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.

The role of Atg16 in autophagy, anthocyanin biosynthesis, and programmed cell death in leaves of the lace plant (Aponogeton madagascariensis).

Aponogeton madagascariensis, commonly known as the lace plant, produces leaves that form perforations by programmed cell death (PCD). Leaf development is divided into several stages beginning with "pre-perforation" furled leaves enriched with red pigmentation from anthocyanins. The leaf blade is characterized by a series of grids known as areoles bounded by veins. As leaves develop into the "window stage", anthocyanins recede from the center of the areole towards the vasculature creating a gradient of pigmentation and cell death. Cells in the middle of the areole that lack anthocyanins undergo PCD (PCD cells), while cells that retain anthocyanins (non-PCD cells) maintain homeostasis and persist in the mature leaf. Autophagy has reported roles in survival or PCD promotion across different plant cell types. However, the direct involvement of autophagy in PCD and anthocyanin levels during lace plant leaf development has not been determined. Previous RNA sequencing analysis revealed the upregulation of autophagy-related gene Atg16 transcripts in pre-perforation and window stage leaves, but how Atg16 affects PCD in lace plant leaf development is unknown. In this study, we investigated the levels of Atg16 in lace plant PCD by treating whole plants with either an autophagy promoter rapamycin or inhibitors concanamycin A (ConA) or wortmannin. Following treatments, window and mature stage leaves were harvested and analyzed using microscopy, spectrophotometry, and western blotting. Western blotting showed significantly higher Atg16 levels in rapamycin-treated window leaves, coupled with lower anthocyanin levels. Wortmannin-treated leaves had significantly lower Atg16 protein and higher anthocyanin levels compared to the control. Mature leaves from rapamycin-treated plants generated significantly fewer perforations compared to control, while wortmannin had the opposite effect. However, ConA treatment did not significantly change Atg16 levels, nor the number of perforations compared to the control, but anthocyanin levels did increase significantly in window leaves. We propose autophagy plays a dual role in promoting cell survival in NPCD cells by maintaining optimal anthocyanin levels and mediating a timely cell death in PCD cells in developing lace plant leaves. How autophagy specifically affects anthocyanin levels remained unexplained.

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.

The role of auxin in developmentally regulated programmed cell death in lace plant.

PREMISE: Lace plant (Aponogeton madagascariensis) leaves are remodeled via developmental programmed cell death (PCD) to produce perforations located equidistantly between longitudinal and transverse veins. Auxin has been implicated in other developmental PCD processes in plants; however, the role of auxin in perforation formation in lace plant is unknown. Here the role of auxin in developmental PCD in lace plant was studied using two auxin inhibitors N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, and auxinole, a potent auxin antagonist. METHODS: Sterile cultures of lace plants were propagated and treated with NPA or auxinole. Leaf length, leaf width, and number of perforations were then analyzed. Vein patterning and perforation area were further examined in NPA-treated plants. Downstream PCD transduction events were investigated via spectrophotometric assays, histochemical staining, and immuno-probing. RESULTS: Lace plants treated with NPA or auxinole produced leaves with fewer perforations compared to their respective controls. Although NPA treatment was insufficient to completely alter vein patterning, NPA-treated leaves did have significantly more atypical areoles compared to control leaves. Events involved in perforation formation in lace plant leaves were altered following treatment with NPA, including anthocyanin production, reactive oxygen species (ROS) accumulation, and the release of mitochondrial cytochrome c. CONCLUSIONS: Our results indicated that inhibition of auxin signaling disrupts several downstream features of the lace plant PCD signaling cascade and results in fewer or no perforations. Therefore, we concluded that auxin signaling is important for developmentally regulated PCD in lace plant leaves.

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.

Determining the effect of calcium on cell death rate and perforation formation during leaf development in the novel model system, the lace plant (Aponogeton madagascariensis).

