High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato.

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.

Stress Knowledge Map: A knowledge graph resource for systems biology analysis of plant stress responses.

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.

Calcium signaling: an emerging player in plant antiviral defense.

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.

Organelles and phytohormones: a network of interactions in plant stress responses.

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.

Perturbations in plant energy homeostasis prime lateral root initiation via SnRK1-bZIP63-ARF19 signaling.

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.

Chloroplast Isolation and Enrichment of Low-Abundance Proteins by Affinity Chromatography for Identification in Complex Proteomes.

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.

Adjustment of photosynthetic activity to drought and fluctuating light in wheat.

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.

The High Light Response in Arabidopsis Requires the Calcium Sensor Protein CAS, a Target of STN7- and STN8-Mediated Phosphorylation.

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.

Sumoylation and phosphorylation: hidden and overt links.

Post-translational modifications are essential mediators between stimuli from development or the environment and adaptive transcriptional patterns. Recent data allow a first glimpse at how two modifications, phosphorylation and sumoylation, act interdependently to modulate stress responses. In particular, many components of the SUMO conjugation system are phosphoproteins, and some regulators and enzymes of protein phosphorylation can be sumoylated. Equally important, however, a number of proteins can be subject to both modifications. These substrates also have the capacity to connect stimuli transmitted via sumoylation with those transmitted via phosphorylation. As a prime example, we review data suggesting that nitrate reductase is a hub that integrates cues from these two modifications. Powerful proteomics approaches allowed the identification of additional common substrates, paving the way for studies to understand, on a broader basis, the cross-talk of phosphorylation with sumoylation and how it contributes to plant growth.

Snf1-RELATED KINASE1-Controlled C/S1-bZIP Signaling Activates Alternative Mitochondrial Metabolic Pathways to Ensure Plant Survival in Extended Darkness.

Sustaining energy homeostasis is of pivotal importance for all living organisms. In Arabidopsis thaliana, evolutionarily conserved SnRK1 kinases (Snf1-RELATED KINASE1) control metabolic adaptation during low energy stress. To unravel starvation-induced transcriptional mechanisms, we performed transcriptome studies of inducible knockdown lines and found that S1-basic leucine zipper transcription factors (S1-bZIPs) control a defined subset of genes downstream of SnRK1. For example, S1-bZIPs coordinate the expression of genes involved in branched-chain amino acid catabolism, which constitutes an alternative mitochondrial respiratory pathway that is crucial for plant survival during starvation. Molecular analyses defined S1-bZIPs as SnRK1-dependent regulators that directly control transcription via binding to G-box promoter elements. Moreover, SnRK1 triggers phosphorylation of group C-bZIPs and the formation of C/S1-heterodimers and, thus, the recruitment of SnRK1 directly to target promoters. Subsequently, the C/S1-bZIP-SnRK1 complex interacts with the histone acetylation machinery to remodel chromatin and facilitate transcription. Taken together, this work reveals molecular mechanisms underlying how energy deprivation is transduced to reprogram gene expression, leading to metabolic adaptation upon stress.

Redox state-dependent modulation of plant SnRK1 kinase activity differs from AMPK regulation in animals.

The evolutionarily highly conserved SNF1-related protein kinase (SnRK1) protein kinase is a metabolic master regulator in plants, balancing the critical energy consumption between growth- and stress response-related metabolic pathways. While the regulation of the mammalian [AMP-activated protein kinase (AMPK)] and yeast (SNF1) orthologues of SnRK1 is well-characterised, the regulation of SnRK1 kinase activity in plants is still an open question. Here we report that the activity and T-loop phosphorylation of AKIN10, the kinase subunit of the SnRK1 complex, is regulated by the redox status. Although this regulation is dependent on a conserved cysteine residue, the underlying mechanism is different to the redox regulation of animal AMPK and has functional implications for the regulation of the kinase complex in plants under stress conditions.

Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation.

Since years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a snrk1α1 knock out background (snrk1α1/α2) to abolish SnRK1 activity to understand major systemic functions of SnRK1 signalling under energy deprivation triggered by extended night treatment. We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants.

An evolutionary perspective of AMPK-TOR signaling in the three domains of life.

AMPK and TOR protein kinases are the major control points of energy signaling in eukaryotic cells and organisms. They form the core of a complex regulatory network to co-ordinate metabolic activities in the cytosol with those in the mitochondria and plastids. Despite its relevance, it is still unclear when and how this regulatory pathway was formed during evolution, and to what extent its representations in the major eukaryotic lineages resemble each other. Here we have traced 153 essential proteins forming the human AMPK-TOR pathways across 412 species representing all three domains of life-prokaryotes (bacteria, archaea) and eukaryotes-and reconstructed their evolutionary history. The resulting phylogenetic profiles indicate the presence of primordial core pathways including seven proto-kinases in the last eukaryotic common ancestor. The evolutionary origins of the oldest components of the AMPK pathway, however, extend into the pre-eukaryotic era, and descendants of these ancient proteins can still be found in contemporary prokaryotes. The TOR complex in turn appears as a eukaryotic invention, possibly to aid in retrograde signaling between the mitochondria and the remainder of the cell. Within the eukaryotes, AMPK/TOR showed both a highly conserved core structure and a considerable plasticity. Most notably, KING1, a protein originally assigned as the γ subunit of AMPK in plants, is more closely related to the yeast SDS23 gene family than to the γ subunits in animals or fungi. This suggests its functional difference from a canonical AMPK γ subunit.

