Vacuolar Degradation of Plant Organelles.

Plants continuously remodel and degrade their organelles due to damage from their metabolic activities and environmental stressors, as well as an integral part of their cell differentiation programs. Whereas certain organelles use local hydrolytic enzymes for limited remodeling, most of pathways that control the partial or complete dismantling of organelles rely on vacuolar degradation. Specifically, selective autophagic pathways play a crucial role in recognizing and sorting plant organelle cargo for vacuolar clearance, especially under cellular stress conditions induced by factors like heat, drought, and damaging light. In these short reviews, we discuss the mechanisms that control the vacuolar degradation of chloroplasts, mitochondria, endoplasmic reticulum, Golgi, and peroxisomes, with an emphasis on autophagy, recently discovered selective autophagy receptors for plant organelles, and crosstalk with other catabolic pathways.

NAI2 and TSA1 Drive Differentiation of Constitutive and Inducible ER Body Formation in Brassicaceae.

Brassicaceae and closely related species develop unique endoplasmic reticulum (ER)-derived structures called ER bodies, which accumulate ß-glucosidases/myrosinases that are involved in chemical defense. There are two different types of ER bodies: ER bodies constitutively present in seedlings (cER bodies) and ER bodies in rosette leaves induced by treatment with the wounding hormone jasmonate (JA) (iER bodies). Here, we show that At-α whole-genome duplication (WGD) generated the paralogous genes NAI2 and TSA1, which consequently drive differentiation of cER bodies and iER bodies in Brassicaceae plants. In Arabidopsis, NAI2 is expressed in seedlings where cER bodies are formed, whereas TSA1 is expressed in JA-treated leaves where iER bodies are formed. We found that the expression of NAI2 in seedlings and the JA inducibility of TSA1 are conserved across other Brassicaceae plants. The accumulation of NAI2 transcripts in Arabidopsis seedlings is dependent on the transcription factor NAI1, whereas the JA induction of TSA1 in rosette leaves is dependent on MYC2, MYC3 and MYC4. We discovered regions of microsynteny, including the NAI2/TSA1 genes, but the promoter regions are differentiated between TSA1 and NAI2 genes in Brassicaceae. This suggests that the divergence of function between NAI2 and TSA1 occurred immediately after WGD in ancestral Brassicaceae plants to differentiate the formation of iER and cER bodies. Our findings indicate that At-α WGD enabled diversification of defense strategies, which may have contributed to the massive diversification of Brassicaceae plants.

Vacuolar processing enzymes in the plant life cycle.

Vacuolar processing enzyme (VPE) is a cysteine-type endopeptidase that has a substrate-specificity for asparagine or aspartic acid residues and cleaves peptide bonds at their carboxyl-terminal side. Various vacuolar proteins are synthesized as larger proprotein precursors, and VPE is an important initiator of maturation and activation of these proteins. It mediates programmed cell death (PCD) by provoking vacuolar rupture and initiating the proteolytic cascade leading to PCD. Vacuolar processing enzyme also possesses a peptide ligation activity, which is responsible for producing cyclic peptides in several plant species. These unique functions of VPE support developmental and environmental responses in plants. The number of VPE homologues is higher in angiosperm species, indicating that there has been differentiation and specialization of VPE function over the course of evolution. Angiosperm VPEs are separated into two major types: the γ-type VPEs, which are expressed mainly in vegetative organs, and the ß-type VPEs, whose expression occurs mainly in storage organs; in eudicots, the δ-type VPEs are further separated within γ-type VPEs. This review also considers the importance of processing and peptide ligation by VPE in vacuolar protein maturation.

Dynamics of the Light-Dependent Transition of Plant Peroxisomes.

Peroxisomes are present in almost all plant cells. These organelles are involved in various metabolic processes, such as lipid catabolism and photorespiration. A notable feature of plant peroxisomes is their flexible adaptive responses to environmental conditions such as light. When plants shift from heterotrophic to autotrophic growth during the post-germinative stage, peroxisomes undergo a dynamic response, i.e. enzymes involved in lipid catabolism are replaced with photorespiratory enzymes. Although the detailed molecular mechanisms underlying the functional transition of peroxisomes have previously been unclear, recent analyses at the cellular level have enabled this detailed machinery to be characterized. During the functional transition, obsolete enzymes are degraded inside peroxisomes by Lon protease, while newly synthesized enzymes are transported into peroxisomes. In parallel, mature and oxidized peroxisomes are eliminated via autophagy; this functional transition occurs in an efficient manner. Moreover, it has become clear that quality control mechanisms are important for the peroxisomal response to environmental stimuli. In this review, we highlight recent advances in elucidating the molecular mechanisms required for the regulation of peroxisomal roles in response to changes in environmental conditions.

Chaperone and protease functions of LON protease 2 modulate the peroxisomal transition and degradation with autophagy.

