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It is difficult to obtain reliable shear strength parameters for the stability analysis and evaluation of tailings dams in an unstable state. In this study, the sensitivity of the shear strength index to the safety factor of a tailings dam was evaluated. The cohesion C range of a tailings earth rock dam in an unstable state is determined by the safety factor, and the Morgenstern–Price method is used for inversion. During parameter inversion, uncertainty reasoning is established based on cloud theory, which overcomes the problem that the fuzziness and randomness of the quantitative cohesion value are transferred to the qualitative concept of the safety factor. The results show that the change in cohesion C has a greater influence on the safety factor Fs of the tailings dam, and the value of parameter inversion is 8.6901 kPa. The deformation and failure of tailings dams under extreme rainfall conditions are analyzed by using the modified cohesion C value. The dam toe becomes the main response area of plastic deformation and slowly expands to the interior, showing creep deformation. The displacement field gradually transfers from the accumulated tailings to the tailings dam with the flow direction, causing erosion damage. This study provides a new idea and method for parameter inversion of the shear strength index of tailings dams and provides a reference for the disaster prediction and prevention of tailings dams subjected to extreme rainfall.

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.

Computed tomography (CT) is widely used in clinical medicine, and low-dose CT (LDCT) has become popular to reduce potential patient harm during CT acquisition. However, LDCT aggravates the problem of noise and artifacts in CT images, increasing diagnosis difficulty. Through deep learning, denoising CT images by artificial neural network has aroused great interest for medical imaging and has been hugely successful. We propose a framework to achieve excellent LDCT noise reduction using independent operation search cells, inspired by neural architecture search, and introduce the Laplacian to further improve image quality. Employing patch-based training, the proposed method can effectively eliminate CT image noise while retaining the original structure and details, hence significantly improving diagnosis efficiency and promoting LDCT clinical applications.

The roles of Rho family guanosine triphosphatases (GTPases) of plants (ROPs) in modulating plant growth and development have been well characterized. However, little is known about the roles of ROP signaling pathways in regulating plant autophagy and autophagosome formation. In this study, we identify a unique ROP signaling mechanism, which mediates developmental to autophagic transition under stress conditions in the model plant Arabidopsis. Loss-of-function mutants of ROP8 showed stress-induced hypersensitive phenotypes and compromised autophagic flux. Similar to other ROPs in the ROP/RAC family, ROP8 exhibits both plasma membrane and cytosolic punctate localization patterns. Upon autophagic induction, active ROP8 puncta colocalize with autophagosomal markers and are degraded inside the vacuole. In human cells, RalB, an RAS subfamily GTPase, engages its effector Exo84 for autophagosome assembly. However, a RalB counterpart is missing in the plant lineage. Intriguingly, we discovered that plant ROP8 promotes autophagy via its downstream effector Sec5. Live-cell super-resolution imaging showed that ROP8 and Sec5 reside on phagophores for autophagosome formation. Taken together, our findings highlight a previously unappreciated role of an ROP8-Sec5 signaling axis in autophagy promotion, providing new insights into how plants utilize versatile ROP signaling networks to coordinate developmental and autophagic responses depending on environmental changes.

In multicellular and even single-celled organisms, individual components are interconnected at multiscale levels to produce enormously complex biological networks that help these systems maintain homeostasis for development and environmental adaptation. Systems biology studies initially adopted network analysis to explore how relationships between individual components give rise to complex biological processes. Network analysis has been applied to dissect the complex connectivity of mammalian brains across different scales in time and space in The Human Brain Project. In plant science, network analysis has similarly been applied to study the connectivity of plant components at the molecular, subcellular, cellular, organic, and organism levels. Analysis of these multiscale networks contributes to our understanding of how genotype determines phenotype. In this review, we summarized the theoretical framework of plant multiscale networks and introduced studies investigating plant networks by various experimental and computational modalities. We next discussed the currently available analytic methodologies and multi-level imaging techniques used to map multiscale networks in plants. Finally, we highlighted some of the technical challenges and key questions remaining to be addressed in this emerging field.

