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Abstract Background In yeast and mammals, many plasma membrane (PM) proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT) machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. Results Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. Conclusions Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route, but it also mediates vacuolar delivery if displayed at the Golgi. In both cases, ubiquitin-tagged proteins travel via early endosomes and multivesicular bodies to the lytic vacuole. This suggests that vacuolar degradation of ubiquitinated proteins is not restricted to PM proteins but might also facilitate the turnover of membrane proteins in the early secretory pathway.

Marginal lands have been proposed to produce non-food crop biomass for energy or green materials. For this purpose, the selection, implementation, and growth optimization of plant species on such lands are key elements to investigate to achieve relevant plant yields. Stinging nettle ( Urtica dioica ) is a herbaceous perennial that grows spontaneously on contaminated lands and was described as suitable to produce fibers for material applications. Two mercury-contaminated soils from industrial wastelands with different properties (grassland soil and sediment landfill) were used in this study to assess the potential growth of stinging nettle in a greenhouse mesocosm experiment. Two organic amendments were studied for their impact on nettle growth. The solid digestate from organic food wastes significantly doubled plant biomass whereas the compost from green wastes had a lower impact. The highest doses of organic amendments significantly increased the number of fibers, which doubled following digestate application, while reducing leaf Hg concentration. Both amendments significantly improved soil respiration and enzymatic activities linked to the microbial biomass in the soil from the sediment landfill by the end of the experiment. In the context of a phytomanagement scenario, solid digestate would be a preferred amendment resource to improve nettle production on industrial wastelands.

High-throughput sequencing has become a prominent tool to assess plant-associated microbial diversity. Still, some technical challenges remain in characterising these communities, notably due to plant and fungal DNA co-amplification. Fungal-specific primers, Peptide Nucleic Acid (PNA) clamps, or adjusting PCR conditions are approaches to limit plant DNA contamination. However, a systematic comparison of these factors and their interactions, which could limit plant DNA contamination in the study of plant mycobiota, is still lacking. Here, three primers targeting the ITS2 region were evaluated alone or in combination with PNA clamps both on nettle ( Urtica dioica ) root DNA and a mock community. PNA clamps did not improve the richness or diversity of the fungal communities but increased the number of fungal reads. Among the tested factors, the most significant was the primer pair. Specifically, the 5.8S-Fun/ITS4-Fun pair exhibited a higher OTU richness but fewer fungal reads. Our study demonstrates that the choice of primers is critical for limiting plant and fungal DNA co-amplification. PNA clamps increase the number of fungal reads when ITS2 is targeted but do not result in higher fungal diversity recovery at high sequencing depth. At lower read depths, PNA clamps might enhance microbial diversity quantification for primer pairs lacking fungal specificity., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

The stinging nettle Urtica dioica L. is a perennial crop with low fertilizer and pesticide requirements, well adapted to a wide range of environmental conditions. It has been successfully grown in most European climatic zones while also promoting local flora and fauna diversity. The cultivation of nettle could help meet the strong increase in demand for raw materials based on plant fibers as a substitute for artificial fibers in sectors as diverse as the textile and automotive industries. In the present review, we present a historical perspective of selection, harvest, and fiber processing features where the state of the art of nettle varietal selection is detailed. A synthesis of the general knowledge about its biology, adaptability, and genetics constituents, highlighting gaps in our current knowledge on interactions with other organisms, is provided. We further addressed cultivation and processing features, putting a special emphasis on harvesting systems and fiber extraction processes to improve fiber yield and quality. Various uses in industrial processes and notably for the restoration of marginal lands and avenues of future research on this high-value multi-use plant for the global fiber market are described.