Programmed cell death (PCD) is the destruction of unwanted cells through an intracellularly mediated process. Perforation formation in the lace plant (Aponogeton madagascariensis) provides an excellent model for studying developmentally regulated PCD. Ca2+ fluxes have previously been identified as important signals for PCD in plants and mammals. The fundamental goal of this project was to determine the influence of Ca2+ on the rate of cell death and perforation formation during leaf development in the lace plant. This was investigated using the application of various known calcium modulators including lanthanum III chloride (LaCl3 ), ruthenium red and calcium ionophore A23187. Detached lace plant leaves at an early stage of development were treated with these modulators in both short- and long-term exposure assays and analysed using live cell imaging. Results from this study indicate that calcium plays a vital role in developmentally regulated PCD in the lace plant as application of the modulators significantly altered the rate of cell death and perforation formation during leaf development. In conclusion, this study exemplifies the suitability of the lace plant for live cell imaging and detached leaf experiments to study cell death and provides insight into the importance of Ca2+ in developmentally regulated PCD in planta.

Hsp70 plays a role in programmed cell death during the remodelling of leaves of the lace plant (Aponogeton madagascariensis).

Lace plant leaves utilize programmed cell death (PCD) to form perforations during development. The role of heat shock proteins (Hsps) in PCD during lace plant leaf development is currently unknown. Hsp70 amounts were measured throughout lace plant leaf development, and the results indicate that it is highest before and during PCD. Increased Hsp70 amounts correlate with raised anthocyanin content and caspase-like protease (CLP) activity. To investigate the effects of Hsp70 on leaf development, whole plants were treated with either of the known regulators of PCD [reactive oxygen species (ROS) or antioxidants] or an Hsp70 inhibitor, chlorophenylethynylsulfonamide (PES-Cl). ROS treatment significantly increased Hsp70 2-fold and CLP activity in early developing leaves, but no change in anthocyanin and the number of perforations formed was observed. Antioxidant treatment significantly decreased Hsp70, anthocyanin, and CLP activity in early leaves, resulting in the fewest perforations. PES-Cl (25 µM) treatment significantly increased Hsp70 4-fold in early leaves, while anthocyanin, superoxide, and CLP activity significantly declined, leading to fewer perforations. Results show that significantly increased (4-fold) or decreased Hsp70 amounts lead to lower anthocyanin and CLP activity, inhibiting PCD induction. Our data support the hypothesis that Hsp70 plays a role in regulating PCD at a threshold in lace plant leaf development. Hsp70 affects anthocyanin content and caspase-like protease activity, and helps regulate PCD during the remodelling of leaves of lace plant, Aponogeton madagascariensis.

The Function of Autophagy in Lace Plant Programmed Cell Death.

The lace plant (Aponogeton madagascariensis) is an aquatic monocot that utilizes programmed cell death (PCD) to form perforations throughout its mature leaves as part of normal development. The lace plant is an emerging model system representing a unique form of developmental PCD. The role of autophagy in lace plant PCD was investigated using live cell imaging, transmission electron microscopy (TEM), immunolocalization, and in vivo pharmacological experimentation. ATG8 immunostaining and acridine orange staining revealed that autophagy occurs in both healthy and dying cells. Autophagosome-like vesicles were also found in healthy and dying cells through ultrastructural analysis with TEM. Following autophagy modulation, there was a noticeable increase in vesicles and vacuolar aggregates. A novel cell death assay utilizing lace plant leaves revealed that autophagy enhancement with rapamycin significantly decreased cell death rates compared to the control, whereas inhibition of autophagosome formation with wortmannin or blocking the degradation of cargoes with concanamycin A had an opposite effect. Although autophagy modulation significantly affected cell death rates in cells that are destined to die, neither the promotion nor inhibition of autophagy in whole plants had a significant effect on the number of perforations formed in lace plant leaves. Our data indicate that autophagy predominantly contributes to cell survival, and we found no clear evidence for its direct involvement in the induction of developmental PCD during perforation formation in lace plant leaves.

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.

Discovery of pan autophagy inhibitors through a high-throughput screen highlights macroautophagy as an evolutionarily conserved process across 3 eukaryotic kingdoms.

