future crop breeding strategies.

Reversible phosphorylation of thylakoid proteins contributes to photoacclimation responses in photosynthetic organisms, enabling the fine-tuning of light harvesting under changing light conditions and promoting the onset of photoprotective processes. However, the precise functional role of many of the described phosphorylation events on thylakoid proteins remains elusive. The calcium sensor receptor protein (CAS) has previously been indicated as one of the targets of the state transition kinase 8 (STN8). Here we show that in Arabidopsis thaliana, CAS is also phosphorylated by the state transition kinase 7 (STN7), as well as by another, so-far unknown, Ca2+-dependent kinase. Phosphoproteomics analysis and in vitro phosphorylation assays on CAS variants identified the phylogenetically conserved residues Thr-376, Ser-378, and Thr-380 as the major phosphorylation sites of the STN kinases. Spectroscopic analyses of chlorophyll fluorescence emission at 77K further showed that, while the cas mutant is not affected in state transition, it displays a persistent strong excitation of PSI under high light exposure, similar to the phenotype previously observed in other mutants defective in photoacclimation mechanisms. Together with the observation of a strong concomitant phosphorylation of light harvesting complex II (LHCII) and photosynthetic core proteins under high irradiance in the cas mutant this suggests a role for CAS in the STN7/STN8/TAP38 network of phosphorylation-mediated photoacclimation processes in Arabidopsis.