Novel connections in plant organellar signalling link different stress responses and signalling pathways.

To coordinate growth, development and responses to environmental stimuli, plant cells need to communicate the metabolic state between different sub-compartments of the cell. This requires signalling pathways, including protein kinases, secondary messengers such as Ca(2+) ions or reactive oxygen species (ROS) as well as metabolites and plant hormones. The signalling networks involved have been intensively studied over recent decades and have been elaborated more or less in detail. However, it has become evident that these signalling networks are also tightly interconnected and often merge at common targets such as a distinct group of transcription factors, most prominently ABI4, which are amenable to regulation by phosphorylation, potentially also in a Ca(2+)- or ROS-dependent fashion. Moreover, the signalling pathways connect several organelles or subcellular compartments, not only in functional but also in physical terms, linking for example chloroplasts to the nucleus or peroxisomes to chloroplasts thereby enabling physical routes for signalling by metabolite exchange or even protein translocation. Here we briefly discuss these novel findings and try to connect them in order to point out the remaining questions and emerging developments in plant organellar signalling.

Know where your clients are: subcellular localization and targets of calcium-dependent protein kinases.

Calcium-dependent protein kinases (CDPKs) are at the forefront of decoding transient Ca(2+) signals into physiological responses. They play a pivotal role in many aspects of plant life starting from pollen tube growth, during plant development, and in stress response to senescence and cell death. At the cellular level, Ca(2+) signals have a distinct, narrow distribution, thus requiring a conjoined localization of the decoders. Accordingly, most CDPKs have a distinct subcellular distribution which enables them to 'sense' the local Ca(2+) concentration and to interact specifically with their targets. Here we present a comprehensive overview of identified CDPK targets and discuss them in the context of kinase-substrate specificity and subcellular distribution of the CDPKs. This is particularly relevant for calcium-mediated phosphorylation where different CDPKs, as well as other kinases, were frequently reported to be involved in the regulation of the same target. However, often these studies were not performed in an in situ context. Thus, considering the specific expression patterns, distinct subcellular distribution, and different Ca(2+) affinities of CDPKs will narrow down the number of potential CDPKs for one given target. A number of aspects still remain unresolved, giving rise to pending questions for future research.

An evolutionary view on thylakoid protein phosphorylation uncovers novel phosphorylation hotspots with potential functional implications.

The regulation of photosynthetic light reactions by reversible protein phosphorylation is well established today, but functional studies have so far mostly been restricted to processes affecting light-harvesting complex II and the core proteins of photosystem II. Virtually no functional data are available on regulatory effects at the other photosynthetic complexes despite the identification of multiple phosphorylation sites. Therefore we summarize the available data from 50 published phospho-proteomics studies covering the main complexes involved in photosynthetic light reactions in the 'green lineage' (i.e. green algae and land plants) as well as its cyanobacterial counterparts. In addition, we performed an extensive orthologue search for the major photosynthetic thylakoid proteins in 41 sequenced genomes and generated sequence alignments to survey the phylogenetic distribution of phosphorylation sites and their evolutionary conservation from green algae to higher plants. We observed a number of uncharacterized phosphorylation hotspots at photosystem I and the ATP synthase with potential functional relevance as well as an unexpected divergence of phosphosites. Although technical limitations might account for a number of those differences, we think that many of these phosphosites have important functions. This is particularly important for mono- and dicot plants, where these sites might be involved in regulatory processes such as stress acclimation.

SUMOylation represses SnRK1 signaling in Arabidopsis.

The SnRK1 protein kinase balances cellular energy levels in accordance with extracellular conditions and is thereby key for plant stress tolerance. In addition, SnRK1 has been implicated in numerous growth and developmental processes from seed filling and maturation to flowering and senescence. Despite its importance, the mechanisms that regulate SnRK1 activity are poorly understood. Here, we demonstrate that the SnRK1 complex is SUMOylated on multiple subunits and identify SIZ1 as the E3 Small Ubiquitin-like Modifier (SUMO) ligase responsible for this modification. We further show that SnRK1 is ubiquitinated in a SIZ1-dependent manner, causing its degradation through the proteasome. In consequence, SnRK1 degradation is deficient in siz1-2 mutants, leading to its accumulation and hyperactivation of SnRK1 signaling. Finally, SnRK1 degradation is strictly dependent on its activity, as inactive SnRK1 variants are aberrantly stable but recover normal degradation when expressed as SUMO mimetics. Altogether, our data suggest that active SnRK1 triggers its own SUMOylation and degradation, establishing a negative feedback loop that attenuates SnRK1 signaling and prevents detrimental hyperactivation of stress responses.