RESUMEN
Seeds of dicotyledonous plants store proteins in dedicated membrane-bounded organelles called protein storage vacuoles (PSVs). Formed during seed development through morphological and functional reconfiguration of lytic vacuoles in embryos [M. Feeney et al., Plant Physiol. 177, 241-254 (2018)], PSVs undergo division during the later stages of seed maturation. Here, we study the biophysical mechanism of PSV morphogenesis in vivo, discovering that micrometer-sized liquid droplets containing storage proteins form within the vacuolar lumen through phase separation and wet the tonoplast (vacuolar membrane). We identify distinct tonoplast shapes that arise in response to membrane wetting by droplets and derive a simple theoretical model that conceptualizes these geometries. Conditions of low membrane spontaneous curvature and moderate contact angle (i.e., wettability) favor droplet-induced membrane budding, thereby likely serving to generate multiple, physically separated PSVs in seeds. In contrast, high membrane spontaneous curvature and strong wettability promote an intricate and previously unreported membrane nanotube network that forms at the droplet interface, allowing molecule exchange between droplets and the vacuolar interior. Furthermore, our model predicts that with decreasing wettability, this nanotube structure transitions to a regime with bud and nanotube coexistence, which we confirmed in vitro. As such, we identify intracellular wetting [J. Agudo-Canalejo et al., Nature 591, 142-146 (2021)] as the mechanism underlying PSV morphogenesis and provide evidence suggesting that interconvertible membrane wetting morphologies play a role in the organization of liquid phases in cells.
Asunto(s)
Magnoliopsida/metabolismo , Semillas/crecimiento & desarrollo , Vacuolas/metabolismo , Membranas Intracelulares/metabolismo , Nanotubos , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Semillas/metabolismo , HumectabilidadRESUMEN
Precise measurements of dynamic changes in free Ca2+ concentration in the lumen of the plant endoplasmic reticulum (ER) have been lacking so far, despite increasing evidence for the contribution of this intracellular compartment to Ca2+ homeostasis and signalling in the plant cell. In the present study, we targeted an aequorin chimera with reduced Ca2+ affinity to the ER membrane and facing the ER lumen. To this aim, the cDNA for a low-Ca2+ -affinity aequorin variant (AEQmut) was fused to the nucleotide sequence encoding a non-cleavable N-terminal ER signal peptide (fl2). The correct targeting of fl2-AEQmut was confirmed by immunocytochemical analyses in transgenic Arabidopsis thaliana (Arabidopsis) seedlings. An experimental protocol well-established in animal cells - consisting of ER Ca2+ depletion during photoprotein reconstitution followed by ER Ca2+ refilling - was applied to carry out ER Ca2+ measurements in planta. Rapid and transient increases of the ER luminal Ca2+ concentration ([Ca2+ ]ER ) were recorded in response to different environmental stresses, displaying stimulus-specific Ca2+ signatures. The comparative analysis of ER and chloroplast Ca2+ dynamics indicates a complex interplay of these organelles in shaping cytosolic Ca2+ signals during signal transduction events. Our data highlight significant differences in basal [Ca2+ ]ER and Ca2+ handling by plant ER compared to the animal counterpart. The set-up of an ER-targeted aequorin chimera extends and complements the currently available toolkit of organelle-targeted Ca2+ indicators by adding a reporter that improves our quantitative understanding of Ca2+ homeostasis in the plant endomembrane system.
Asunto(s)
Aequorina/metabolismo , Arabidopsis/metabolismo , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Aequorina/genética , Animales , Arabidopsis/genética , Cloroplastos/metabolismo , Citosol/metabolismo , Homeostasis , Proteínas Luminiscentes/metabolismo , Plantones/metabolismoRESUMEN
Accumulating evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synthesized around the chloroplasts of many photosynthetic eukaryotes, from glaucophyte algae to early-diverging land plants including pteridophyte ferns, but the biosynthetic pathway has not been demonstrated. Here, we employed mass spectrometry and enzymology in a two-fold approach to characterize the synthesis of peptidoglycan in chloroplasts of the moss Physcomitrium (Physcomitrella) patens. To drive the accumulation of peptidoglycan pathway intermediates, P. patens was cultured with the antibiotics fosfomycin, D-cycloserine, and carbenicillin, which inhibit key peptidoglycan pathway proteins in bacteria. Mass spectrometry of the trichloroacetic acid-extracted moss metabolome revealed elevated levels of five of the predicted intermediates from uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) through the uridine diphosphate N-acetylmuramic acid (UDP-MurNAc)-D,L-diaminopimelate (DAP)-pentapeptide. Most Gram-negative bacteria, including cyanobacteria, incorporate meso-diaminopimelic acid (D,L-DAP) into the third residue of the stem peptide of peptidoglycan, as opposed to L-lysine, typical of most Gram-positive bacteria. To establish the specificity of D,L-DAP incorporation into the P. patens precursors, we analyzed the recombinant protein UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase (MurE) from both P. patens and the cyanobacterium Anabaena sp. (Nostoc sp. strain PCC 7120). Both ligases incorporated D,L-DAP in almost complete preference to L-Lys, consistent with the mass spectrophotometric data, with catalytic efficiencies similar to previously documented Gram-negative bacterial MurE ligases. We discuss how these data accord with the conservation of active site residues common to DL-DAP-incorporating bacterial MurE ligases and of the probability of a horizontal gene transfer event within the plant peptidoglycan pathway.