In forests, bacteria and fungi are key players in wood degradation. Still, studies focusing on bacterial and fungal successions during the decomposition process depending on the wood types (i.e. sapwood and heartwood) remain scarce. This study aimed to understand the effect of wood type on the dynamics of microbial ecological guilds in wood decomposition. Using Illumina metabarcoding, bacterial and fungal communities were monitored every 3 months for 3 years from Quercus petraea wood discs placed on forest soil. Wood density and microbial enzymes involved in biopolymer degradation were measured. We observed rapid changes in the bacterial and fungal communities and microbial ecological guilds associated with wood decomposition throughout the experiment. Bacterial and fungal succession dynamics were very contrasted between sapwood and heartwood. The initial microbial communities were quickly replaced by new bacterial and fungal assemblages in the sapwood. Conversely, some initial functional guilds (i.e. endophytes and yeasts) persisted all along the experiment in heartwood and finally became dominant, possibly limiting the development of saprotrophic fungi. Our data also suggested a significant role of bacteria in nitrogen cycle during wood decomposition., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Stinging nettle (Urtica dioica L.) raises growing interest in phytomanagement because it commonly grows under poplar Short Rotation Coppices (SRC) set up at trace-metal (TM) contaminated sites and provides high-quality herbaceous fibres. The mycobiome of this non-mycorhizal plant and its capacity to adapt to TM-contaminated environments remains unknown. This study aimed at characterizing the mycobiome associated with nettle and poplar roots co-occurring at a TM-contaminated site. Plant root barcoding using the fungi-specific ITS1F-ITS2 primers and Illumina MiSeq technology revealed that nettle and poplar had distinct root fungal communities. The nettle mycobiome was dominated by Pezizomycetes from known endophytic taxa and from the supposedly saprotrophic genus Kotlabaea (which was the most abundant). Several ectomycorrhizal fungi such as Inocybe (Agaricomycetes) and Tuber (Pezizomycetes) species were associated with the poplar roots. Most of the Pezizomycetes taxa were present in the highly TM-contaminated area whereas Agaricomycetes tended to be reduced. Despite being a known non-mycorrhizal plant, nettle was associated with a significant proportion of ectomycorrhizal OTU (9.7%), suggesting some connexions between the poplar and the nettle root mycobiomes. Finally, our study raised the interest in reconsidering the fungal networking beyond known mycorrhizal interactions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

RNA silencing is a major antiviral defense mechanism in plants and invertebrates. Plant ARGONAUTE1 (AGO1) is pivotal in RNA silencing, and hence is a major target for counteracting viral suppressors of RNA-silencing proteins (VSRs). P0 from Turnip yellows virus (TuYV) is a VSR that was previously shown to trigger AGO1 degradation via an autophagy-like process. However, the identity of host proteins involved and the cellular site at which AGO1 and P0 interact were unknown. Here we report that P0 and AGO1 associate on the endoplasmic reticulum (ER), resulting in their loading into ER-associated vesicles that are mobilized to the vacuole in an ATG5- and ATG7-dependent manner. We further identified ATG8-Interacting proteins 1 and 2 (ATI1 and ATI2) as proteins that associate with P0 and interact with AGO1 on the ER up to the vacuole. Notably, ATI1 and ATI2 belong to an endogenous degradation pathway of ER-associated AGO1 that is significantly induced following P0 expression. Accordingly, ATI1 and ATI2 deficiency causes a significant increase in posttranscriptional gene silencing (PTGS) activity. Collectively, we identify ATI1 and ATI2 as components of an ER-associated AGO1 turnover and proper PTGS maintenance and further show how the VSR P0 manipulates this pathway., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Brassinosteroid (BR) hormone signaling controls multiple processes during plant growth and development and is initiated at the plasma membrane through the receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) together with co-receptors such as BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). BRI1 abundance is regulated by endosomal recycling and vacuolar targeting, but the role of vacuole-related proteins in BR receptor dynamics and BR responses remains elusive. Here, we show that the absence of two DUF300 domain-containing tonoplast proteins, LAZARUS1 (LAZ1) and LAZ1 HOMOLOG1 (LAZ1H1), causes vacuole morphology defects, growth inhibition, and constitutive activation of BR signaling. Intriguingly, tonoplast accumulation of BAK1 was substantially increased and appeared causally linked to enhanced BRI1 trafficking and degradation in laz1 laz1h1 plants. Since unrelated vacuole mutants exhibited normal BR responses, our findings indicate that DUF300 proteins play distinct roles in the regulation of BR signaling by maintaining vacuole integrity required to balance subcellular BAK1 pools and BR receptor distribution., (Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.)

