RoPod, a customizable toolkit for non-invasive root imaging, reveals cell type-specific dynamics of plant autophagy.

Arabidopsis root is a classic model system in plant cell and molecular biology. The sensitivity of plant roots to local environmental perturbation challenges data reproducibility and incentivizes further optimization of imaging and phenotyping tools. Here we present RoPod, an easy-to-use toolkit for low-stress live time-lapse imaging of Arabidopsis roots. RoPod comprises a dedicated protocol for plant cultivation and a customizable 3D-printed vessel with integrated microscopy-grade glass that serves simultaneously as a growth and imaging chamber. RoPod reduces impact of sample handling, preserves live samples for prolonged imaging sessions, and facilitates application of treatments during image acquisition. We describe a protocol for RoPods fabrication and provide illustrative application pipelines for monitoring root hair growth and autophagic activity. Furthermore, we showcase how the use of RoPods advanced our understanding of plant autophagy, a major catabolic pathway and a key player in plant fitness. Specifically, we obtained fine time resolution for autophagy response to commonly used chemical modulators of the pathway and revealed previously overlooked cell type-specific changes in the autophagy response. These results will aid a deeper understanding of the physiological role of autophagy and provide valuable guidelines for choosing sampling time during end-point assays currently employed in plant autophagy research.

Thermoprotection by a cell membrane-localized metacaspase in a green alga.

Caspases are restricted to animals, while other organisms, including plants, possess metacaspases (MCAs), a more ancient and broader class of structurally related yet biochemically distinct proteases. Our current understanding of plant MCAs is derived from studies in streptophytes, and mostly in Arabidopsis (Arabidopsis thaliana) with 9 MCAs with partially redundant activities. In contrast to streptophytes, most chlorophytes contain only 1 or 2 uncharacterized MCAs, providing an excellent platform for MCA research. Here we investigated CrMCA-II, the single type-II MCA from the model chlorophyte Chlamydomonas (Chlamydomonas reinhardtii). Surprisingly, unlike other studied MCAs and similar to caspases, CrMCA-II dimerizes both in vitro and in vivo. Furthermore, activation of CrMCA-II in vivo correlated with its dimerization. Most of CrMCA-II in the cell was present as a proenzyme (zymogen) attached to the plasma membrane (PM). Deletion of CrMCA-II by genome editing compromised thermotolerance, leading to increased cell death under heat stress. Adding back either wild-type or catalytically dead CrMCA-II restored thermoprotection, suggesting that its proteolytic activity is dispensable for this effect. Finally, we connected the non-proteolytic role of CrMCA-II in thermotolerance to the ability to modulate PM fluidity. Our study reveals an ancient, MCA-dependent thermotolerance mechanism retained by Chlamydomonas and probably lost during the evolution of multicellularity.

SPIRO - the automated Petri plate imaging platform designed by biologists, for biologists.

Phenotyping of model organisms grown on Petri plates is often carried out manually, despite the procedures being time-consuming and laborious. The main reason for this is the limited availability of automated phenotyping facilities, whereas constructing a custom automated solution can be a daunting task for biologists. Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photographs of up to four vertically oriented Petri plates in a single experiment, corresponding to 192 seedlings for a typical root growth assay and up to 2500 seeds for a germination assay. SPIRO is catered specifically to biologists' needs, requiring no engineering or programming expertise for assembly and operation. Its small footprint is optimized for standard incubators, the inbuilt green LED enables imaging under dark conditions, and remote control provides access to the data without interfering with sample growth. SPIRO's excellent image quality is suitable for automated image processing, which we demonstrate on the example of seed germination and root growth assays. Furthermore, the robot can be easily customized for specific uses, as all information about SPIRO is released under open-source licenses. Importantly, uninterrupted imaging allows considerably more precise assessment of seed germination parameters and root growth rates compared with manual assays. Moreover, SPIRO enables previously technically challenging assays such as phenotyping in the dark. We illustrate the benefits of SPIRO in proof-of-concept experiments which yielded a novel insight on the interplay between autophagy, nitrogen sensing, and photoblastic response.

Interactome of Arabidopsis ATG5 Suggests Functions beyond Autophagy.