The factors and mechanisms involved in vacuolar transport in plants, and in particular those directing vesicles to their target endomembrane compartment, remain largely unknown. To identify components of the vacuolar trafficking machinery, we searched for Arabidopsis modified transport to the vacuole (mtv) mutants that abnormally secrete the synthetic vacuolar cargo VAC2. We report here on the identification of 17 mtv mutations, corresponding to mutant alleles of MTV2/VSR4, MTV3/PTEN2A MTV7/EREL1, MTV8/ARFC1, MTV9/PUF2, MTV10/VPS3, MTV11/VPS15, MTV12/GRV2, MTV14/GFS10, MTV15/BET11, MTV16/VPS51, MTV17/VPS54, and MTV18/VSR1. Eight of the MTV proteins localize at the interface between the trans-Golgi network (TGN) and the multivesicular bodies (MVBs), supporting that the trafficking step between these compartments is essential for segregating vacuolar proteins from those destined for secretion. Importantly, the GARP tethering complex subunits MTV16/VPS51 and MTV17/VPS54 were found at endoplasmic reticulum (ER)- and microtubule-associated compartments (EMACs). Moreover, MTV16/VPS51 interacts with the motor domain of kinesins, suggesting that, in addition to tethering vesicles, the GARP complex may regulate the motors that transport them. Our findings unveil a previously uncharacterized compartment of the plant vacuolar trafficking pathway and support a role for microtubules and kinesins in GARP-dependent transport of soluble vacuolar cargo in plants., This work was supported by the Spanish Ministry of Economy and Competitivity and European Regional Development Fund (FEDER) funds (BIO2018-094257-B Ministerio de Ciencia e Innovación/FEDER to E.R., M. Sanmartín, and J.J.S.-S.) and by the Ministry of Education, Youth and Sports of the Czech Republic (LQ1601/CEITEC 2020 grant to J.Z.). G.R. and A.L. were recipients of Formación de Personal Investigador scholarships from the Spanish Ministry of Economy and Competitivity. We thank Dr. Niko Gelner, Dr. Takashi Ueda, Dr. Daniel Van Damme, Dr. Lorenzo Frigerio, Dr. Karin Schumacher, and Dr. Vicente Pallas for kindly providing compartment markers.

Computed Tomography (CT) is widely used in medicine, which has an irreplaceable role compared with other medical imaging methods because of its fast imaging speed, low cost and good imaging effect on bone and lung. But X-rays are harmful to the human body. In order to reduce the harm caused by the process of obtaining CT, low-dose CT is gaining popularity in recent years. Guided by deep learning, low-does CT denoising is successful using artificial neural network. This paper will use the convolutional neural network (CNN), combined the attention block and perceptual loss, to achieve excellent low-does CT denoising performance while preserving more details. Experimental results show that our method achieves good results at different noise levels.

All eukaryotic cells contain an endomembrane system with functionally distinct membrane-bound organelles, including the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN), prevacuolar compartment or multivesicular body (PVC/MVB) and vacuole or lysosome. These organelles are generated accurately and efficiently in a well-organized way so as to exert their essential functions in protein transport, plant growth and responses to the environment. Since the year 2000, great progress has been achieved by the Chinese scientists toward our understanding on organelle biogenesis and function in plants. In this review, representative research findings in plant protein trafficking contributed by Chinese scientists are first introduced. Due to the functional importance of PVCs/MVBs, we then focus on the identification and characterization of PVCs/MVBs as well as the mechanisms underlying their biogenesis as regulated by the ESCRT (endosomal sorting complexes required for transport) machinery, especially by the plant-unique ESCRT component, FREE1 (FYVE domain protein required for endosomal sorting 1). In addition, some Chinese scientists recently perform a whole-cell electron tomography analysis in the endomembrane system of root cells to first propose a new MVB-derived vacuole biogenesis model for future investigation of the underlying mechanisms of vacuole formation. Finally, we summarize current research progress on organelle (PVC/MVB and vacuole) biogenesis and function in plants and present future perspectives in this research field.

Eukaryotic cells consist of numerous membrane-bound organelles, which compartmentalize cellular materials to fulfil a variety of vital functions. In the post-genomic era, it is widely recognized that identification of the subcellular organelle localization and transport mechanisms of the encoded proteins are necessary for a fundamental understanding of their biological functions and the organization of cellular activity. Multiple experimental approaches are now available to determine the subcellular localizations and dynamics of proteins. In this review, we provide an overview of the current methods and organelle markers for protein subcellular localization and trafficking studies in plants, with a focus on the organelles of the endomembrane system. We also discuss the limitations of each method in terms of protein colocalization studies.