Polar nuclear migration is crucial during the development of diverse eukaryotes. In plants, root hair growth requires polar nuclear migration into the outgrowing hair. However, knowledge about the dynamics and the regulatory mechanisms underlying nuclear movements in root epidermal cells remains limited. Here, we show that both auxin and Rho-of-Plant (ROP) signaling modulate polar nuclear position at the inner epidermal plasma membrane domain oriented to the cortical cells during cell elongation as well as subsequent polar nuclear movement to the outer domain into the emerging hair bulge in Arabidopsis ( Arabidopsis thaliana ). Auxin signaling via the nuclear AUXIN RESPONSE FACTOR7 (ARF7)/ARF19 and INDOLE ACETIC ACID7 pathway ensures correct nuclear placement toward the inner membrane domain. Moreover, precise inner nuclear placement relies on SPIKE1 Rho-GEF, SUPERCENTIPEDE1 Rho-GDI, and ACTIN7 (ACT7) function and to a lesser extent on VTI11 vacuolar SNARE activity. Strikingly, the directionality and/or velocity of outer polar nuclear migration into the hair outgrowth along actin strands also are ACT7 dependent, auxin sensitive, and regulated by ROP signaling. Thus, our findings provide a founding framework revealing auxin and ROP signaling of inner polar nuclear position with some contribution by vacuolar morphology and of actin-dependent outer polar nuclear migration in root epidermal hair cells., (© 2018 The author(s). All Rights Reserved.)

The epidermis of aerial plant organs is thought to be limiting for growth, because it acts as a continuous load-bearing layer, resisting tension. Leaf epidermis contains jigsaw puzzle piece-shaped pavement cells whose shape has been proposed to be a result of subcellular variations in expansion rate that induce local buckling events. Paradoxically, such local compressive buckling should not occur given the tensile stresses across the epidermis. Using computational modeling, we show that the simplest scenario to explain pavement cell shapes within an epidermis under tension must involve mechanical wall heterogeneities across and along the anticlinal pavement cell walls between adjacent cells. Combining genetics, atomic force microscopy, and immunolabeling, we demonstrate that contiguous cell walls indeed exhibit hybrid mechanochemical properties. Such biochemical wall heterogeneities precede wall bending. Altogether, this provides a possible mechanism for the generation of complex plant cell shapes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The outermost cell layer of plants, the epidermis, and its outer (lateral) membrane domain facing the environment are continuously challenged by biotic and abiotic stresses. Therefore, the epidermis and the outer membrane domain provide important selective and protective barriers. However, only a small number of specifically outer membrane-localized proteins are known. Similarly, molecular mechanisms underlying the trafficking and the polar placement of outer membrane domain proteins require further exploration. Here, we demonstrate that ACTIN7 (ACT7) mediates trafficking of the PENETRATION3 (PEN3) outer membrane protein from the trans-Golgi network (TGN) to the plasma membrane in the root epidermis of Arabidopsis (Arabidopsis thaliana) and that actin function contributes to PEN3 endocytic recycling. In contrast to such generic ACT7-dependent trafficking from the TGN, the EXOCYST84b (EXO84b) tethering factor mediates PEN3 outer-membrane polarity. Moreover, precise EXO84b placement at the outer membrane domain itself requires ACT7 function. Hence, our results uncover spatially and mechanistically distinct requirements for ACT7 function during outer lateral membrane cargo trafficking and polarity establishment. They further identify an exocyst tethering complex mediator of outer lateral membrane cargo polarity., (© 2016 The author(s). All Rights Reserved.)

ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane.

Secretion is the cellular process present in every organism that delivers soluble proteins and cargoes to the extracellular space. In eukaryotes, conventional protein secretion (CPS) is the trafficking route that secretory proteins undertake when are transported from the endoplasmic reticulum (ER) to the Golgi apparatus (GA), and subsequently to the plasma membrane (PM) via secretory vesicles or secretory granules. This book chapter recalls the fundamental steps in cell biology research contributing to the elucidation of CPS; it describes the most prominent examples of conventionally secreted proteins in eukaryotic cells and the molecular mechanisms necessary to regulate each step of this process.

