high-throughput-image-based-phenotyping-for-determining-morphological

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 ...

University of Bonn

The translocon on the outer membrane of mitochondria (TOM) facilitates the import of nuclear-encoded proteins. The principal machinery of mitochondrial protein transport seems conserved in eukaryotes; however, divergence in the composition and structure of TOM components has been observed between mammals, yeast, and plants. TOM9, the plant homolog of yeast Tom22, is significantly smaller due to a truncation in the cytosolic receptor domain, and its precise function is not understood. Here we provide evidence showing that TOM9.2 from Arabidopsis thaliana is involved in the formation of mature TOM complex, most likely by influencing the assembly of the pore-forming subunit TOM40. Dexamethasone-induced RNAi gene silencing of TOM9.2 results in a severe reduction in the mature TOM complex, and the assembly of newly imported TOM40 into the complex is impaired. Nevertheless, mutant plants are fully viable and no obvious downstream effects of the loss of TOM complex, i.e., on mitochondrial import capacity, were observed. Furthermore, we found that TOM9.2 can bind calmodulin (CaM) in vitro and that CaM impairs the assembly of TOM complex in the isolated wild-type mitochondria, suggesting a regulatory role of TOM9.2 and a possible integration of TOM assembly into the cellular calcium signaling network.

TOM9.2 Is a Calmodulin-Binding Protein Critical for TOM Complex Assembly but Not for Mitochondrial Protein Import in Arabidopsis thaliana.

Chloroplasts require a fine-tuned control of their internal Ca concentration, which is crucial for many aspects of photosynthesis and for other chloroplast-localized processes. Increasing evidence suggests that calcium regulation within chloroplasts also may influence Ca signaling pathways in the cytosol. To investigate the involvement of thylakoids in Ca homeostasis and in the modulation of chloroplast Ca signals in vivo, we targeted the bioluminescent Ca reporter aequorin as a YFP fusion to the lumen and the stromal surface of thylakoids in Arabidopsis (). Thylakoid localization of aequorin-based probes in stably transformed lines was confirmed by confocal microscopy, immunogold labeling, and biochemical analyses. In resting conditions in the dark, free Ca levels in the thylakoid lumen were maintained at about 0.5 μm, which was a 3- to 5-fold higher concentration than in the stroma. Monitoring of chloroplast Ca dynamics in different intrachloroplast subcompartments (stroma, thylakoid membrane, and thylakoid lumen) revealed the occurrence of stimulus-specific Ca signals, characterized by unique kinetic parameters. Oxidative and salt stresses initiated pronounced free Ca changes in the thylakoid lumen. Localized Ca increases also were observed on the thylakoid membrane surface, mirroring transient Ca changes observed for the bulk stroma, but with specific Ca dynamics. Moreover, evidence was obtained for dark-stimulated intrathylakoid Ca changes, suggesting a new scenario for light-to-dark-induced Ca fluxes inside chloroplasts. Hence, thylakoid-targeted aequorin reporters can provide new insights into chloroplast Ca storage and signal transduction. These probes represent novel tools with which to investigate the role of thylakoids in Ca signaling networks within chloroplasts and plant cells.

Chloroplast Ca Fluxes into and across Thylakoids Revealed by Thylakoid-Targeted Aequorin Probes.

Ca is a potent signalling molecule that regulates many cellular processes. In cyanobacteria, Ca has been linked to cell growth, stress response and photosynthesis, and to the development of specialist heterocyst cells in certain nitrogen-fixing species. Despite this, the pathways of Ca signal transduction in cyanobacteria are poorly understood, and very few protein components are known. The current study describes a previously unreported Ca-binding protein which was called the Ca Sensor EF-hand (CSE), which is conserved in filamentous, nitrogen-fixing cyanobacteria. CSE is shown to bind Ca, which induces a conformational change in the protein structure. Poor growth of a strain of Anabaena sp. PCC 7120 overexpressing CSE was attributed to diminished photosynthetic performance. Transcriptomics, biophysics and proteomics analyses revealed modifications in the light-harvesting phycobilisome and photosynthetic reaction centre protein complexes.

A novel Ca-binding protein influences photosynthetic electron transport in Anabaena sp. PCC 7120.