In eukaryotes, secretory proteins traffic from the endoplasmic reticulum (ER) to the Golgi apparatus via coat protein complex II (COPII) vesicles. Intriguingly, during nutrient starvation, the COPII machinery acts constructively as a membrane source for autophagosomes during autophagy to maintain cellular homeostasis by recycling intermediate metabolites. In higher plants, essential roles of autophagy have been implicated in plant development and stress responses. Nonetheless, the membrane sources of autophagosomes, especially the participation of the COPII machinery in the autophagic pathway and autophagosome biogenesis, remains elusive in plants. Here, we provided evidence in support of a novel role of a specific Sar1 homolog AtSar1d in plant autophagy in concert with a unique Rab1/Ypt1 homolog AtRabD2a. First, proteomic analysis of the plant ATG (autophagy-related gene) interactome uncovered the mechanistic connections between ATG machinery and specific COPII components including AtSar1d and Sec23s, while a dominant negative mutant of AtSar1d exhibited distinct inhibition on YFP-ATG8 vacuolar degradation upon autophagic induction. Second, a transfer DNA insertion mutant of AtSar1d displayed starvation-related phenotypes. Third, AtSar1d regulated autophagosome progression through specific recognition of ATG8e by a noncanonical motif. Fourth, we demonstrated that a plant-unique Rab1/Ypt1 homolog AtRabD2a coordinates with AtSar1d to function as the molecular switch in mediating the COPII functions in the autophagy pathway. AtRabD2a appears to be essential for bridging the specific AtSar1d-positive COPII vesicles to the autophagy initiation complex and therefore contributes to autophagosome formation in plants. Taken together, we identified a plant-specific nexus of AtSar1d-AtRabD2a in regulating autophagosome biogenesis.

Bamboo is an important model plant to study the molecular mechanisms of rapid shoot growth and flowering once in a lifetime. However, bamboo research about protein functional characterization is largely lagged behind, mainly due to the lack of gene transformation platforms. In this study, a protoplast transient gene expression system in moso bamboo has been first established. Using this reliable and efficient system, we have generated a set of multicolored fluorescent markers based on the targeting sequences from endogenous proteins, which have been validated by their comparative localization with Arabidopsis organelle markers, in a combination with pharmaceutical treatments. Moreover, we further demonstrated the power of this multicolor marker set for rapid, combinatorial analysis of the subcellular localization of uncharacterized proteins, which may play potential functions in moso bamboo flowering and fast growth of shoots. Finally, this protoplast transient gene expression system has been elucidated for functional analysis in protein–protein interaction by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. Taken together, in combination with the set of moso bamboo organelle markers, the protoplast transient gene expression system could be used for subcellular localization and functional study of unknown proteins in bamboo and will definitely promote rapid progress in diverse areas of research in bamboo plants.

The plant hormone auxin controls many aspects of plant development. Membrane trafficking processes, such as secretion, endocytosis and recycling, regulate the polar localization of auxin transporters in order to establish an auxin concentration gradient. Here, we investigate the function of the Arabidopsis thaliana R-SNAREs VESICLE-ASSOCIATED MEMBRANE PROTEIN 721 (VAMP721) and VAMP722 in the post-Golgi trafficking required for proper auxin distribution and seedling growth. We show that multiple growth phenotypes, such as cotyledon development, vein patterning and lateral root growth, were defective in the double homozygous vamp721 vamp722 mutant. Abnormal auxin distribution and root patterning were also observed in the mutant seedlings. Fluorescence imaging revealed that three auxin transporters, PIN-FORMED 1 (PIN1), PIN2 and AUXIN RESISTANT 1 (AUX1), aberrantly accumulate within the cytoplasm of the double mutant, impairing the polar localization at the plasma membrane (PM). Analysis of intracellular trafficking demonstrated the involvement of VAMP721 and VAMP722 in the endocytosis of FM4-64 and the secretion and recycling of the PIN2 transporter protein to the PM, but not its trafficking to the vacuole. Furthermore, vamp721 vamp722 mutant roots display enlarged trans-Golgi network (TGN) structures, as indicated by the subcellular localization of a variety of marker proteins and the ultrastructure observed using transmission electron microscopy. Thus, our results suggest that the R-SNAREs VAMP721 and VAMP722 mediate the post-Golgi trafficking of auxin transporters to the PM from the TGN subdomains, substantially contributing to plant growth.