Spatial regulation of the plant hormone indole-3-acetic acid (IAA, or auxin) is essential for plant development. Auxin gradient establishment is mediated by polarly localized auxin transporters, including PIN-FORMED (PIN) proteins. Their localization and abundance at the plasma membrane are tightly regulated by endomembrane machinery, especially the endocytic and recycling pathways mediated by the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM. We assessed the role of the early secretory pathway in establishing PIN1 polarity in Arabidopsis thaliana by pharmacological and genetic approaches. We identified the compound endosidin 8 (ES8), which selectively interferes with PIN1 basal polarity without altering the polarity of apical proteins. ES8 alters the auxin distribution pattern in the root and induces a strong developmental phenotype, including reduced root length. The ARF-GEF-defective mutants gnom-like 1 (gnl1-1) and gnom (van7) are significantly resistant to ES8. The compound does not affect recycling or vacuolar trafficking of PIN1 but leads to its intracellular accumulation, resulting in loss of PIN1 basal polarity at the plasma membrane. Our data confirm a role for GNOM in endoplasmic reticulum (ER)-Golgi trafficking and reveal that a GNL1/GNOM-mediated early secretory pathway selectively regulates PIN1 basal polarity establishment in a manner essential for normal plant development.

The endoplasmic reticulum (ER) represents the gateway for intracellular trafficking of membrane proteins, soluble cargoes and lipids. In all eukaryotes, the best described mechanism of exiting the ER is via COPII-coated vesicles, which transport both membrane proteins and soluble cargoes to the cis-Golgi. The vacuole, together with the plasma membrane, is the most distal point of the secretory pathway, and many vacuolar proteins are transported from the ER through intermediate compartments. However, past results and recent findings demonstrate the presence of alternative transport routes from the ER towards the tonoplast, which are independent of Golgi- and post-Golgi trafficking. Moreover, the transport mechanism of the vacuolar proton pumps VHA-a3 and AVP1 challenges the current model of vacuole biogenesis, pointing to the endoplasmic reticulum for being the main membrane source for the biogenesis of the plant lytic compartment. This review gives an overview of the current knowledge on the transport routes towards the vacuole and discusses the possible mechanism of vacuole biogenesis in plants.

Vacuoles are multifunctional organelles essential for the sessile lifestyle of plants. Despite their central functions in cell growth, storage, and detoxification, knowledge about mechanisms underlying their biogenesis and associated protein trafficking pathways remains limited. Here, we show that in meristematic cells of the Arabidopsis thaliana root, biogenesis of vacuoles as well as the trafficking of sterols and of two major tonoplast proteins, the vacuolar H(+)-pyrophosphatase and the vacuolar H(+)-adenosinetriphosphatase, occurs independently of endoplasmic reticulum (ER)-Golgi and post-Golgi trafficking. Instead, both pumps are found in provacuoles that structurally resemble autophagosomes but are not formed by the core autophagy machinery. Taken together, our results suggest that vacuole biogenesis and trafficking of tonoplast proteins and lipids can occur directly from the ER independent of Golgi function.

The subcellular localization of the sorting nexins (SNXs) in higher plants is a matter of controversy. Previous confocal laser scanning microscopy (CLSM studies on root cells from a transgenic Arabidopsis line expressing SNX1-GFP have suggested that this SNX is present on an endosome having characteristics of both the trans-Golgi network (TGN) and the multivesicular body (MVB). In contrast, SNX2a locates exclusively to the TGN when transiently expressed in tobacco mesophyll protoplasts. By performing immunogold electron microscopy on cryofixed Arabidopsis roots, we have tried to clarify the situation. Both SNX1-GFP and endogenous SNX2a locate principally to the TGN. Labeling of MVBs could not be confirmed with any certainty.

PDMP (D-L-threo-1-phenyl-2-decanoyl amino-3-morpholino-1-propanol) is a well-known inhibitor of glucosylceramide synthase (GCS), a key enzyme in sphingolipid biosynthesis. Through the resultant increase in ceramides which interact with mTOR and Beclin1 (Atg6), this drug is also known to induce macroautophagy in mammalian cells. This study investigated the response of Arabidopsis root cells to PDMP, and what are probably numerous tightly packed small vacuoles in the control cells appear to fuse to form a single globular-shaped vacuole. However, during this fusion process, cytoplasm channels between the individual vacuoles become trapped in deep invaginations of the tonoplast. In both optical sections in the confocal laser scanning microscope and in ultrathin sections in the electron microscope, these invaginations have the appearance of cytoplasmic inclusions in the vacuole lumen. These changes in vacuole morphology are rapid (occurring within minutes after application of PDMP) and are independent of ongoing protein synthesis. The tonoplast invaginations remain visible for hours, but after 24h almost all disappear. Experiments designed to examine whether ceramide levels might be the cause of the PDMP effect have not proved conclusive. On the other hand, this study has been able to rule out the release of Ca(2+) ions from intracellular stores as a contributing factor.