Balancing repair and degradation is essential for maintaining organellar and cellular homeostasis. Peroxisomes are ubiquitous organelles in eukaryotic cells that play pivotal roles in cell survival. However, the quality control mechanism used to maintain peroxisomes is unclear. Here, we demonstrate that LON protease 2 (LON2), which is encoded by ABERRANT PEROXISOME MORPHOLOGY 10 (APEM10), is responsible for the functional transition of peroxisomes with autophagy. The Arabidopsis apem10 mutant displayed accelerated peroxisome degradation and a dramatically reduced number of peroxisomes. LON2 deficiency caused enhanced peroxisome degradation by autophagy, and peroxisomal proteins accumulated in the cytosol due to a decrease in the number of peroxisomes. We also show the proteolytic consequence of LON2 for the degradation of peroxisomal proteins, and we demonstrated that unnecessary proteins are eliminated by LON2- and autophagy-dependent degradation pathways during the functional transition of peroxisomes. LON2 plays dual roles as an ATP-dependent protease and a chaperone. We show that the chaperone domain of LON2 is essential for the suppression of autophagy, whereas its peptidase domain interferes with this chaperone function, indicating that intramolecular modulation between the proteolysis and chaperone functions of LON2 regulates degradation of peroxisomes by autophagy.

Pexophagy in plants: a mechanism to remit cells from oxidative damage caused under high-intensity light.

Light is essential for plant growth, but excessive light energy produces reactive oxygen species (ROS), which can seriously damage cells. Mutants defective in ATG (autophagy related) genes show light intensity-dependent leaf damage and ROS accumulation. We found that autophagy is one of the crucial systems in protecting plants from ROS-induced damage by removing oxidative peroxisomes. Damaged peroxisomes are targeted by the PtdIns3P marker and specifically engulfed by phagophores labeled by ATG18a-GFP. Under high-intensity light, huge peroxisome aggregates are induced and captured by vacuolar membranes. Research provides a deeper understanding of plant stress response to light irradiation.

Image-Based Analysis Revealing the Molecular Mechanism of Peroxisome Dynamics in Plants.

Peroxisomes are present in eukaryotic cells and have essential roles in various biological processes. Plant peroxisomes proliferate by de novo biosynthesis or division of pre-existing peroxisomes, degrade, or replace metabolic enzymes, in response to developmental stages, environmental changes, or external stimuli. Defects of peroxisome functions and biogenesis alter a variety of biological processes and cause aberrant plant growth. Traditionally, peroxisomal function-based screening has been employed to isolate Arabidopsis thaliana mutants that are defective in peroxisomal metabolism, such as lipid degradation and photorespiration. These analyses have revealed that the number, subcellular localization, and activity of peroxisomes are closely related to their efficient function, and the molecular mechanisms underlying peroxisome dynamics including organelle biogenesis, protein transport, and organelle interactions must be understood. Various approaches have been adopted to identify factors involved in peroxisome dynamics. With the development of imaging techniques and fluorescent proteins, peroxisome research has been accelerated. Image-based analyses provide intriguing results concerning the movement, morphology, and number of peroxisomes that were hard to obtain by other approaches. This review addresses image-based analysis of peroxisome dynamics in plants, especially A. thaliana and Marchantia polymorpha.

Pexophagy suppresses ROS-induced damage in leaf cells under high-intensity light.

Although light is essential for photosynthesis, it has the potential to elevate intracellular levels of reactive oxygen species (ROS). Since high ROS levels are cytotoxic, plants must alleviate such damage. However, the cellular mechanism underlying ROS-induced leaf damage alleviation in peroxisomes was not fully explored. Here, we show that autophagy plays a pivotal role in the selective removal of ROS-generating peroxisomes, which protects plants from oxidative damage during photosynthesis. We present evidence that autophagy-deficient mutants show light intensity-dependent leaf damage and excess aggregation of ROS-accumulating peroxisomes. The peroxisome aggregates are specifically engulfed by pre-autophagosomal structures and vacuolar membranes in both leaf cells and isolated vacuoles, but they are not degraded in mutants. ATG18a-GFP and GFP-2×FYVE, which bind to phosphatidylinositol 3-phosphate, preferentially target the peroxisomal membranes and pre-autophagosomal structures near peroxisomes in ROS-accumulating cells under high-intensity light. Our findings provide deeper insights into the plant stress response caused by light irradiation.

Microautophagy in Plants: Consideration of Its Molecular Mechanism.