Chloroplasts are integral to sensing biotic and abiotic stress in plants, but their role in transducing Ca-mediated stress signals remains poorly understood. Here we identify cMCU, a member of the mitochondrial calcium uniporter (MCU) family, as an ion channel mediating Ca flux into chloroplasts in vivo. Using a toolkit of aequorin reporters targeted to chloroplast stroma and the cytosol in cMCU wild-type and knockout lines, we provide evidence that stress-stimulus-specific Ca dynamics in the chloroplast stroma correlate with expression of the channel. Fast downstream signalling events triggered by osmotic stress, involving activation of the mitogen-activated protein kinases (MAPK) MAPK3 and MAPK6, and the transcription factors MYB60 and ethylene-response factor 6 (ERF6), are influenced by cMCU activity. Relative to wild-type plants, cMCU knockouts display increased resistance to long-term water deficit and improved recovery on rewatering. Modulation of stromal Ca in specific processing of stress signals identifies cMCU as a component of plant environmental sensing.

A chloroplast-localized mitochondrial calcium uniporter transduces osmotic stress in Arabidopsis.

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 , CAS is also phosphorylated by the state transition kinase 7 (STN7), as well as by another, so-far unknown, Ca-dependent kinase. Phosphoproteomics analysis and  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  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  mutant this suggests a role for CAS in the STN7/STN8/TAP38 network of phosphorylation-mediated photoacclimation processes in Arabidopsis.

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

Mitochondria are crucial bioenergetic organelles for providing different metabolites, including ATP, to sustain cell growth both in animals and in plants. These organelles, delimited by two membranes (outer and inner mitochondrial membrane), maintain their function by an intensive communication with other organelles as well as with the cytosol. Transport of metabolites across the two membranes, but also that of inorganic ions, takes place through specific ion channels and transporters and plays a crucial role in ensuring an adequate ionic milieu within the mitochondria. In the present review we briefly summarize the current knowledge about plant mitochondrial ion channels and transporters in comparison to those of animal mitochondria and examine the possible molecular identity of the so far unidentified transport systems taking into account subcellular targeting predictions and data from literature.

Channels and transporters for inorganic ions in plant mitochondria: Prediction and facts.

Calcium ion (Ca) is a versatile signaling transducer in all eukaryotic organisms. In plants, intracellular changes in free Ca levels act as regulators in many growth and developmental processes. Ca also mediates the cellular responses to environmental stimuli and thus plays an important role in providing stress tolerance to plants. Ca signals are decoded by a tool kit of various families of Ca-binding proteins and their downstream targets, which mediate the transformation of the Ca signal into appropriate cellular response. Early interest and research on Ca signaling focused on its function in the cytosol, however it has become evident that this important regulatory pathway also exists in organelles such as nucleus, chloroplast, mitochondria, peroxisomes and the endomembrane system. In this review, we give an overview on the knowledge about organellar Ca signaling with a focus on recent advances and developments.

Organellar calcium signaling in plants: An update.

Metabolic fluctuations in chloroplasts and mitochondria can trigger retrograde signals to modify nuclear gene expression. Mobile signals likely to be involved are reactive oxygen species (ROS), which can operate protein redox switches by oxidation of specific cysteine residues. Redox buffers, such as the highly reduced glutathione pool, serve as reservoirs of reducing power for several ROS-scavenging and ROS-induced damage repair pathways. Formation of glutathione disulfide and a shift of the glutathione redox potential (EGSH) toward less negative values is considered as hallmark of several stress conditions. Here we used the herbicide methyl viologen (MV) to generate ROS locally in chloroplasts of intact Arabidopsis (Arabidopsis thaliana) seedlings and recorded dynamic changes in EGSH and H2O2 levels with the genetically encoded biosensors Grx1-roGFP2 (for EGSH) and roGFP2-Orp1 (for H2O2) targeted to chloroplasts, the cytosol, or mitochondria. Treatment of seedlings with MV caused rapid oxidation in chloroplasts and, subsequently, in the cytosol and mitochondria. MV-induced oxidation was significantly boosted by illumination with actinic light, and largely abolished by inhibitors of photosynthetic electron transport. MV also induced autonomous oxidation in the mitochondrial matrix in an electron transport chain activity-dependent manner that was milder than the oxidation triggered in chloroplasts by the combination of MV and light. In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provides a basis for understanding how compartment-specific redox dynamics might operate in retrograde signaling and stress acclimation in plants.