In multicellular and even single-celled organisms, individual components are interconnected at multiscale levels to produce enormously complex biological networks that help these systems maintain homeostasis for development and environmental adaptation. Systems biology studies initially adopted network analysis to explore how relationships between individual components give rise to complex biological processes. Network analysis has been applied to dissect the complex connectivity of mammalian brains across different scales in time and space in The Human Brain Project. In plant science, network analysis has similarly been applied to study the connectivity of plant components at the molecular, subcellular, cellular, organic, and organism levels. Analysis of these multiscale networks contributes to our understanding of how genotype determines phenotype. In this review, we summarized the theoretical framework of plant multiscale networks and introduced studies investigating plant networks by various experimental and computational modalities. We next discussed the currently available analytic methodologies and multi-level imaging techniques used to map multiscale networks in plants. Finally, we highlighted some of the technical challenges and key questions remaining to be addressed in this emerging field.

Vesicle trafficking between the membrane-bound organelles in plant cells plays crucial roles in the precise transportation of various materials, and thus supports cell proliferation and cellular polarization. Conventionally, plant prevacuolar compartments (PVCs), identified as multivesicular bodies (MVBs), play important roles in both the secretory pathway as intermediate compartments and the endocytic pathway as late endosomes. In recent years, the PVC/MVBs have been proposed to play important roles in both protein vacuolar delivery and unconventional secretion, but several important questions on the new regulators and environmental cues that coordinate the PVC/MVB–organelle membrane interactions and their biological significances remain. In this review, we first summarize the identity and nature of the plant PVC/MVBs, and then we present an update on our current understanding on the interaction of PVC/MVBs with other organelles in the plant endomembrane system with focus on the vacuole, autophagosome, and plasma membrane (PM) in plant development and stress responses. Finally, we raise some open questions and present future perspectives in the study of PVC/MVB–organelle interactions and associated biological functions.

FYVE domain protein required for endosomal sorting1 (FREE1), a plant-specific endosomal sorting complex required for transport-I component, is essential for the biogenesis of multivesicular bodies (MVBs), vacuolar degradation of membrane protein, cargo vacuolar sorting, autophagic degradation, and vacuole biogenesis in Arabidopsis (Arabidopsis thaliana). Here, we report the characterization of RESURRECTION1 (RST1) as a suppressor of free1 that, when mutated as a null mutant, restores the normal MVB and vacuole formation of a FREE1-RNAi knockdown line and consequently allows survival. RST1 encodes an evolutionarily conserved multicellular organism-specific protein, which contains two Domain of Unknown Function 3730 domains, showing no similarity to known proteins, and predominantly localizes in the cytosol. The depletion of FREE1 causes substantial accumulation of RST1, and transgenic Arabidopsis plants overexpressing RST1 display retarded seedling growth with dilated MVBs, and inhibition of endocytosed FM4-64 dye to the tonoplast, suggesting that RST1 has a negative role in vacuolar transport. Consistently, enhanced endocytic degradation of membrane vacuolar cargoes occurs in the rst1 mutant. Further transcriptomic comparison of rst1 with free1 revealed a negative association between gene expression profiles, demonstrating that FREE1 and RST1 have antagonistic functions. Thus, RST1 is a negative regulator controlling membrane protein homeostasis and FREE1-mediated functions in plants.

FREE1 (FYVE domain protein required for endosomal sorting 1), a newly identified component of endosomal sorting complex required for transport I (ESCRT I), plays multiple roles in regulating protein trafficking and organelle biogenesis in Arabidopsis. Similar to other ESCRT components, FREE1 is essential for plant growth and development because free1 mutant is seedling lethal. To identify key components that genetically interact with FREE1, we performed forward genetic suppressor screening using a dexamethasone (DEX)-inducible FREE1-RNAi line. Here we describe the detailed protocol of identifying novel FREE1 regulators using an inducible FREE1-RNAi line for the genetic suppressor screen. This protocol represents a whole procedure for identifying ESCRT genetic interactors in Arabidopsis thaliana.

