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.

FLZ13 interacts with FLC and ABI5 to negatively regulate flowering time in Arabidopsis.

The transition from vegetative to reproductive growth, known as flowering, is a critical developmental process in flowering plants to ensure reproductive success. This process is strictly controlled by various internal and external cues; however, the underlying molecular regulatory mechanisms need to be further characterized. Here, we report a plant-specific protein, FCS-LIKE ZINC FINGER PROTEIN 13 (FLZ13), which functions as a hitherto unknown negative modulator of flowering time in Arabidopsis thaliana. Biochemical analysis showed that FLZ13 directly interacts with FLOWERING LOCUS C (FLC), a major flowering repressor, and that FLZ13 largely depends on FLC to repress the transcription of two core flowering integrators: FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1. In addition, FLZ13 works together with ABSCISIC ACID INSENSITIVE 5 to activate FLC expression to delay flowering. Taken together, our findings suggest that FLZ13 is an important component of the gene regulatory network for flowering time control in plants.

Endocytosis-mediated entry of a caterpillar effector into plants is countered by Jasmonate.

Insects and pathogens release effectors into plant cells to weaken the host defense or immune response. While the imports of some bacterial and fungal effectors into plants have been previously characterized, the mechanisms of how caterpillar effectors enter plant cells remain a mystery. Using live cell imaging and real-time protein tracking, we show that HARP1, an effector from the oral secretions of cotton bollworm (Helicoverpa armigera), enters plant cells via protein-mediated endocytosis. The entry of HARP1 into a plant cell depends on its interaction with vesicle trafficking components including CTL1, PATL2, and TET8. The plant defense hormone jasmonate (JA) restricts HARP1 import by inhibiting endocytosis and HARP1 loading into endosomes. Combined with the previous report that HARP1 inhibits JA signaling output in host plants, it unveils that the effector and JA establish a defense and counter-defense loop reflecting the robust arms race between plants and insects.

Autophagy contributes to positive feedback regulation of SnRK1 signaling in plants.

SnRK1 (SNF1-related protein kinase 1) is a plant ortholog of yeast Snf1 and mammalian adenosine monophosphate-activated protein kinase (AMPK) that acts as a positive regulator of macroautophagy/autophagy. However, whether and how the autophagy pathway modulates SnRK1 activity remains elusive. Recently, we identified a clade of plant-specific FLZ (FCS-like zinc finger) proteins as novel ATG8 (autophagy-related 8)-interacting partners in Arabidopsis thaliana. These AtFLZs, which mainly localize on the surface of mitochondria, can inhibit SnRK1 signaling by repressing the T-loop phosphorylation of its catalytic α subunits, thereby negatively regulating carbon starvation-induced autophagy and plant tolerance to energy deprivation. Upon energy starvation, autophagy is activated to mediate the degradation of these AtFLZs, thus relieving their repression of SnRK1. More importantly, the ATG8-FLZ-SnRK1 regulatory axis appears to be functionally conserved during seed plant evolution. These findings highlight the positive role of autophagy in SnRK1 signaling activation under energy-limiting conditions in plants.Abbreviations: ADS, AIMs docking site; AIM, ATG8-interacting motif; AMPK, adenosine monophosphate-activated protein kinase; ATG, autophagy-related; ESCRT, endosomal sorting complexes required for transport; FLZ, FCS-like zinc finger protein; FREE1, FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1; RAPTOR, REGULATORY-ASSOCIATED PROTEIN OF TOR; Snf1, SUCROSE NON-FERMENTING 1; SnRK1, SNF1-related kinase 1; TOR, TARGET OF RAPAMYCIN.

A positive feedback regulation of SnRK1 signaling by autophagy in plants.