Microautophagy is a type of autophagy. It is characterized by direct enclosing with the vacuolar/lysosomal membrane, which completes the isolation and uptake of cell components in the vacuole. Several publications present evidence that plants exhibit microautophagy. Plant microautophagy is involved in anthocyanin accumulation in the vacuole, eliminating damaged chloroplasts and degrading cellular components during starvation. However, information on the molecular mechanism of microautophagy is less available than that on the general macroautophagy, because the research focusing on microautophagy has not been widely reported. In yeast and animals, it is suggested that microautophagy can be classified into several types depending on morphology and the requirements of autophagy-related (ATG) genes. This review summarizes the studies on plant microautophagy and discusses possible techniques for a future study in this field while taking into account the information on microautophagy obtained from yeast and animals.

Endoplasmic reticulum-derived bodies enable a single-cell chemical defense in Brassicaceae plants.

Brassicaceae plants have a dual-cell type of chemical defense against herbivory. Here, we show a novel single-cell defense involving endoplasmic reticulum (ER)-derived organelles (ER bodies) and the vacuoles. We identify various glucosinolates as endogenous substrates of the ER-body ß-glucosidases BGLU23 and BGLU21. Woodlice strongly prefer to eat seedlings of bglu23 bglu21 or a glucosinolate-deficient mutant over wild-type seedlings, confirming that the ß-glucosidases have a role in chemical defense: production of toxic compounds upon organellar damage. Deficiency of the Brassicaceae-specific protein NAI2 prevents ER-body formation, which results in a loss of BGLU23 and a loss of resistance to woodlice. Hence, NAI2 that interacts with BGLU23 is essential for sequestering BGLU23 in ER bodies and preventing its degradation. Artificial expression of NAI2 and BGLU23 in non-Brassicaceae plants results in the formation of ER bodies, indicating that acquisition of NAI2 by Brassicaceae plants is a key step in developing their single-cell defense system.

Gateway binary vectors with organelle-targeted fluorescent proteins for highly sensitive reporter assay in gene expression analysis of plants.

Fluorescent proteins are valuable tools in the bioscience field especially in subcellular localization analysis of proteins and expression analysis of genes. Fusion with organelle-targeting signal accumulates fluorescent proteins in specific organelles, increases local brightness, and highlights the signal of fluorescent proteins even in tissues emitting a high background of autofluorescence. For these advantages, organelle-targeted fluorescent proteins are preferably used for promoter:reporter assay to define organ-, tissue-, or cell-specific expression pattern of genes in detail. In this study, we have developed a new series of Gateway cloning technology-compatible binary vectors, pGWBs (attR1-attR2 acceptor sites) and R4L1pGWB (attR4-attL1 acceptor sites), carrying organelle-targeted synthetic green fluorescent protein with S65T mutation (sGFP) (ER-, nucleus-, peroxisome-, and mitochondria-targeted sGFP) and organelle-targeted tag red fluorescent protein (TagRFP) (nucleus-, peroxisome-, and mitochondria-targeted TagRFP). These are available for preparation of promoter:reporter constructs by an LR reaction with a promoter entry clone attL1-promoter-attL2 (for pGWBs) or attL4-promoter-attR1 (for R4L1pGWBs), respectively. A transient expression experiment with particle bombardment using cauliflower mosaic virus 35S promoter-driven constructs has confirmed the correct localization of newly developed organelle-targeted TagRFPs by a co-localization analysis with the previously established organelle-targeted sGFPs. More intense and apparent fluorescence signals were detected by the nucleus- and peroxisome-targeted sGFPs than by the normal sGFPs in the promoter assay using transgenic Arabidopsis thaliana. The new pGWBs and R4L1pGWBs developed here are highly efficient and may serve as useful platforms for more accurate observation of GFP and RFP signals in gene expression analyses of plants.

Sucrose Starvation Induces Microautophagy in Plant Root Cells.

Autophagy is an essential system for degrading and recycling cellular components for survival during starvation conditions. Under sucrose starvation, application of a papain protease inhibitor E-64d to the Arabidopsis root and tobacco BY-2 cells induced the accumulation of vesicles, labeled with a fluorescent membrane marker FM4-64. The E-64d-induced vesicle accumulation was reduced in the mutant defective in autophagy-related genes ATG2, ATG5, and ATG7, suggesting autophagy is involved in the formation of these vesicles. To clarify the formation of these vesicles in detail, we monitored time-dependent changes of tonoplast, and vesicle accumulation in sucrose-starved cells. We found that these vesicles were derived from the tonoplast and produced by microautophagic process. The tonoplast proteins were excluded from the vesicles, suggesting that the vesicles are generated from specific membrane domains. Concanamycin A treatment in GFP-ATG8a transgenic plants showed that not all FM4-64-labeled vesicles, which were derived from the tonoplast, contained the ATG8a-containing structure. These results suggest that ATG8a may not always be necessary for microautophagy.

Bimolecular Fluorescence Complementation with Improved Gateway-Compatible Vectors to Visualize Protein-Protein Interactions in Plant Cells.

