Endomembrane-biased dimerization of ABCG16 and ABCG25 transporters determines their substrate selectivity in ABA-regulated plant growth and stress responses

ATP-binding cassette(ABC)transporters are integral membrane proteins that have evolved diverse func-tions fulfilled via the transport of various substrates.In Arabidopsis,the G subfamily of ABC proteins is particularly abundant and participates in multiple signaling pathways during plant development and stress responses.In this study,we revealed that two Arabidopsis ABCG transporters,ABCG16 and ABCG25,engage in ABA-mediated stress responses and early plant growth through endomembrane-specific dimerization-coupled transport of ABA and ABA-glucosyl ester(ABA-GE),respectively.We first revealed that ABCG16 contributes to osmotic stress tolerance via ABA signaling.More specifically,ABCG16 induces cellular ABA efflux in both yeast and plant cells.Using FRET analysis,we showed that ABCG16 forms oblig-atory homodimers for ABA export activity and that the plasma membrane-resident ABCG16 homodimers specifically respond to ABA,undergoing notable conformational changes.Furthermore,we demonstrated that ABCG16 heterodimerizes with ABCG25 at the endoplasmic reticulum(ER)membrane and facilitates the ER entry of ABA-GE in both Arabidopsis and tobacco cells.The specific responsiveness of the ABCG16-ABCG25 heterodimer to ABA-GE and the superior growth of their double mutant support an inhib-itory role of these twoABCGs in early seedling establishment via regulation of ABA-GE translocation across the ER membrane.Our endomembrane-specific analysis of the FRET signals derived from the homo-or heterodimerized ABcG complexes allowed us to link endomembrane-biased dimerization to the transloca-tion of distinct substrates by ABcG transporters,providing a prototypic framework for understanding the omnipotence of ABcG transporters in plant development and stress responses.

Organelle pH in the Arabidopsis Endomembrane System

The pH of intracellular compartments is essential for the viability of cells. Despite its relevance, little is known about the pH of these compartments. To measure pH in vivo, we have first generated two pH sensors by combining the improved-solubility feature of solubility-modified green fluorescent protein （GFP） （smGFP） with the pH-sensing capabil- ity of the pHluorins and codon optimized for expression in Arabidopsis. PEpHluorin （plant-solubility-modified ecliptic pHluorin） gradually loses fluorescence as pH is lowered with fluorescence vanishing at pH 6.2 and PRpHluorin （plant- solubility-modified ratiomatric pHluorin）, a dual-excitation sensor, allowing for precise measurements. Compartment- specific sensors were generated by further fusing specific sorting signals to PEpHluorin and PRpHluorin. Our results show that the pH of cytosol and nucleus is similar （pH 7.3 and 7.2）, while peroxisomes, mitochondrial matrix, and plastidial stroma have alkaline pH. Compartments of the secretory pathway reveal a gradual acidification, spanning from pH 7.1 in the endoplasmic reticulum （ER） to pH 5.2 in the vacuole. Surprisingly, pH in the trans-Golgi network （TGN） and mul- tivesicular body （MVB） is, with pH 6.3 and 6.2, quite similar. The inhibition of vacuolar-type H＋-ATPase （V-ATPase） with concanamycin A （ConcA） caused drastic increase in pH in TGN and vacuole. Overall, the PEpHluorin and PRpHluorin are excellent pH sensors for visualization and quantification of pH in vivo, respectively.