SnRK1, an evolutionarily conserved heterotrimeric kinase complex that acts as a key metabolic sensor in maintaining energy homeostasis in plants, is an important upstream activator of autophagy that serves as a cellular degradation mechanism for the healthy growth of plants. However, whether and how the autophagy pathway is involved in regulating SnRK1 activity remains unknown. In this study, we identified a clade of plant-specific and mitochondria-localized FCS-like zinc finger (FLZ) proteins as currently unknown ATG8-interacting partners that actively inhibit SnRK1 signaling by repressing the T-loop phosphorylation of the catalytic α subunits of SnRK1, thereby negatively modulating autophagy and plant tolerance to energy deprivation caused by long-term carbon starvation. Interestingly, these AtFLZs are transcriptionally repressed by low-energy stress, and AtFLZ proteins undergo a selective autophagy-dependent pathway to be delivered to the vacuole for degradation, thereby constituting a positive feedback regulation to relieve their repression of SnRK1 signaling. Bioinformatic analyses show that the ATG8-FLZ-SnRK1 regulatory axis first appears in gymnosperms and seems to be highly conserved during the evolution of seed plants. Consistent with this, depletion of ATG8-interacting ZmFLZ14 confers enhanced tolerance, whereas overexpression of ZmFLZ14 leads to reduced tolerance to energy deprivation in maize. Collectively, our study reveals a previously unknown mechanism by which autophagy contributes to the positive feedback regulation of SnRK1 signaling, thereby enabling plants to better adapt to stressful environments.

Recent advances in plant endomembrane research and new microscopical techniques.

The endomembrane system consists of various membrane-bound organelles including the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN), endosomes, and the lysosome/vacuole. Membrane trafficking between distinct compartments is mainly achieved by vesicular transport. As the endomembrane compartments and the machineries regulating the membrane trafficking are largely conserved across all eukaryotes, our current knowledge on organelle biogenesis and endomembrane trafficking in plants has mainly been shaped by corresponding studies in mammals and yeast. However, unique perspectives have emerged from plant cell biology research through the characterization of plant-specific regulators as well as the development and application of the state-of-the-art microscopical techniques. In this review, we summarize our current knowledge on the plant endomembrane system, with a focus on several distinct pathways: ER-to-Golgi transport, protein sorting at the TGN, endosomal sorting on multivesicular bodies, vacuolar trafficking/vacuole biogenesis, and the autophagy pathway. We also give an update on advanced imaging techniques for the plant cell biology research.

Advances in omics research on peanut response to biotic stresses.

Peanut growth, development, and eventual production are constrained by biotic and abiotic stresses resulting in serious economic losses. To understand the response and tolerance mechanism of peanut to biotic and abiotic stresses, high-throughput Omics approaches have been applied in peanut research. Integrated Omics approaches are essential for elucidating the temporal and spatial changes that occur in peanut facing different stresses. The integration of functional genomics with other Omics highlights the relationships between peanut genomes and phenotypes under specific stress conditions. In this review, we focus on research on peanut biotic stresses. Here we review the primary types of biotic stresses that threaten sustainable peanut production, the multi-Omics technologies for peanut research and breeding, and the recent advances in various peanut Omics under biotic stresses, including genomics, transcriptomics, proteomics, metabolomics, miRNAomics, epigenomics and phenomics, for identification of biotic stress-related genes, proteins, metabolites and their networks as well as the development of potential traits. We also discuss the challenges, opportunities, and future directions for peanut Omics under biotic stresses, aiming sustainable food production. The Omics knowledge is instrumental for improving peanut tolerance to cope with various biotic stresses and for meeting the food demands of the exponentially growing global population.

ABI5-FLZ13 module transcriptionally represses growth-related genes to delay seed germination in response to ABA.

The bZIP transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5) is a master regulator of seed germination and post-germinative growth in response to abscisic acid (ABA), but the detailed molecular mechanism by which it represses plant growth remains unclear. In this study, we used proximity labeling to map the neighboring proteome of ABI5 and identified FCS-LIKE ZINC FINGER PROTEIN 13 (FLZ13) as a novel ABI5 interaction partner. Phenotypic analysis of flz13 mutants and FLZ13-overexpressing lines demonstrated that FLZ13 acts as a positive regulator of ABA signaling. Transcriptomic analysis revealed that both FLZ13 and ABI5 downregulate the expression of ABA-repressed and growth-related genes involved in chlorophyll biosynthesis, photosynthesis, and cell wall organization, thereby repressing seed germination and seedling establishment in response to ABA. Further genetic analysis showed that FLZ13 and ABI5 function together to regulate seed germination. Collectively, our findings reveal a previously uncharacterized transcriptional regulatory mechanism by which ABA mediates inhibition of seed germination and seedling establishment.