KEA4, KEA5, and KEA6 are members of the Arabidopsis thaliana K+ efflux antiporter (KEA) family that share high sequence similarity, but whose function remains unknown. Here, we show their gene expression pattern, subcellular localization, and physiological function in Arabidopsis. KEA4, KEA5, and KEA6 had a similar tissue expression pattern and the three KEA proteins localized to the Golgi, the trans-Golgi network (TGN), and the pre-vacuolar compartment (PVC)/multivesicular bodies (MVB), suggesting overlapping roles of these proteins in the endomembrane system. Phenotypic analyses of single, double, and triple mutants confirmed functional redundancy. The triple mutant kea4 kea5 kea6 had small rosettes, short seedlings, and was sensitive to low K+ availability and to the sodicity imposed by high salinity. Also, the kea4 kea5 kea6 mutant plants had a reduced luminal pH in the Golgi, TGN, PVC, and vacuole, in accordance with the K/H exchange activity of KEA proteins. Genetic analysis indicated that KEA4, KEA5, KEA6 and endosomal Na+/H+exchanger (NHX) 5 and NHX6 acted coordinately to facilitate the endosomal pH homeostasis and salt tolerance. Neither cancelling nor overexpressing the vacuolar antiporters NHX1 and NHX2 in the kea4 kea5 kea6 mutant background altered the salt-sensitive phenotype. The NHX1 and NHX2 proteins in the kea4 kea5 kea6 mutant background could not suppress the acidity of the endomembrane system but brought the vacuolar pH close to wild-type values. Together, these data signify that KEA4, KEA5, and KEA6 are endosomal K+ transporters functioning in maintaining pH and ion homeostasis in the endomembrane network., This work was supported by the National Natural Science Foundation of China (31571464, 31371438, and 31070222 to Q.-S.Q.), the National Basic Research Program of China (973 project, 2013CB429904 to Q.-S.Q.), and the Research Fund for the Doctoral Program of Higher Education of China (20130211110001 to Q.-S.Q.). F.J.Q. and J.M.P. were supported by grants from the Spanish Ministry MINECO cofinanced by the European Regional Development Fund (BIO2015-70946-R and BFU2015-64671-R) and by the Rural Development Administration, Republic of Korea, SSAC (PJ01318205).

KEA4, KEA5, and KEA6 are members of the Arabidopsis (Arabidopsis thaliana) K(+) efflux antiporter (KEA) family that share high sequence similarity but whose function remains unknown. Here, we show their gene expression pattern, subcellular localization, and physiological function in Arabidopsis. KEA4, KEA5, and KEA6 had similar tissue expression patterns, and the three KEA proteins localized to the Golgi, the trans-Golgi network, and the prevacuolar compartment/multivesicular bodies, suggesting overlapping roles of these proteins in the endomembrane system. Phenotypic analyses of single, double, and triple mutants confirmed functional redundancy. The triple mutant kea4 kea5 kea6 had small rosettes, short seedlings, and was sensitive to low K(+) availability and to the sodicity imposed by high salinity. Also, the kea4 kea5 kea6 mutant plants had a reduced luminal pH in the Golgi, trans-Golgi network, prevacuolar compartment, and vacuole, in accordance with the K/H exchange activity of KEA proteins. Genetic analysis indicated that KEA4, KEA5, and KEA6 as well as endosomal Na(+)/H(+)exchanger5 (NHX5) and NHX6 acted coordinately to facilitate endosomal pH homeostasis and salt tolerance. Neither cancelling nor overexpressing the vacuolar antiporters NHX1 and NHX2 in the kea4 kea5 kea6 mutant background altered the salt-sensitive phenotype. The NHX1 and NHX2 proteins in the kea4 kea5 kea6 mutant background could not suppress the acidity of the endomembrane system but brought the vacuolar pH close to wild-type values. Together, these data signify that KEA4, KEA5, and KEA6 are endosomal K(+) transporters functioning in maintaining pH and ion homeostasis in the endomembrane network.