Due to the involvement of macroautophagy/autophagy in different pathophysiological conditions such as infections, neurodegeneration and cancer, identification of novel small molecules that modulate the process is of current research and clinical interest. In this work, we developed a luciferase-based sensitive and robust kinetic high-throughput screen (HTS) of small molecules that modulate autophagic degradation of peroxisomes in the budding yeast Saccharomyces cerevisiae. Being a pathway-specific rather than a target-driven assay, we identified small molecule modulators that acted at key steps of autophagic flux. Two of the inhibitors, Bay11 and ZPCK, obtained from the screen were further characterized using secondary assays in yeast. Bay11 inhibited autophagy at a step before fusion with the vacuole whereas ZPCK inhibited the cargo degradation inside the vacuole. Furthermore, we demonstrated that these molecules altered the process of autophagy in mammalian cells as well. Strikingly, these molecules also modulated autophagic flux in a novel model plant, Aponogeton madagascariensis. Thus, using small molecule modulators identified by using a newly developed HTS autophagy assay, our results support that macroautophagy is a conserved process across fungal, animal and plant kingdoms.

Remodelling of lace plant leaves: antioxidants and ROS are key regulators of programmed cell death.

MAIN CONCLUSION: Antioxidants and reactive oxygen species are integral for programmed cell death signaling during perforation formation in the lace plant ( Aponogeton madagascariensis ). The lace plant is an excellent model system for studying developmentally regulated programmed cell death (PCD). During early lace plant leaf development, PCD systematically deletes cells resulting in a perforated leaf morphology that is unique in planta. A distinct feature in young lace plant leaves is an abundance of anthocyanins, which have antioxidant properties. The first sign of PCD induction is the loss of anthocyanin pigmentation in cells that are targeted for destruction, which results in a visible gradient of cell death. The cellular dynamics and time course of lace plant PCD are well documented; however, the signals involved in the pathway remain elusive. This study investigates the roles of antioxidants and ROS in developmental PCD signaling during lace plant perforation formation. The involvement of antioxidants and ROS in the pathway was determined using a variety of techniques including pharmacological whole plant experimentation, long-term live cell imaging, the 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid anti-radical activity assay, and western blot analysis. Results indicate that antioxidants and ROS are key regulators of PCD during the remodelling of lace plant leaves.

Methods to Study Plant Programmed Cell Death.

Programmed cell death (PCD) is a critical component of plant development, defense against invading pathogens, and response to environmental stresses. In this chapter, we provide detailed technical methods for studying PCD associated with plant development or induced by abiotic stress. A root hair assay or electrolyte leakage assay are excellent techniques for the quantitative determination of PCD and/or cellular injury induced in response to abiotic stress, whereas the lace plant provides a unique model that facilitates the study of genetically regulated PCD during leaf development.

A comparison of induced and developmental cell death morphologies in lace plant (Aponogeton madagascariensis) leaves.

BACKGROUND: Programmed cell death (PCD) is an important process for the development and maintenance of multicellular eukaryotes. In animals, there are three morphologically distinct cell death types: apoptosis, autophagic cell death, and necrosis. The search for an all-encompassing classification system based on plant cell death morphology continues. The lace plant is a model system for studying PCD as leaf perforations form predictably via this process during development. This study induced death in cells that do not undergo developmental PCD using various degrees and types of stress (heat, salt, acid and base). Cell death was observed via live cell imaging and compared to the developmental PCD pathway. RESULTS: Morphological similarities between developmental and induced PCD included: disappearance of anthocyanin from the vacuole, increase in vesicle formation, nuclear condensation, and fusing of vesicles containing organelles to the vacuole prior to tonoplast collapse. Plasma membrane retraction was a key feature of developmental PCD but did not occur in all induced modes of cell death. CONCLUSIONS: Regardless of the causal agent in cell death, the vacuole appeared to play a central role in dying cells. The results indicated that within a single system, various types and intensities of stress will influence cell death morphology. In order to establish a plant cell death classification system, future research should combine morphological data with biochemical and molecular data.