Autophagy is a catabolic pathway capable of degrading cellular components ranging from individual molecules to organelles. Autophagy helps cells cope with stress by removing superfluous or hazardous material. In a previous work, we demonstrated that transcriptional upregulation of two autophagy-related genes, ATG5 and ATG7, in Arabidopsis thaliana positively affected agronomically important traits: biomass, seed yield, tolerance to pathogens and oxidative stress. Although the occurrence of these traits correlated with enhanced autophagic activity, it is possible that autophagy-independent roles of ATG5 and ATG7 also contributed to the phenotypes. In this study, we employed affinity purification and LC-MS/MS to identify the interactome of wild-type ATG5 and its autophagy-inactive substitution mutant, ATG5K128R Here we present the first interactome of plant ATG5, encompassing not only known autophagy regulators but also stress-response factors, components of the ubiquitin-proteasome system, proteins involved in endomembrane trafficking, and potential partners of the nuclear fraction of ATG5. Furthermore, we discovered post-translational modifications, such as phosphorylation and acetylation present on ATG5 complex components that are likely to play regulatory functions. These results strongly indicate that plant ATG5 complex proteins have roles beyond autophagy itself, opening avenues for further investigations on the complex roles of autophagy in plant growth and stress responses.

Abscisic acid signaling activates distinct VND transcription factors to promote xylem differentiation in Arabidopsis.

Plants display remarkable abilities to adjust growth and development to environmental conditions, such as the amount of available water. This developmental plasticity is apparent not only in root and shoot growth rates, but also in tissue patterning and cell morphology.1,2 We have previously shown that in response to limited water availability, Arabidopsis thaliana root displays changes in xylem morphology, mediated by the non-cell-autonomous action of abscisic acid, ABA.2 Here, we show, through analyses of ABA response reporters and tissue-specific suppression of ABA signaling, that xylem cells themselves act as primary signaling centers governing both xylem cell fate and xylem differentiation rate, revealing the cell-autonomous control of multiple aspects of xylem development by ABA. ABA rapidly activates the expression of genes encoding VASCULAR-RELATED NAC DOMAIN (VND) transcription factors. Molecular and genetic analyses revealed that the two ABA-mediated xylem developmental changes are regulated by distinct members of this transcription factor family, with VND2 and VND3 promoting differentiation rate of metaxylem cells, while VND7 promotes the conversion of metaxylem toward protoxylem morphology. This phenomenon shows how different aspects of developmental plasticity can be interlinked, yet genetically separable. Moreover, similarities in phenotypic and molecular responses to ABA in diverse species indicate evolutionary conservation of the ABA-xylem development regulatory network among eudicots. Hence, this study gives molecular insights into how environmental stress modifies plant vascular anatomy and has potential relevance for water use optimization and adaptation to drought conditions.

Apoptosis is not conserved in plants as revealed by critical examination of a model for plant apoptosis-like cell death.

BACKGROUND: Animals and plants diverged over one billion years ago and evolved unique mechanisms for many cellular processes, including cell death. One of the most well-studied cell death programmes in animals, apoptosis, involves gradual cell dismantling and engulfment of cellular fragments, apoptotic bodies, through phagocytosis. However, rigid cell walls prevent plant cell fragmentation and thus apoptosis is not applicable for executing cell death in plants. Furthermore, plants are devoid of the key components of apoptotic machinery, including phagocytosis as well as caspases and Bcl-2 family proteins. Nevertheless, the concept of plant "apoptosis-like programmed cell death" (AL-PCD) is widespread. This is largely due to superficial morphological resemblances between plant cell death and apoptosis, and in particular between protoplast shrinkage in plant cells killed by various stimuli and animal cell volume decrease preceding fragmentation into apoptotic bodies. RESULTS: Here, we provide a comprehensive spatio-temporal analysis of cytological and biochemical events occurring in plant cells subjected to heat shock at 40-55 °C and 85 °C, the experimental conditions typically used to trigger AL-PCD and necrotic cell death, respectively. We show that cell death under both conditions was not accompanied by membrane blebbing or formation of apoptotic bodies, as would be expected during apoptosis. Instead, we observed instant and irreversible permeabilization of the plasma membrane and ATP depletion. These processes did not depend on mitochondrial functionality or the presence of Ca2+ and could not be prevented by an inhibitor of ferroptosis. We further reveal that the lack of protoplast shrinkage at 85 °C, the only striking morphological difference between cell deaths induced by 40-55 °C or 85 °C heat shock, is a consequence of the fixative effect of the high temperature on intracellular contents. CONCLUSIONS: We conclude that heat shock-induced cell death is an energy-independent process best matching definition of necrosis. Although the initial steps of this necrotic cell death could be genetically regulated, classifying it as apoptosis or AL-PCD is a terminological misnomer. Our work supports the viewpoint that apoptosis is not conserved across animal and plant kingdoms and demonstrates the importance of focusing on plant-specific aspects of cell death pathways.