Posttranscriptional gene silencing (PTGS) mediated by siRNAs is an evolutionarily conserved antiviral defense mechanism in higher plants and invertebrates. In this mechanism, viral-derived siRNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation of the corresponding viral RNAs. In Arabidopsis, a key component of RISC is ARGONAUTE1 (AGO1), which not only binds to siRNAs but also carries the RNA slicer activity. At present little is known about posttranslational mechanisms regulating AGO1 turnover. Here we report that the viral suppressor of RNA silencing protein P0 triggers AGO1 degradation by the autophagy pathway. Using a P0-inducible transgenic line, we observed that AGO1 degradation is blocked by inhibition of autophagy. The engineering of a functional AGO1 fluorescent reporter protein further indicated that AGO1 colocalizes with autophagy-related (ATG) protein 8a (ATG8a) positive bodies when degradation is impaired. Moreover, this pathway also degrades AGO1 in a nonviral context, especially when the production of miRNAs is impaired. Our results demonstrate that a selective process such as ubiquitylation can lead to the degradation of a key regulatory protein such as AGO1 by a degradation process generally believed to be unspecific. We anticipate that this mechanism will not only lead to degradation of AGO1 but also of its associated proteins and eventually small RNAs.

Retromer is a cytosolic protein complex which binds to post-Golgi organelles involved in the trafficking of proteins to the lytic compartment of the cell. In non-plant organisms, retromer mediates the recycling of acid hydrolase receptors from early endosomal (EE) compartments. In plants, retromer components are required for the targeting of vacuolar storage proteins, and for the recycling of endocytosed PIN proteins. However, there are contradictory reports as to the localization of the sorting nexins and the core subunit of retromer. There is also uncertainty as to the identity of the organelles from which vacuolar sorting receptors (VSRs) and endocytosed plasma membrane (PM) proteins are recycled. In this review we try to resolve some of these conflicting observations., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The plant trans-Golgi network/early endosome (TGN/EE) is a major hub for secretory and endocytic trafficking with complex molecular mechanisms controlling sorting and transport of cargo. Vacuolar transport from the TGN/EE to multivesicular bodies/late endosomes (MVBs/LEs) is assumed to occur via clathrin-coated vesicles, although direct proof for their participation is missing. Here, we present evidence that post-TGN transport toward lytic vacuoles occurs independently of clathrin and that MVBs/LEs are derived from the TGN/EE through maturation. We show that the V-ATPase inhibitor concanamycin A significantly reduces the number of MVBs and causes TGN and MVB markers to colocalize in Arabidopsis thaliana roots. Ultrastructural analysis reveals the formation of MVBs from the TGN/EE and their fusion with the vacuole. The localization of the ESCRT components VPS28, VPS22, and VPS2 at the TGN/EE and MVBs/LEs indicates that the formation of intraluminal vesicles starts already at the TGN/EE. Accordingly, a dominant-negative mutant of VPS2 causes TGN and MVB markers to colocalize and blocks vacuolar transport. RNA interference-mediated knockdown of the annexin ANNAT3 also yields the same phenotype. Together, these data indicate that MVBs originate from the TGN/EE in a process that requires the action of ESCRT for the formation of intraluminal vesicles and annexins for the final step of releasing MVBs as a transport carrier to the vacuole.

Interphase nuclear architecture is disrupted and rapidly reformed with each cell division cycle. Successive cell generations exhibit a "memory" of this nuclear architecture, as well as for gene expression. Furthermore, many features of nuclear and mitotic chromosome structure are recognizably species and tissue specific. We wish to know what properties of the underlying chromatin structure may determine these conserved features of nuclear architecture. Employing a particular mouse autoimmune anti-nucleosome monoclonal antibody (PL2-6), combined with deconvolution immunofluorescence microscopy, we present evidence for a unique epitope (involving a ternary complex of histones H2A and H2B and DNA) which is localized only at the exterior chromatin surface of interphase nuclei and mitotic chromosomes in mammalian, invertebrate and plant systems. As only the surface chromatin region is identified with antibody PL2-6, we have assigned it the name "epichromatin". We describe an "epichromatin hypothesis", suggesting that epichromatin may have a unique evolutionary conserved conformation which facilitates interaction with the reforming post-mitotic nuclear envelope and a rapid return of interphase nuclear architecture.