An emerging role of non-canonical conjugation of ATG8 proteins in plant response to heat stress.

Members of the ATG8 (autophagy-related protein 8) protein family can be non-canonically conjugated to single membrane-bound organelles. The exact function of ATG8 on these single membranes remains poorly understood. Recently, using Arabidopsis thaliana as a model system, we identified a non-canonical conjugation of ATG8 pathway involved in the reconstruction of the Golgi apparatus upon heat stress. Short acute heat stress resulted in rapid vesiculation of the Golgi, which was accompanied with the translocation of ATG8 proteins (ATG8a to ATG8i) to the dilated cisternae. More importantly, we found that ATG8 proteins can recruit clathrin to facilitate Golgi reassembly by stimulating the budding of ATG8-positive vesicles from dilated cisternae. These findings provide new insight into one of the possible functions of ATG8 translocation onto single membrane organelles, and will contribute to a better understanding of non-canonical conjugation of ATG8 in eukaryotic cells.Abbreviations: ADS, AIMs docking site; AIM, ATG8-interacting motif; ATG, autophagy-related; CLC2, Clathrin light chain 2; ConcA, concanamycin A; HS, heat stress; PE, phosphatidylethanolamine; PM, plasma membrane; PS, phosphatidylserine; TGN, trans-Golgi network; V-ATPase, vacuolar-type ATPase.

Arabidopsis AUTOPHAGY-RELATED2 is essential for ATG18a and ATG9 trafficking during autophagosome closure.

As a fundamental metabolic pathway, autophagy plays important roles in plant growth and development, particularly under stress conditions. A set of autophagy-related (ATG) proteins is recruited for the formation of a double-membrane autophagosome. Among them, the essential roles of ATG2, ATG18, and ATG9 have been well established in plant autophagy via genetic analysis; however, the underlying molecular mechanism for ATG2 in plant autophagosome formation remains poorly understood. In this study, we focused on the specific role of ATG2 in the trafficking of ATG18a and ATG9 during autophagy in Arabidopsis (Arabidopsis thaliana). Under normal conditions, YFP-ATG18a proteins are partially localized on late endosomes and translocated to ATG8e-labeled autophagosomes upon autophagic induction. Real-time imaging analysis revealed sequential recruitment of ATG18a on the phagophore membrane, showing that ATG18a specifically decorated the closing edges and finally disassociated from the completed autophagosome. However, in the absence of ATG2, most of the YFP-ATG18a proteins are arrested on autophagosomal membranes. Ultrastructural and 3D tomography analysis showed that unclosed autophagosome structures are accumulated in the atg2 mutant, displaying direct connections with the endoplasmic reticulum membrane and vesicular structures. Dynamic analysis of ATG9 vesicles suggested that ATG2 depletion also affects the association between ATG9 vesicles and the autophagosomal membrane. Furthermore, using interaction and recruitment analysis, we mapped the interaction relationship between ATG2 and ATG18a, implying a possible role of ATG18a in recruiting ATG2 and ATG9 to the membrane. Our findings unveil a specific role of ATG2 in coordinating ATG18a and ATG9 trafficking to mediate autophagosome closure in Arabidopsis.

A non-canonical role of ATG8 in Golgi recovery from heat stress in plants.

Above-optimal growth temperatures, usually referred to as heat stress (HS), pose a challenge to organisms' survival as they interfere with essential physiological functions and disrupt cellular organization. Previous studies have elucidated the complex transcriptional regulatory networks involved in plant HS responses, but the mechanisms of organellar remodelling and homeostasis during plant HS adaptations remain elusive. Here we report a non-canonical function of ATG8 in regulating the restoration of plant Golgi damaged by HS. Short-term acute HS causes vacuolation of the Golgi apparatus and translocation of ATG8 to the dilated Golgi membrane. The inactivation of the ATG conjugation system, but not of the upstream autophagic initiators, abolishes the targeting of ATG8 to the swollen Golgi, causing a delay in Golgi recovery after HS. Using TurboID-based proximity labelling, we identified CLATHRIN LIGHT CHAIN 2 (CLC2) as an interacting partner of ATG8 via the AIM-LDS interface. CLC2 is recruited to the cisternal membrane by ATG8 to facilitate Golgi reassembly. Collectively, our study reveals a hitherto unanticipated process of Golgi stack recovery from HS in plant cells and uncovers a previously unknown mechanism of organelle resilience involving ATG8.