The Basal Level Ethylene Response is Important to the Wall and Endomembrane Structure in the Hypocotyl Cells of Etiolated Arabidopsis Seedlings

The sub-cellular events that occur during the ethylene-modulated cell elongation were characterized by examining the ultra-structure of etiolated Arabidopsis seedling hypocotyl cells. Preventing the basal level ethylene response facilitated cell elongation, and the cells exhibited wall loosening and separation phenotype. Nearby the wall separation sites were frequently associated with an increase in the cortical rough endoplasmic reticulum （rER） membranes, the presence of paramural bodies, and the circular Golgi formation. The cortical rER proliferation and circular Golgi phenotype were reverted by the protein biosynthesis inhibitor cycloheximide. The cortical rER membranes were longer when the ethylene response was prevented and shortened with elevated ethylene responses. Proteomic changes between wild type and the ethylene-insensitive mutant ethylene insensitive2 （ein2） seedling hypocotyls indicated that distinct subsets of proteins involving endomembrane trafficking, remodeling, and wall modifications were differentially expressed. FM4-64 staining supported the proteomic changes, which indicated reduced endocytosis activity with alleviation of the ethylene response. The basal level ethylene response has an important role in endomembrane trafficking, biological materials transport and maintenance of the endomembrane organization. It is possible that endomembrane alterations may partly associate with the wall modifications, though the biological significance of the alterations should be addressed in future studies.

An Approach to Quantify Endomembrane Dynamics in Pollen Utilizing Bioactive Chemicals

Tip growth of pollen tubes and root hairs occurs via rapid polar growth. These rapidly elongating cells require tip-focused endomembrane trafficking for the deposition and recycling of proteins, membranes, and cell wall materials. Most of the image-based data published to date are subjective and non-quantified. Quantitative and com- parative descriptors of these highly dynamic processes have been a major challenge, but are highly desirable for genetic and chemical genomics approaches to dissect this biological network. To address this problem, we screened for small molecules that perturbed the localization of a marker for the Golgi Ras-like monomeric G-protein RAB2：GFP expressed in transgenic tobacco pollen. Semi-automated high-throughput imaging and image analysis resulted in the identifica- tion of novel compounds that altered pollen tube development and endomembrane trafficking. Six compounds that caused mislocalization and varying degrees of altered movement of RAB2：GFP-labeled endomembrane bodies were used to generate a training set of image data from which to quantify vesicle dynamics. The area, velocity, straightness, and intensity of each body were quantified using semi-automated image analysis tools revealing quantitative differences in the phenotype caused by each compound. A score was then given to each compound enabling quantitative comparisons between compounds. Our results demonstrate that image analysis can be used to quantitatively evaluate dynamic sub- cellular endomembrane phenotypes induced by bioactive chemicals, mutations, or other perturbing agents as part of a strategy to quantitatively dissect the endomembrane network.

Trans-Golgi Network-An Intersection of Trafficking Cell Wall Components

The cell wall, a crucial cell compartment, is composed of a network of polysaccharides and proteins, providing structural support and protection from external stimuli.

The endoplasmic reticulum: A dynamic and well-connected organelle

The endoplasmic reticulum forms the first compart- ment in a series of organelles which comprise the secretory pathway, it takes the form of an extremely dynamic and pleomorphic membrane-bounded network of tubules and cisternae which have numerous different cellular functions. In this review, we discuss the nature of endoplasmic reticulum structure and dynamics, its relationship with closely associated organelles, and its possible function as a highway for the distribution and delivery of a diverse range of structures from metabolic complexes to viral particles.

A Bi-Functional Xyloglucan Galactosyltransferase Is an Indispensable Salt Stress Tolerance Determinant in Arabidopsis

Salinity is an abiotic stress that substantially limits crop production worldwide. To identify salt stress tolerance determinants, we screened for Arabidopsis mutants that are hypersensitive to salt stress and designated these mutants as short root in salt medium （rsa）. One of these mutants, rsa3-1, is hypersensitive to NaCI and LiCI but not to CsCI or to general osmotic stress. Reactive oxygen species （ROS） over-accumulate in rsa3-1 plants under salt stress. Gene expression profiling with Affymetrix microarray analysis revealed that RSA3 controls expression of many genes including genes encoding proteins for ROS detoxification under salt stress. Map-based cloning showed that RSA3 encodes a xyloglucan galactosyltransferase, which is allelic to a gene previously named MUR3/KAM1. The RSA3/ MUR3/KAMl-encoded xylogluscan galactosyltransferase regulates actin microfilament organization （and thereby con- tributes to endomembrane distribution） and is also involved in cell wall biosynthesis. In rsa3-1, actin cannot assemble and form bundles as it does in the wild-type but instead aggregates in the cytoplasm. Furthermore, addition of phal- Ioidin, which prevents actin depolymerization, can rescue salt hypersensitivity of rsa3-1. Together, these results sug- gest that RSA3/MUR3/KAM1 along with other cell wall-associated proteins plays a critical role in salt stress tolerance by maintaining the proper organization of actin microfilaments in order to minimize damage caused by excessive ROS.