Stresses increasing the load of unfolded proteins that enter the endoplasmic reticulum (ER) trigger a protective response termed the unfolded protein response (UPR). Stromal cell-derived factor2 (SDF2)-type proteins are highly conserved throughout the plant and animal kingdoms. In this study we have characterized AtSDF2 as crucial component of the UPR in Arabidopsis thaliana. Using a combination of biochemical and cell biological methods, we demonstrate that SDF2 is induced in response to ER stress conditions causing the accumulation of unfolded proteins. Transgenic reporter plants confirmed induction of SDF2 during ER stress. Under normal growth conditions SDF2 is highly expressed in fast growing, differentiating cells and meristematic tissues. The increased production of SDF2 due to ER stress and in tissues that require enhanced protein biosynthesis and secretion, and its association with the ER membrane qualifies SDF2 as a downstream target of the UPR. Determination of the SDF2 three-dimensional crystal structure at 1.95 A resolution revealed the typical beta-trefoil fold with potential carbohydrate binding sites. Hence, SDF2 might be involved in the quality control of glycoproteins. Arabidopsis sdf2 mutants display strong defects and morphological phenotypes during seedling development specifically under ER stress conditions, thus establishing that SDF2-type proteins play a key role in the UPR.

Plants constantly adjust their repertoire of plasma membrane proteins that mediates transduction of environmental and developmental signals as well as transport of ions, nutrients, and hormones. The importance of regulated secretory and endocytic trafficking is becoming increasingly clear; however, our knowledge of the compartments and molecular machinery involved is still fragmentary. We used immunogold electron microscopy and confocal laser scanning microscopy to trace the route of cargo molecules, including the BRASSINOSTEROID INSENSITIVE1 receptor and the REQUIRES HIGH BORON1 boron exporter, throughout the plant endomembrane system. Our results provide evidence that both endocytic and secretory cargo pass through the trans-Golgi network/early endosome (TGN/EE) and demonstrate that cargo in late endosomes/multivesicular bodies is destined for vacuolar degradation. Moreover, using spinning disc microscopy, we show that TGN/EEs move independently and are only transiently associated with an individual Golgi stack.

Receptor-mediated sorting processes in the secretory pathway of eukaryotic cells rely on mechanisms to recycle the receptors after completion of transport. Based on this principle, plant vacuolar sorting receptors (VSRs) are thought to recycle after dissociating of receptor-ligand complexes in a pre-vacuolar compartment. This recycling is mediated by retromer, a cytosolic coat complex that comprises sorting nexins and a large heterotrimeric subunit. To analyse retromer-mediated VSR recycling, we have used a combination of immunoelectron and fluorescence microscopy to localize the retromer components sorting nexin 1 (SNX1) and sorting nexin 2a (SNX2a) and the vacuolar sorting protein VPS29p. All retromer components localize to the trans-Golgi network (TGN), which is considered to represent the early endosome of plants. In addition, we show that inhibition of retromer function in vivo by expression of SNX1 or SNX2a mutants as well as transient RNAi knockdown of all sorting nexins led to accumulation of the VSR BP80 at the TGN. Quantitative protein transport studies and live-cell imaging using fluorescent vacuolar cargo molecules revealed that arrival of these VSR ligands at the vacuole is not affected under these conditions. Based on these findings, we propose that the TGN is the location of retromer-mediated recycling of VSRs, and that transport towards the lytic vacuole downstream of the TGN is receptor-independent and occurs via maturation, similar to transition of the early endosome into the late endosome in mammalian cells.