Protein trafficking is a fundamental mechanism of subcellular organization and contributes to organellar biogenesis. AtCAP2 is an Arabidopsis homolog of the Mesembryanthemum crystallinum calcium-dependent protein kinase 1 adaptor protein 2 (McCAP2), a member of the syntaxin superfamily. Here, we show that AtCAP2 plays an important role in the conversion to the lytic vacuole (LV) during early plant development. The AtCAP2 loss-of-function mutant atcap2-1 displayed delays in protein storage vacuole (PSV) protein degradation, PSV fusion, LV acidification, and biosynthesis of several vacuolar proteins during germination. At the mature stage, atcap2-1 plants accumulated vacuolar proteins in the prevacuolar compartment (PVC) instead of the LV. In wild-type plants, AtCAP2 localizes to the PVC as a peripheral membrane protein and in the PVC compartment recruits glyceraldehyde-3-phosphate dehydrogenase C2 (GAPC2) to the PVC. We propose that AtCAP2 contributes to LV biogenesis during early plant development by supporting the trafficking of specific proteins involved in the PSV-to-LV transition and LV acidification during early stages of plant development.

High figures of merit is important and necessary for thermoelectric materials in a particular engineering application of thermoelectric generation. Crystalline is an important factor for the high figures of merit of thermoelectric materials. Single-crystalline structure Sb/Bi2Te3 supperlattice nanowires (SLNWs) were synthesised in large quantity by pulsed electrodeposition technology using porous anodic alumina oxide as a template. The X-ray diffraction result indicates that the as-prepared compound consists of rhombohedral phase Bi2Te3 and monoclinic phase Sb. Transmission electron microscope images demonstrate that the segments of SLNWs are built by Bi2Te3 and Sb alternately and exhibit the distinguished interface between two segments. The average diameter and length of each segment are about 80 and 90 nm, respectively. The composition of neighbouring segments was investigated by selected area electron diffraction (SAED), further verifying that the neighbouring segments of SLNWs are comprised of Bi2Te3 and Sb. Meanwhile, the spot pattern of SAED indicates these segments are crystallised well.

Vacuolar sorting receptors (VSRs) are type I integral membrane family proteins in plant cells that can sort cargo proteins at the late Golgi or trans-Golgi network (TGN) for vacuolar transport via the prevacuolar compartment (PVC). However, little is known about VSR cargo proteins in plants. Here, we describe a new method for the identification of VSR cargos, which is based on the premise that the expressed N-terminus of VSRs will be secreted into the culture media along with their corresponding cargo proteins. The protocol described here should be applicable to all VSRs and should be also useful for other receptor cargo identification and protein-protein interaction in vivo.

The maintenance of pH in the intracellular compartments is essential for the viability of cells. Several genetically encoded fluorescent pH sensors have been developed for noninvasive in vivo analysis. Here, we describe the methods using PEpHluorin (plant-solubility-modified ecliptic pHluorin) and PRpHluorin (plant-solubility-modified ratiometric pHluorin) as pH sensors for in vivo visualization and quantification organelle pH of in plant secretory pathway.

In plant secretory pathways, the Golgi apparatus serves as the major sorting hub to receive de novo synthesized protein from the endoplasmic reticulum for further sorting to post-Golgi compartments or for residence in the cisternae of Golgi stacks. Meanwhile, Golgi functions as a pivotal biochemical factory to make modifications of N-glycans and to produce mature glycoproteins. Fluorescent tag-based confocal microscopy in combination with the brefeldin A drug or the genetic tools to disturb Golgi function have been shown as powerful approaches to analyze Golgi-mediated protein traffic in transiently expressed plant protoplasts or in stably expressed transgenic plants. Various endoglycosidases like Endo H and PNGase F have been widely used to monitor Golgi-mediated glycosylation of secretory proteins. Here, using fluorescently tagged Golgi-resident proteins and known glycosylated proteins as examples, we describe detailed protocols to analyze Golgi-mediated protein traffic and glycosylation in transiently expressed protoplasts derived from Arabidopsis suspension culture cells and in stably expressed transgenic plants.