The plant unique ESCRT component FREE1 regulates autophagosome closure.

The energy sensor AMP-activated protein kinase (AMPK) can activate autophagy when cellular energy production becomes compromised. However, the degree to which nutrient sensing impinges on the autophagosome closure remains unknown. Here, we provide the mechanism underlying a plant unique protein FREE1, upon autophagy-induced SnRK1α1-mediated phosphorylation, functions as a linkage between ATG conjugation system and ESCRT machinery to regulate the autophagosome closure upon nutrient deprivation. Using high-resolution microscopy, 3D-electron tomography, and protease protection assay, we showed that unclosed autophagosomes accumulated in free1 mutants. Proteomic, cellular and biochemical analysis revealed the mechanistic connection between FREE1 and the ATG conjugation system/ESCRT-III complex in regulating autophagosome closure. Mass spectrometry analysis showed that the evolutionary conserved plant energy sensor SnRK1α1 phosphorylates FREE1 and recruits it to the autophagosomes to promote closure. Mutagenesis of the phosphorylation site on FREE1 caused the autophagosome closure failure. Our findings unveil how cellular energy sensing pathways regulate autophagosome closure to maintain cellular homeostasis.

A plant-unique protein BLISTER coordinates with core retromer to modulate endosomal sorting of plasma membrane and vacuolar proteins.

The retromer is a heteromeric protein complex that localizes to endosomal membranes and drives the formation of endosomal tubules that recycle membrane protein cargoes. In plants, the retromer plays essential and canonical functions in regulating the transport of vacuolar storage proteins and the recycle of endocytosed plasma membrane proteins (PM); however, the mechanisms underlying the regulation of assembly, protein stability, and membrane recruitment of the plant retromer complex remain to be elucidated. In this study, we identify a plant-unique endosomal regulator termed BLISTER (BLI), which colocalizes and associates with the retromer complex by interacting with the retromer core subunits VPS35 and VPS29. Depletion of BLI perturbs the assembly and membrane recruitment of the retromer core VPS26-VPS35-VPS29 trimer. Consequently, depletion of BLI disrupts retromer-regulated endosomal trafficking function, including transport of soluble vacuolar proteins and recycling of endocytosed PIN-FORMED (PIN) proteins from the endosomes back to the PM. Moreover, genetic analysis in Arabidopsis thaliana mutants reveals BLI and core retromer interact genetically in the regulation of endosomal trafficking. Taken together, we identified BLI as a plant-specific endosomal regulator, which functions in retromer pathway to modulate the recycling of endocytosed PM proteins and the trafficking of soluble vacuolar cargoes.

The plant FYVE domain-containing protein FREE1 associates with microprocessor components to repress miRNA biogenesis.

FYVE domain protein required for endosomal sorting 1 (FREE1), originally identified as a plant-specific component of the endosomal sorting complex required for transport (ESCRT) machinery, plays diverse roles either in endosomal sorting in the cytoplasm or in transcriptional regulation of abscisic acid signaling in the nucleus. However, to date, a role for FREE1 or other ESCRT components in the regulation of plant miRNA biology has not been discovered. Here, we demonstrate a nuclear function of FREE1 as a cofactor in miRNA biogenesis in plants. FREE1 directly interacts with the plant core microprocessor component CPL1 in nuclear bodies and disturbs the association between HYL1, SE and CPL1. Inactivation of FREE1 in the nucleus increases the binding affinity between HYL1, SE, and CPL1 and causes a transition of HYL1 from the inactive hyperphosphorylated version to the active hypophosphorylated form, thereby promoting miRNA biogenesis. Our results suggest that FREE1 has evolved as a negative regulator of miRNA biogenesis and provides evidence for a link between FYVE domain-containing proteins and miRNA biogenesis in plants.

Engineered ATG8-binding motif-based selective autophagy to degrade proteins and organelles in planta.