Overexpression of the Golgi and endoplasmic reticulum (ER) syntaxins SYP31 and SYP81 strongly inhibits constitutive secretion. By comparing the secreted reporter alpha-amylase with the ER-retained reporter alpha-amylase-HDEL, it was concluded that SYP81 overexpression inhibits both retrograde and anterograde transport, while SYP31 overexpression mainly affected anterograde transport. Of the other interacting SNAREs investigated, only the overexpression of MEMB11 led to an inhibition of protein secretion. Although the position of a fluorescent tag does not influence the correct localization of the fusion protein, only N-terminal-tagged SYP31 retained the ability of the untagged SNARE to inhibit transport. C-terminal-tagged SYP31 failed to exhibit this effect. Overexpression of both wild-type and N-terminal-tagged syntaxins caused standard Golgi marker proteins to redistribute into the ER. Nevertheless, green fluorescent protein (GFP)-SYP31 was still visible as fluorescent punctae, which, unlike SYP31-GFP, were resistant to brefeldin A treatment. Immunogold electron microscopy showed that endogenous SYP81 is not only present at the ER but also in the cis Golgi, indicating that this syntaxin cycles between these two organelles. However, when expressed at non-inhibitory levels, YFP-SYP81 was seen to locate principally to subdomains of the ER. These punctate structures were physically separated from the Golgi, suggesting that they might possibly reflect the position of ER import sites.

The maize seed comprises two major compartments, the embryo and the endosperm, both originating from the double fertilization event. The embryogenetic process allows the formation of a well-differentiated embryonic axis, surrounded by a single massive cotyledon, the scutellum. The mature endosperm constitutes the bulk of the seed and comprises specific regions containing reserve proteins, complex carbohydrates, and oils. To gain more insight into molecular events that underlie seed development, three monogenic mutants were characterized, referred to as emp (empty pericarp) on the basis of their extreme endosperm reduction, first recognizable at about 12 d after pollination. Their histological analysis reveals a partial development of the endosperm domains as well as loss of adhesion between pedicel tissues and the basal transfer layer. In the endosperm, programmed cell death (PCD) is delayed. The embryo appears retarded in its growth, but not impaired in its morphogenesis. The mutants can be rescued by culturing immature embryos, even though the seedlings appear retarded in their growth. The analysis of seeds with discordant embryo-endosperm phenotype (mutant embryo, normal endosperm and vice-versa), obtained using B-A translocations, suggests that emp expression in the embryo is necessary, but not sufficient, for proper seed development. In all three mutants the picture emerging is one of a general delay in processes related to growth, as a result of a mutation affecting endosperm development as a primary event.

Proton pump interactor, isoform 1 (PPI1) is a novel interactor of the C-terminus of Arabidopsis thaliana plasma membrane H(+)-ATPase (EC 3.6.3.6). We produced two fusion proteins consisting of, respectively, the first 88 amino acids or the entire protein deleted of the last 24 hydrophobic amino acids, and we show that the latter protein has a threefold higher affinity for the H(+)-ATPase. PPI1-induced stimulation of H(+)-ATPase activity dramatically decreased with the increase of pH above pH 6.8, but became largely pH-independent when the enzyme C-terminus was displaced by fusicoccin-induced binding of 14-3-3 proteins. The latter treatment did not affect PPI1 affinity for the H(+)-ATPase. These results indicate that PPI1 can bind the H(+)-ATPase independently of the C-terminus conformation, but is not able to suppress the C-terminus auto-inhibitory action.

Antagonism is the ability of a modified antigenic peptide (altered peptide ligand, APL) to prevent CD4 T cell activation by the original peptide. Here we show that antagonistic activity can be conferred to peptides of HIV envelope glycoprotein gp120 and reverse transcriptase p66 by adding flanking polypeptide sequences at the C or at the N terminus by genetic engineering, rather than by introducing substitutions by synthesis. The glutathione S-transferase (GST)-peptide system has been used to produce molecules that display the peptide at the appropriate end of the GST carrier. When the gp120 peptide 191-205 (pep24) was expressed at the C terminus of GST (GST-24), antigenicity of specific human CD4 T cells was maintained. In contrast, when the peptide was expressed at the N terminus of GST (24-GST), antigenicity was abolished and antagonistic activity was introduced. Similar results were obtained with a p66-derived peptide at the C terminus of the GST carrier. Antagonism was (1) specific; proliferation of a CD4 T cell line from the same donor responding to the envelope glycoprotein of another retrovirus, HTLV-1, was not affected; (2) reversible; proliferative response was rescued in T cells exposed to antigen-presenting cells (APC) pulsed with the antagonist; (3) dominant; T cells cultured with APC pulsed with the agonist and with APC pulsed with the antagonist did not proliferate. The carrier could be cleaved by proteolysis while the antagonistic activity was preserved. Thus a minimal sequence that confers antagonistic activity can be engineered or synthesized with peptides to antagonize undesired CD4 responses as an alternative to the use of APL.