Plant development, defense, and many physiological processes rely on the endosomal sorting complex required for transport (ESCRT) machinery to control the homeostasis of membrane proteins by selective vacuolar degradation. Although ESCRT core components are conserved among higher eukaryotes, the regulators that control the function of the ESCRT machinery remain elusive. We recently identified a plant-specific ESCRT component, FREE1, that is essential for multivesicular body/prevacuolar compartment (MVB/PVC) biogenesis and vacuolar sorting of membrane proteins. Here we identify a plant-specific Bro1-domain protein BRAF, which regulates FREE1 recruitment to the MVB/PVC membrane by competitively binding to the ESCRT-I component Vps23. Altogether, we have successfully identified a role for BRAF, whose function as a unique evolutionary ESCRT regulator in orchestrating intraluminal vesicle formation in MVB/PVCs and the sorting of membrane proteins for degradation in plants makes it an important regulatory mechanism underlying the ESCRT machinery in higher eukaryotes.

AtNHX5 and AtNHX6 are endosomal Na

The endosomal sorting complex required for transport (ESCRT) machinery is an ancient system that deforms membrane and severs membrane necks from the inside. Extensive evidence has accumulated to demonstrate the conserved functions of plant ESCRTs in multivesicular body (MVB) biogenesis and MVB-mediated membrane protein sorting. In addition, recent exciting findings have uncovered unique plant ESCRT components and point to emerging roles for plant ESCRTs in non-endosomal sorting events such as autophagy, cytokinesis, and viral replication. Plant-specific processes, such as abscisic acid (ABA) signaling and chloroplast turnover, provide further evidence for divergences in the functions of plant ESCRTs during evolution. We summarize the multiple roles and current working models for plant ESCRT machinery and speculate on future ESCRT studies in the plant field.

In plant secretory pathways, the Golgi apparatus serves as the major sorting hub to receive de novo synthesized protein from the endoplasmic reticulum for further sorting to post-Golgi compartments or for residence in the cisternae of Golgi stacks. Meanwhile, Golgi functions as a pivotal biochemical factory to make modifications of N-glycans and to produce mature glycoproteins. Fluorescent tag-based confocal microscopy in combination with the brefeldin A drug or the genetic tools to disturb Golgi function have been shown as powerful approaches to analyze Golgi-mediated protein traffic in transiently expressed plant protoplasts or in stably expressed transgenic plants. Various endoglycosidases like Endo H and PNGase F have been widely used to monitor Golgi-mediated glycosylation of secretory proteins. Here, using fluorescently tagged Golgi-resident proteins and known glycosylated proteins as examples, we describe detailed protocols to analyze Golgi-mediated protein traffic and glycosylation in transiently expressed protoplasts derived from Arabidopsis suspension culture cells and in stably expressed transgenic plants.

Vacuolar sorting receptors (VSRs) are type I integral membrane family proteins in plant cells that can sort cargo proteins at the late Golgi or trans-Golgi network (TGN) for vacuolar transport via the prevacuolar compartment (PVC). However, little is known about VSR cargo proteins in plants. Here, we describe a new method for the identification of VSR cargos, which is based on the premise that the expressed N-terminus of VSRs will be secreted into the culture media along with their corresponding cargo proteins. The protocol described here should be applicable to all VSRs and should be also useful for other receptor cargo identification and protein-protein interaction in vivo.

The maintenance of pH in the intracellular compartments is essential for the viability of cells. Several genetically encoded fluorescent pH sensors have been developed for noninvasive in vivo analysis. Here, we describe the methods using PEpHluorin (plant-solubility-modified ecliptic pHluorin) and PRpHluorin (plant-solubility-modified ratiometric pHluorin) as pH sensors for in vivo visualization and quantification organelle pH of in plant secretory pathway.

Summary Vacuolar sorting receptors (VSRs) in Arabidopsis mediate the sorting of soluble proteins to vacuoles in the secretory pathway. The VSRs are post-translationally modified by the attachment of N-glycans, but the functional significance of such a modification remains unknown. Here we have studied the role(s) of glycosylation in the stability, trafficking and vacuolar protein transport of AtVSR1 in Arabidopsis protoplasts. AtVSR1 harbors three complex-type N-glycans, which are located in the N-terminal ‘PA domain’, the central region and the C-terminal epidermal growth factor repeat domain, respectively. We have demonstrated that: (i) the N-glycans do not affect the targeting of AtVSR1 to pre-vacuolar compartments (PVCs) and its vacuolar degradation; and (ii) N-glycosylation alters the binding affinity of AtVSR1 to cargo proteins and affects the transport of cargo into the vacuole. Hence, N-glycosylation of AtVSR1 plays a critical role in its function as a VSR in plants.