Protein-targeting technologies represent essential approaches in biological research. Protein knockdown tools developed recently in mammalian cells by exploiting natural degradation mechanisms allow for precise determination of protein function and discovery of degrader-type drugs. However, no method to directly target endogenous proteins for degradation is currently available in plants. Here, we describe a novel method for targeted protein clearance by engineering an autophagy receptor with a binder to provide target specificity and an ATG8-binding motif (AIM) to link the targets to nascent autophagosomes, thus harnessing the autophagy machinery for degradation. We demonstrate its specificity and broad potentials by degrading various fluorescence-tagged proteins, including cytosolic mCherry, the nucleus-localized bZIP transcription factor TGA5, and the plasma membrane-anchored brassinosteroid receptor BRI1, as well as fluorescence-coated peroxisomes, using a tobacco-based transient expression system. Stable expression of AIM-based autophagy receptors in Arabidopsis further confirms the feasibility of this approach in selective autophagy of endogenous proteins. With its wide substrate scope and its specificity, our concept of engineered AIM-based selective autophagy could provide a convenient and robust research tool for manipulating endogenous proteins in plants and may open an avenue toward degradation of cytoplasmic components other than proteins in plant research.

VAMP724 and VAMP726 are involved in autophagosome formation in Arabidopsis thaliana.

Macroautophagy/autophagy, an evolutionarily conserved degradative process essential for cell homeostasis and development in eukaryotes, involves autophagosome formation and fusion with a lysosome/vacuole. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play important roles in regulating autophagy in mammals and yeast, but relatively little is known about SNARE function in plant autophagy. Here we identified and characterized two Arabidopsis SNAREs, AT4G15780/VAMP724 and AT1G04760/VAMP726, involved in plant autophagy. Phenotypic analysis showed that mutants of VAMP724 and VAMP726 are sensitive to nutrient-starved conditions. Live-cell imaging on mutants of VAMP724 and VAMP726 expressing YFP-ATG8e showed the formation of abnormal autophagic structures outside the vacuoles and compromised autophagic flux. Further immunogold transmission electron microscopy and electron tomography (ET) analysis demonstrated a direct connection between the tubular autophagic structures and the endoplasmic reticulum (ER) in vamp724-1 vamp726-1 double mutants. Further transient co-expression, co-immunoprecipitation and double immunogold TEM analysis showed that ATG9 (autophagy related 9) interacts and colocalizes with VAMP724 and VAMP726 in ATG9-positive vesicles during autophagosome formation. Taken together, VAMP724 and VAMP726 regulate autophagosome formation likely working together with ATG9 in Arabidopsis.Abbreviations: ATG, autophagy related; BTH, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester; Conc A, concanamycin A; EM, electron microscopy; ER, endoplasmic reticulum; FRET, Förster/fluorescence resonance energy transfer; MS, Murashige and Skoog; MVB, multivesicular body; PAS, phagophore assembly site; PM, plasma membrane; PVC, prevacuolar compartment; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TEM, transmission electron microscopy; TGN, trans-Golgi network; WT, wild-type.

Arabidopsis flowering integrator SOC1 transcriptionally regulates autophagy in response to long-term carbon starvation.

Autophagy is a highly conserved, self-digestion process that is essential for plant adaptations to various environmental stresses. Although the core components of autophagy in plants have been well established, the molecular basis for its transcriptional regulation remains to be fully characterized. In this study, we demonstrate that SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), a MADS-box family transcription factor that determines flowering transition in Arabidopsis, functions as a transcriptional repressor of autophagy. EMSAs, ChIP-qPCR assays, and dual-luciferase receptor assays showed that SOC1 can bind to the promoters of ATG4b, ATG7, and ATG18c via the conserved CArG box. qRT-PCR analysis showed that the three ATG genes ATG4b, ATG7, and ATG18c were up-regulated in the soc1-2 mutant. In line with this, the mutant also displayed enhanced autophagy activity, as revealed by increased autophagosome formation and elevated autophagic flux compared with the wild type. More importantly, SOC1 negatively affected the tolerance of plants to long-term carbon starvation, and this process requires a functional autophagy pathway. Finally, we found that SOC1 was repressed upon carbon starvation at both the transcriptional and protein levels. Overall, our study not only uncovers an important transcriptional mechanism that contributes to the regulation of plant autophagy in response to nutrient starvation, but also highlights novel cellular functions of the flowering integrator SOC1.