Subcellular Localization of Acyl-CoA: Lysophosphatidylethanolamine Acyltransferases (LPEATs) and the Effects of Knocking-Out and Overexpression of Their Genes on Autophagy Markers Level and Life Span of A. thaliana.

Arabidopsis thaliana possesses two acyl-CoA:lysophosphatidylethanolamine acyltransferases, LPEAT1 and LPEAT2, which are encoded by At1g80950 and At2g45670 genes, respectively. Both single lpeat2 mutant and double lpeat1 lpeat2 mutant plants exhibit a variety of conspicuous phenotypes, including dwarfed growth. Confocal microscopic analysis of tobacco suspension-cultured cells transiently transformed with green fluorescent protein-tagged versions of LPEAT1 or LPEAT2 revealed that LPEAT1 is localized to the endoplasmic reticulum (ER), whereas LPEAT2 is localized to both Golgi and late endosomes. Considering that the primary product of the reaction catalyzed by LPEATs is phosphatidylethanolamine, which is known to be covalently conjugated with autophagy-related protein ATG8 during a key step of the formation of autophagosomes, we investigated the requirements for LPEATs to engage in autophagic activity in Arabidopsis. Knocking out of either or both LPEAT genes led to enhanced accumulation of the autophagic adaptor protein NBR1 and decreased levels of both ATG8a mRNA and total ATG8 protein. Moreover, we detected significantly fewer membrane objects in the vacuoles of lpeat1 lpeat2 double mutant mesophyll cells than in vacuoles of control plants. However, contrary to what has been reported on autophagy deficient plants, the lpeat mutants displayed a prolonged life span compared to wild type, including delayed senescence.

Suppression of Metacaspase- and Autophagy-Dependent Cell Death Improves Stress-Induced Microspore Embryogenesis in Brassica napus.

Microspore embryogenesis is a biotechnological process that allows us to rapidly obtain doubled-haploid plants for breeding programs. The process is initiated by the application of stress treatment, which reprograms microspores to embark on embryonic development. Typically, a part of the microspores undergoes cell death that reduces the efficiency of the process. Metacaspases (MCAs), a phylogenetically broad group of cysteine proteases, and autophagy, the major catabolic process in eukaryotes, are critical regulators of the balance between cell death and survival in various organisms. In this study, we analyzed the role of MCAs and autophagy in cell death during stress-induced microspore embryogenesis in Brassica napus. We demonstrate that this cell death is accompanied by the transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), an increase in MCA proteolytic activity and the activation of autophagy. Accordingly, inhibition of autophagy and MCA activity, either individually or in combination, suppressed cell death and increased the number of proembryos, indicating that both components play a pro-cell death role and account for decreased efficiency of early embryonic development. Therefore, MCAs and/or autophagy can be used as new biotechnological targets to improve in vitro embryogenesis in Brassica species and doubled-haploid plant production in crop breeding and propagation programs.

Tandem Tag Assay Optimized for Semi-automated in vivo Autophagic Activity Measurement in Arabidopsis thaliana roots.

Autophagy is the main catabolic process in eukaryotes and plays a key role in cell homeostasis. In vivo measurement of autophagic activity (flux) is a powerful tool for investigating the role of the pathway in organism development and stress responses. Here we describe a significant optimization of the tandem tag assay for detection of autophagic flux in planta in epidermal root cells of Arabidopsis thaliana seedlings. The tandem tag consists of TagRFP and mWasabi fluorescent proteins fused to ATG8a, and is expressed in wildtype or autophagy-deficient backgrounds to obtain reporter and control lines, respectively. Upon autophagy activation, the TagRFP-mWasabi-ATG8a fusion protein is incorporated into autophagosomes and delivered to the lytic vacuole. Ratiometric quantification of the low pH-tolerant TagRFP and low pH-sensitive mWasabi fluorescence in the vacuoles of control and reporter lines allows for a reliable estimation of autophagic activity. We provide a step by step protocol for plant growth, imaging and semi-automated data analysis. The protocol presents a rapid and robust method that can be applied for any studies requiring in planta quantification of autophagic flux.

Remove, Recycle, Degrade: Regulating Plasma Membrane Protein Accumulation.