Chloroplast-derived photo-oxidative stress causes changes in H2O2 and EGSH in other subcellular compartments.

Precise measurements of dynamic changes in free Ca concentration in the lumen of the plant endoplasmic reticulum (ER) have been lacking so far, despite increasing evidence for the contribution of this intracellular compartment to Ca homeostasis and signalling in the plant cell. In the present study, we targeted an aequorin chimera with reduced Ca affinity to the ER membrane and facing the ER lumen. To this aim, the cDNA for a low-Ca -affinity aequorin variant (AEQmut) was fused to the nucleotide sequence encoding a non-cleavable N-terminal ER signal peptide (fl2). The correct targeting of fl2-AEQmut was confirmed by immunocytochemical analyses in transgenic Arabidopsis thaliana (Arabidopsis) seedlings. An experimental protocol well-established in animal cells - consisting of ER Ca depletion during photoprotein reconstitution followed by ER Ca refilling - was applied to carry out ER Ca measurements in planta. Rapid and transient increases of the ER luminal Ca concentration ([Ca ] ) were recorded in response to different environmental stresses, displaying stimulus-specific Ca signatures. The comparative analysis of ER and chloroplast Ca dynamics indicates a complex interplay of these organelles in shaping cytosolic Ca signals during signal transduction events. Our data highlight significant differences in basal [Ca ] and Ca handling by plant ER compared to the animal counterpart. The set-up of an ER-targeted aequorin chimera extends and complements the currently available toolkit of organelle-targeted Ca indicators by adding a reporter that improves our quantitative understanding of Ca homeostasis in the plant endomembrane system.

Monitoring calcium handling by the plant endoplasmic reticulum with a low-Ca -affinity targeted aequorin reporter.

In this study, we investigated Arabidopsis thaliana plants with altered levels of the enzyme JASMONATE RESISTANT 1 (JAR1), which converts jasmonic acid (JA) to jasmonoyl-l-isoleucine (JA-Ile). Analysis of a newly generated overexpression line (35S::JAR1) revealed that constitutively increased JA-Ile production in 35S::JAR1 alters plant development, resulting in stunted growth and delayed flowering. Under drought-stress conditions, 35S::JAR1 plants showed reduced wilting and recovered better from desiccation than the wild type. By contrast, jar1-11 plants with a strong reduction in JA-Ile content were hypersensitive to drought. RNA-sequencing analysis and hormonal profiling of plants under normal and drought conditions provided insights into the molecular reprogramming caused by the alteration in JA-Ile content. Especially 35S::JAR1 plants displayed changes in expression of developmental genes related to growth and flowering. Further transcriptional differences pertained to drought-related adaptive systems, including stomatal density and aperture, but also reactive oxygen species production and detoxification. Analysis of wild type and jar1-11 plants carrying the roGFP-Orp1 sensor support a role of JA-Ile in the alleviation of methyl viologen-induced H O production. Our data substantiate a role of JA-Ile in abiotic stress response and suggest that JAR1-mediated increase in JA-Ile content primes Arabidopsis towards improved drought stress tolerance.

Constitutive expression of JASMONATE RESISTANT 1 induces molecular changes that prime the plants to better withstand drought.

Plants are continuously exposed to changing environmental conditions and biotic attacks that affect plant growth. In crops, the inability to respond appropriately to stress has strong detrimental effects on agricultural production and yield. Ca signalling plays a fundamental role in the response of plants to most abiotic and biotic stresses. However, research on stimulus-specific Ca signals has mostly been pursued in Arabidopsis thaliana, while in other species these events are little investigated .

Comparative analysis of stress-induced calcium signals in the crop species barley and the model plant Arabidopsis thaliana.

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.

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

Chloroplast calcium homeostasis plays an important role in modulating the response of plants to abiotic and biotic stresses. One of the greatest challenges is to understand how chloroplast calcium-permeable pathways and sensors are regulated in a concerted manner to translate specific information into a calcium signature and to elucidate the downstream effects of specific chloroplast calcium dynamics. One of the six homologs of the mitochondrial calcium uniporter (MCU) was found to be located in chloroplasts in the leaves and to crucially contribute to drought- and oxidative stress-triggered uptake of calcium into this organelle.

Comparative analysis of wild-type and chloroplast MCU-deficient plants reveals multiple consequences of chloroplast calcium handling under drought stress.