Protein trafficking in plant cells:tools and markers

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 theorganization 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.

Fast-Suppressor Screening for New Components in Protein Trafficking,Organelle Biogenesis and Silencing Pathway in Arabidopsis thaliana Using DEX-Inducible FREE1-RNAi Plants

Membrane trafficking is essential for plant growth and responses to external signals.The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex（endosomal sorting complex required for transport）.FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body（MVB）,vacuolar protein transport and vacuole biogenesis,and autophagic degradation.FREE1 knockout plants show defective MVB formation,abnormal vacuolar transport,fragmented vacuoles,accumulated autophagosomes,and seedling lethality.To further uncover the underlying mechanisms of FREE1 function in plants,we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants.The obtained mutants are termed as suppressors of free1（sof）.To date,229 putative sof mutants have been identified.Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors.Also145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants.Through next-generation sequencing（NGS）of bulked DNA from F2 mapping population of two RNAi-related sof mutants,FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly.These FREE1-and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis,as well as for uncovering the new components involved in the regulation of silencing pathways in plants.

Sub-Compartmental Organization of Golgi-Resident N-Glycan Processing Enzymes in Plants

In all eukaryotes, the Golgi apparatus is the main site of protein glycosylation. It is widely accepted that the glycosidases and glycosyltransferases involved in N-glycan processing are found concentrated within the Golgi stack where they provide their function. This means that enzymes catalyzing early steps in the processing pathway are located mainly at the cis-side, whereas late-acting enzymes mostly locate to the trans-side of the stacks, creating a non-uniform distribution along the cis-trans axis of the Golgi. There is compelling evidence that the information for their sorting to specific Golgi cisternae depends on signals encoded in the proteins themselves as well as on the trafficking machinery that recognizes these signals and it is believed that cisternal sub-compartmentalization is achieved and maintained by a combination of retention and retrieval mechanisms. Yet, the signals, mechanism（s）, and molecular factors involved are still unknown. Here, we address recent findings and summarize the current understanding of this fundamental process in plant cell biology.

AtSYP51/52 Functions Diverge in the Post-Golgi Traffic and Differently Affect Vacuolar Sorting

Plant sensitive factor attachment protein receptors （SNAREs） encoded by genes of the same sub-family are generally considered as redundant in promoting vesicle-associated membrane fusion events. Nonetheless, the application of innovative experimental approaches highlighted that members of the same gene sub-family often have different functional specificities. In this work, two closely related Qc-SNAREs--the AtSYP51 and the AtSYP52--are compared in their ability to influence different secretory pathways. Their role in the vesicle sorting to the central vacuole has been revised and they were found to have a novel inhibitory function. When transiently overexpressed, the SYP51 and the SYP52 distributed between the TGN and the tonoplast. Our data demonstrate that these SYPs （syntaxin of plants） act as t-SNARE when present on the membrane of TGN/PVC, whereas they behave as inhibitory or interfering SNAREs （i-SNAREs） when they accumulate on the tonoplast. Moreover, the performed functional analysis indicated that the AtSYP51 and the AtSYP52 roles differ in the traffic to the vacuole. The findings are a novel contribution to the functional characterization of plant SNAREs that reveals additional non-fusogenic roles.