The bimolecular fluorescence complementation (BiFC) assay is a powerful, flexible, and simple tool to study protein-protein interactions in living cells. To accelerate the production and assessment of BiFC constructs, Gateway-compatible multicolor BiFC vectors were generated to enable the simultaneous production of multiple fusion genes that have the split N- or C-terminal fragment of fluorescent protein with the gene of interest in a high-throughput manner. Two different transient expression techniques for the assessment of BiFC in plant cells are described.

Gateway Vectors for Simultaneous Detection of Multiple Protein-Protein Interactions in Plant Cells Using Bimolecular Fluorescence Complementation.

Bimolecular fluorescence complementation (BiFC) is widely used to detect protein-protein interactions, because it is technically simple, convenient, and can be adapted for use with conventional fluorescence microscopy. We previously constructed enhanced yellow fluorescent protein (EYFP)-based Gateway cloning technology-compatible vectors. In the current study, we generated new Gateway cloning technology-compatible vectors to detect BiFC-based multiple protein-protein interactions using N- and C-terminal fragments of enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), and monomeric red fluorescent protein (mRFP1). Using a combination of N- and C-terminal fragments from ECFP, EGFP and EYFP, we observed a shift in the emission wavelength, enabling the simultaneous detection of multiple protein-protein interactions. Moreover, we developed these vectors as binary vectors for use in Agrobacterium infiltration and for the generate transgenic plants. We verified that the binary vectors functioned well in tobacco cells. The results demonstrate that the BiFC vectors facilitate the design of various constructions and are convenient for the detection of multiple protein-protein interactions simultaneously in plant cells.

Vacuolar processing enzyme in plant programmed cell death.

Vacuolar processing enzyme (VPE) is a cysteine proteinase originally identified as the proteinase responsible for the maturation and activation of vacuolar proteins in plants, and it is known to be an ortholog of animal asparaginyl endopeptidase (AEP/VPE/legumain). VPE has been shown to exhibit enzymatic properties similar to that of caspase 1, which is a cysteine protease that mediates the programmed cell death (PCD) pathway in animals. Although there is limited sequence identity between VPE and caspase 1, their predicted three-dimensional structures revealed that the essential amino-acid residues for these enzymes form similar pockets for the substrate peptide YVAD. In contrast to the cytosolic localization of caspases, VPE is localized in vacuoles. VPE provokes vacuolar rupture, initiating the proteolytic cascade leading to PCD in the plant immune response. It has become apparent that the VPE-dependent PCD pathway is involved not only in the immune response, but also in the responses to a variety of stress inducers and in the development of various tissues. This review summarizes the current knowledge on the contribution of VPE to plant PCD and its role in vacuole-mediated cell death, and it also compares VPE with the animal cell death executor caspase 1.

Interaction between chaperone and protease functions of LON2, and autophagy during the functional transition of peroxisomes.

Functional transition of glyoxysomes to leaf peroxisomes is observed in greening cotyledons. Glyoxysomal proteins are rapidly degraded and leaf-peroxisomal proteins are transported into peroxisomes after cotyledons are exposed to light, but the molecular mechanisms underlying these processes remain unclear. We recently discovered that two degradation pathways are involved in the functional transition of peroxisomes using Arabidopsis thaliana. Lon protease 2 (LON2) is responsible for the degradation of glyoxysomal proteins inside peroxisomes, and, in parallel, autophagy eliminates damaged or obsolete peroxisomes. A double mutant defective in both the LON2- and autophagy-dependent degradation pathways accumulated glyoxysomal proteins after the cotyledons became green. Our study also demonstrated that the LON2- and autophagy-dependent pathways are interdependent, with the chaperone function of LON2 suppressing autophagic peroxisome degradation. Moreover, the peptidase domain of LON2 interferes with the suppression of autophagy, indicating that autophagy is regulated by intramolecular modulation between the proteolysis and chaperone functions of LON2.

Plant autophagy is responsible for peroxisomal transition and plays an important role in the maintenance of peroxisomal quality.

In photosynthetic cells, a large amount of hydrogen peroxide is produced in peroxisomes through photorespiration, which is a metabolic pathway related to photosynthesis. Hydrogen peroxide, a reactive oxygen species, oxidizes peroxisomal proteins and membrane lipids, resulting in a decrease in peroxisomal quality. We demonstrate that the autophagic system is responsible for the elimination of oxidized peroxisomes in plant. We isolated Arabidopsis mutants that accumulated oxidized peroxisomes, which formed large aggregates. We revealed that these mutants were defective in autophagy-related (ATG) genes and that the aggregated peroxisomes were selectively targeted by the autophagic machinery. These findings suggest that autophagy plays an important role in the quality control of peroxisomes by the selective degradation of oxidized peroxisomes. In addition, the results suggest that autophagy is also responsible for the functional transition of glyoxysomes to leaf peroxisomes.