Interactions between plant cells and the environment rely on modulation of protein receptors, transporters, channels, and lipids at the plasma membrane (PM) to facilitate intercellular communication, nutrient uptake, environmental sensing, and directional growth. These functions are fine-tuned by cellular pathways maintaining or reducing particular proteins at the PM. Proteins are endocytosed, and their fate is decided between recycling and degradation to modulate localization, abundance, and activity. Selective autophagy is another pathway regulating PM protein accumulation in response to specific conditions or developmental signals. The mechanisms regulating recycling, degradation, and autophagy have been studied extensively, yet we are just now addressing their regulation and coordination. Here, we (1) provide context concerning regulation of protein accumulation, recycling, or degradation by overviewing endomembrane trafficking; (2) discuss pathways regulating recycling and degradation in terms of cellular roles and cargoes; (3) review plant selective autophagy and its physiological significance; (4) focus on two decision-making mechanisms: regulation of recycling versus degradation of PM proteins and coordination between autophagy and vacuolar degradation; and (5) identify future challenges.

Chemical Screening Pipeline for Identification of Specific Plant Autophagy Modulators.

Autophagy is a major catabolic process in eukaryotes with a key role in homeostasis, programmed cell death, and aging. In plants, autophagy is also known to regulate agronomically important traits such as stress resistance, longevity, vegetative biomass, and seed yield. Despite its significance, there is still a shortage of reliable tools modulating plant autophagy. Here, we describe the first robust pipeline for identification of specific plant autophagy-modulating compounds. Our screening protocol comprises four phases: (1) high-throughput screening of chemical compounds in cell cultures of tobacco (Nicotiana tabacum); (2) confirmation of the identified hits in planta using Arabidopsis (Arabidopsis thaliana); (3) further characterization of the effect using conventional molecular biology methods; and (4) verification of chemical specificity on autophagy in planta. The methods detailed here streamline the identification of specific plant autophagy modulators and aid in unraveling the molecular mechanisms of plant autophagy.

Transcriptome analysis of embryonic domains in Norway spruce reveals potential regulators of suspensor cell death.

The terminal differentiation and elimination of the embryo-suspensor is the earliest manifestation of programmed cell death (PCD) during plant ontogenesis. Molecular regulation of suspensor PCD remains poorly understood. Norway spruce (Picea abies) embryos provide a powerful model for studying embryo development because of their large size, sequenced genome, and the possibility to obtain a large number of embryos at a specific developmental stage through somatic embryogenesis. Here, we have carried out global gene expression analysis of the Norway spruce embryo-suspensor versus embryonal mass (a gymnosperm analogue of embryo proper) using RNA sequencing. We have identified that suspensors have enhanced expression of the NAC domain-containing transcription factors, XND1 and ANAC075, previously shown to be involved in the initiation of developmental PCD in Arabidiopsis. The analysis has also revealed enhanced expression of Norway spruce homologues of the known executioners of both developmental and stress-induced cell deaths, such as metacaspase 9 (MC9), cysteine endopeptidase-1 (CEP1) and ribonuclease 3 (RNS3). Interestingly, a spruce homologue of bax inhibitor-1 (PaBI-1, for Picea abies BI-1), an evolutionarily conserved cell death suppressor, was likewise up-regulated in the embryo-suspensor. Since Arabidopsis BI-1 so far has been implicated only in the endoplasmic reticulum (ER)-stress induced cell death, we investigated its role in embryogenesis and suspensor PCD using RNA interference (RNAi). We have found that PaBI-1-deficient lines formed a large number of abnormal embryos with suppressed suspensor elongation and disturbed polarity. Cytochemical staining of suspensor cells has revealed that PaBI-1 deficiency suppresses vacuolar cell death and induces necrotic type of cell death previously shown to compromise embryo development. This study demonstrates that a large number of cell-death components are conserved between angiosperms and gymnosperms and establishes a new role for BI-1 in the progression of vacuolar cell death.

Bacteria Exploit Autophagy for Proteasome Degradation and Enhanced Virulence in Plants.