Unveiling interactions among mitochondria, caspase-like proteases, and the actin cytoskeleton during plant programmed cell death (PCD).

Aponogeton madagascariensis produces perforations over its leaf surface via programmed cell death (PCD). PCD begins between longitudinal and transverse veins at the center of spaces regarded as areoles, and continues outward, stopping several cells from these veins. The gradient of PCD that exists within a single areole of leaves in an early stage of development was used as a model to investigate cellular dynamics during PCD. Mitochondria have interactions with a family of proteases known as caspases, and the actin cytoskeleton during metazoan PCD; less is known regarding these interactions during plant PCD. This study employed the actin stain Alexa Fluor 488 phalloidin, the actin depolymerizer Latrunculin B (Lat B), a synthetic caspase peptide substrate and corresponding specific inhibitors, as well as the mitochondrial pore inhibitor cyclosporine A (CsA) to analyze the role of these cellular constituents during PCD. Results depicted that YVADase (caspase-1) activity is higher during the very early stages of perforation formation, followed by the bundling and subsequent breakdown of actin. Actin depolymerization using Lat B caused no change in YVADase activity. In vivo inhibition of YVADase activity prevented PCD and actin breakdown, therefore substantiating actin as a likely substrate for caspase-like proteases (CLPs). The mitochondrial pore inhibitor CsA significantly decreased YVADase activity, and prevented both PCD and actin breakdown; therefore suggesting the mitochondria as a possible trigger for CLPs during PCD in the lace plant. To our knowledge, this is the first in vivo study using either caspase-1 inhibitor (Ac-YVAD-CMK) or CsA, following which the actin cytoskeleton was examined. Overall, our findings suggest the mitochondria as a possible upstream activator of YVADase activity and implicate these proteases as potential initiators of actin breakdown during perforation formation via PCD in the lace plant.

The pathway of cell dismantling during programmed cell death in lace plant (Aponogeton madagascariensis) leaves.

BACKGROUND: Developmentally regulated programmed cell death (PCD) is the controlled death of cells that occurs throughout the life cycle of both plants and animals. The lace plant (Aponogeton madagascariensis) forms perforations between longitudinal and transverse veins in spaces known as areoles, via developmental PCD; cell death begins in the center of these areoles and develops towards the margin, creating a gradient of PCD. This gradient was examined using both long- and short-term live cell imaging, in addition to histochemical staining, in order to establish the order of cellular events that occur during PCD. RESULTS: The first visible change observed was the reduction in anthocyanin pigmentation, followed by initial chloroplast changes and the bundling of actin microfilaments. At this stage, an increased number of transvacuolar strands (TVS) was evident. Perhaps concurrently with this, increased numbers of vesicles, small mitochondrial aggregates, and perinuclear accumulation of both chloroplasts and mitochondria were observed. The invagination of the tonoplast membrane and the presence of vesicles, both containing organelle materials, suggested evidence for both micro- and macro-autophagy, respectively. Mitochondrial aggregates, as well as individual chloroplasts were subsequently seen undergoing Brownian motion in the vacuole. Following these changes, fragmentation of nuclear DNA, breakdown of actin microfilaments and early cell wall changes were detected. The vacuole then swelled, causing nuclear displacement towards the plasma membrane (PM) and tonoplast rupture followed closely, indicating mega-autophagy. Subsequent to tonoplast rupture, cessation of Brownian motion occurred, as well as the loss of mitochondrial membrane potential (ΔΨm), nuclear shrinkage and PM collapse. Timing from tonoplast rupture to PM collapse was approximately 20 minutes. The entire process from initial chlorophyll reduction to PM collapse took approximately 48 hours. Approximately six hours following PM collapse, cell wall disappearance began and was nearly complete within 24 hours. CONCLUSION: Results showed that a consistent sequence of events occurred during the remodelling of lace plant leaves, which provides an excellent system to study developmental PCD in vivo. These findings can be used to compare and contrast with other developmental PCD examples in plants.