In cereal crops, such as barley ( L.), the ability to appropriately respond to environmental cues is an important factor for yield stability and thus for agricultural production. Reactive oxygen species (ROS), such as hydrogen peroxide (HO), are key components of signal transduction cascades involved in plant adaptation to changing environmental conditions. HO-mediated stress responses include the modulation of expression of stress-responsive genes required to cope with different abiotic and biotic stresses. Despite its importance, knowledge of the effects of HO on the barley transcriptome is still scarce. In this study, we identified global transcriptomic changes induced after application of 10 mM HO to five-day-old barley plants. In total, 1883 and 1001 differentially expressed genes (DEGs) were identified in roots and leaves, respectively. Most of these DEGs were organ-specific, with only 209 DEGs commonly regulated and 37 counter-regulated between both plant parts. A GO term analysis further confirmed that different processes were affected in roots and leaves. It revealed that DEGs in leaves mostly comprised genes associated with hormone signaling, response to HO and abiotic stresses. This includes many transcriptions factors and small heat shock proteins. DEGs in roots mostly comprised genes linked to crucial aspects of HO catabolism and oxidant detoxification, glutathione metabolism, as well as cell wall modulation. These categories include many peroxidases and glutathione transferases. As with leaves, the HO response category in roots contains small heat shock proteins, however, mostly different members of this family were affected and they were all regulated in the opposite direction in the two plant parts. Validation of the expression of the selected commonly regulated DEGs by qRT-PCR was consistent with the RNA-seq data. The data obtained in this study provide an insight into the molecular mechanisms of oxidative stress responses in barley, which might also play a role upon other stresses that induce oxidative bursts.

Global transcriptome profiling reveals root- and leaf-specific responses of barley ( L.) to HO.

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.

Article	Year
TOM9.2 Is a Calmodulin-Binding Protein Critical for TOM Complex Assembly but Not for Mitochondrial Protein Import in Arabidopsis thaliana. Molecular plant\| PubMed ID: 28062321	2017
Chloroplast Ca Fluxes into and across Thylakoids Revealed by Thylakoid-Targeted Aequorin Probes. Plant physiology\| PubMed ID: 29559589	2018
A novel Ca-binding protein influences photosynthetic electron transport in Anabaena sp. PCC 7120. Biochimica et biophysica acta. Bioenergetics\| PubMed ID: 31034800	2019
A chloroplast-localized mitochondrial calcium uniporter transduces osmotic stress in Arabidopsis. Nature plants\| PubMed ID: 31182842	2019
The High Light Response in Arabidopsis Requires the Calcium Sensor Protein CAS, a Target of STN7- and STN8-Mediated Phosphorylation. Frontiers in plant science\| PubMed ID: 31417591	2019
Channels and transporters for inorganic ions in plant mitochondria: Prediction and facts. Mitochondrion\| PubMed ID: 32540403	2020
Organellar calcium signaling in plants: An update. Biochimica et biophysica acta. Molecular cell research\| PubMed ID: 33421535	2021
Chloroplast-derived photo-oxidative stress causes changes in H2O2 and EGSH in other subcellular compartments. Plant physiology\| PubMed ID: 33793922	2021
Monitoring calcium handling by the plant endoplasmic reticulum with a low-Ca -affinity targeted aequorin reporter. The Plant journal : for cell and molecular biology\| PubMed ID: 34837294	2022
Constitutive expression of JASMONATE RESISTANT 1 induces molecular changes that prime the plants to better withstand drought. Plant, cell & environment\| PubMed ID: 35864601	2022
Comparative analysis of stress-induced calcium signals in the crop species barley and the model plant Arabidopsis thaliana. BMC plant biology\| PubMed ID: 36114461	2022
Organelles and phytohormones: a network of interactions in plant stress responses. Journal of experimental botany\| PubMed ID: 36169618	2022
Comparative analysis of wild-type and chloroplast MCU-deficient plants reveals multiple consequences of chloroplast calcium handling under drought stress. Frontiers in plant science\| PubMed ID: 37692417	2023
Global transcriptome profiling reveals root- and leaf-specific responses of barley ( L.) to HO. Frontiers in plant science\| PubMed ID: 37771486	2023
Stress Knowledge Map: A knowledge graph resource for systems biology analysis of plant stress responses. Plant communications\| PubMed ID: 38616489	2024

Ute C. Vothknecht

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