Autophagy and the ubiquitin-proteasome system (UPS) are two major protein degradation pathways implicated in the response to microbial infections in eukaryotes. In animals, the contribution of autophagy and the UPS to antibacterial immunity is well documented and several bacteria have evolved measures to target and exploit these systems to the benefit of infection. In plants, the UPS has been established as a hub for immune responses and is targeted by bacteria to enhance virulence. However, the role of autophagy during plant-bacterial interactions is less understood. Here, we have identified both pro- and antibacterial functions of autophagy mechanisms upon infection of Arabidopsis thaliana with virulent Pseudomonas syringae pv tomato DC3000 (Pst). We show that Pst activates autophagy in a type III effector (T3E)-dependent manner and stimulates the autophagic removal of proteasomes (proteaphagy) to support bacterial proliferation. We further identify the T3E Hrp outer protein M1 (HopM1) as a principle mediator of autophagy-inducing activities during infection. In contrast to the probacterial effects of Pst-induced proteaphagy, NEIGHBOR OF BRCA1-dependent selective autophagy counteracts disease progression and limits the formation of HopM1-mediated water-soaked lesions. Together, we demonstrate that distinct autophagy pathways contribute to host immunity and bacterial pathogenesis during Pst infection and provide evidence for an intimate crosstalk between proteasome and autophagy in plant-bacterial interactions.

Autophagy-related approaches for improving nutrient use efficiency and crop yield protection.

Autophagy is a eukaryotic catabolic pathway essential for growth and development. In plants, it is activated in response to environmental cues or developmental stimuli. However, in contrast to other eukaryotic systems, we know relatively little regarding the molecular players involved in autophagy and the regulation of this complex pathway. In the framework of the COST (European Cooperation in Science and Technology) action TRANSAUTOPHAGY (2016-2020), we decided to review our current knowledge of autophagy responses in higher plants, with emphasis on knowledge gaps. We also assess here the potential of translating the acquired knowledge to improve crop plant growth and development in a context of growing social and environmental challenges for agriculture in the near future.

Autophagy in turnover of lipid stores: trans-kingdom comparison.

Lipids and their cellular utilization are essential for life. Not only are lipids energy storage molecules, but their diverse structural and physical properties underlie various aspects of eukaryotic biology, such as membrane structure, signalling, and trafficking. In the ever-changing environment of cells, lipids, like other cellular components, are regularly recycled to uphold the housekeeping processes required for cell survival and organism longevity. The ways in which lipids are recycled, however, vary between different phyla. For example, animals and plants have evolved distinct lipid degradation pathways. The major cell recycling system, autophagy, has been shown to be instrumental for both differentiation of specialized fat storing-cells, adipocytes, and fat degradation in animals. Does plant autophagy play a similar role in storage and degradation of lipids? In this review, we discuss and compare implications of bulk autophagy and its selective route, lipophagy, in the turnover of lipid stores in animals, fungi, and plants.

Transcriptional stimulation of rate-limiting components of the autophagic pathway improves plant fitness.

Autophagy is a major catabolic process whereby autophagosomes deliver cytoplasmic content to the lytic compartment for recycling. Autophagosome formation requires two ubiquitin-like systems conjugating Atg12 with Atg5, and Atg8 with lipid phosphatidylethanolamine (PE), respectively. Genetic suppression of these systems causes autophagy-deficient phenotypes with reduced fitness and longevity. We show that Atg5 and the E1-like enzyme, Atg7, are rate-limiting components of Atg8-PE conjugation in Arabidopsis. Overexpression of ATG5 or ATG7 stimulates Atg8 lipidation, autophagosome formation, and autophagic flux. It also induces transcriptional changes opposite to those observed in atg5 and atg7 mutants, favoring stress resistance and growth. As a result, ATG5- or ATG7-overexpressing plants exhibit increased resistance to necrotrophic pathogens and oxidative stress, delayed aging and enhanced growth, seed set, and seed oil content. This work provides an experimental paradigm and mechanistic insight into genetic stimulation of autophagy in planta and shows its efficiency for improving plant productivity.

Limited and digestive proteolysis: crosstalk between evolutionary conserved pathways.

Contents 958 I. 958 II. 959 III. 960 IV. 962 V. 962 962 References 963 SUMMARY: Proteases can either digest target proteins or perform the so-called 'limited proteolysis' by cleaving polypeptide chains at specific site(s). Autophagy and the ubiquitin-proteasome system (UPS) are two main mechanisms carrying out digestive proteolysis. While the net outcome of digestive proteolysis is the loss of function of protein substrates, limited proteolysis can additionally lead to gain or switch of function. Recent evidence of crosstalk between autophagy, UPS and limited proteolysis indicates that these pathways are parts of the same proteolytic nexus. Here, we focus on three emerging themes within this area: limited proteolysis as a mechanism modulating autophagy; interplay between autophagy and UPS, including autophagic degradation of proteasomes (proteophagy); and specificity of protein degradation during bulk autophagy.