Asunto(s)
Pared Celular , Peptidoglicano , Bacterias/metabolismo , Pared Celular/metabolismo , Cloroplastos/metabolismo , Bacterias Gramnegativas/metabolismo , Ligasas/metabolismo , Lisina/metabolismo , Peptidoglicano/química , Peptidoglicano/genética , Peptidoglicano/metabolismo , Uridina Difosfato/metabolismoRESUMEN
The endoplasmic reticulum (ER) is the entry point to the secretory pathway and, as such, is critical for adaptive responses to biotic stress, when the demand for de novo synthesis of immunity-related proteins and signalling components increases significantly. Successful phytopathogens have evolved an arsenal of small effector proteins which collectively reconfigure multiple host components and signalling pathways to promote virulence; a small, but important, subset of which are targeted to the endomembrane system including the ER. We identified and validated a conserved C-terminal tail-anchor motif in a set of pathogen effectors known to localize to the ER from the oomycetes Hyaloperonospora arabidopsidis and Plasmopara halstedii (downy mildew of Arabidopsis and sunflower, respectively) and used this protein topology to develop a bioinformatic pipeline to identify putative ER-localized effectors within the effectorome of the related oomycete, Phytophthora infestans, the causal agent of potato late blight. Many of the identified P. infestans tail-anchor effectors converged on ER-localized NAC transcription factors, indicating that this family is a critical host target for multiple pathogens.
Asunto(s)
Oomicetos , Phytophthora infestans , Plantas/metabolismo , Factores de Transcripción/metabolismo , Virulencia , Retículo Endoplásmico/metabolismo , Enfermedades de las PlantasRESUMEN
Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron spice owes its red color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up to 10% of the spice dry weight. We developed a general approach, based on coexpression analysis, heterologous expression in yeast (Saccharomyces cerevisiae), and in vitro transportomic assays using yeast microsomes and total plant metabolite extracts, for the identification of putative vacuolar metabolite transporters, and we used it to identify Crocus sativus transporters mediating vacuolar crocin accumulation in stigmas. Three transporters, belonging to both the multidrug and toxic compound extrusion and ATP binding cassette C (ABCC) families, were coexpressed with crocins and/or with the gene encoding the first dedicated enzyme in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCC family, were able to mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectively expressed in C. sativus stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific and cooperative way, and was able to enhance crocin accumulation when expressed in Nicotiana benthamiana leaves.plantcell;31/11/2789/FX1F1fx1.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Carotenoides/metabolismo , Crocus/metabolismo , Proteínas de Plantas/metabolismo , Vacuolas/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Vías Biosintéticas , Clonación Molecular , Crocus/genética , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas , Cinética , Extractos Vegetales , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Distribución Tisular/fisiología , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
Protein storage vacuoles (PSV) are the main repository of protein in dicotyledonous seeds, but little is known about the origins of these transient organelles. PSV are hypothesized to either arise de novo or originate from the preexisting embryonic vacuole (EV) during seed maturation. Here, we tested these hypotheses by studying PSV formation in Arabidopsis (Arabidopsis thaliana) embryos at different stages of seed maturation and recapitulated this process in Arabidopsis leaves reprogrammed to an embryogenic fate by inducing expression of the LEAFY COTYLEDON2 transcription factor. Confocal and immunoelectron microscopy indicated that both storage proteins and tonoplast proteins typical of PSV were delivered to the preexisting EV in embryos or to the lytic vacuole in reprogrammed leaf cells. In addition, sectioning through embryos at several developmental stages using serial block face scanning electron microscopy revealed the 3D architecture of forming PSV. Our results indicate that the preexisting EV is reprogrammed to become a PSV in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Proteínas de Almacenamiento de Semillas/metabolismo , Semillas/citología , Vacuolas/metabolismo , Acuaporinas/genética , Acuaporinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Concentración de Iones de Hidrógeno , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , Proteínas de Almacenamiento de Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Saffron is the dried stigmas of Crocus sativus and is the most expensive spice in the world. Its red color is due to crocins, which are apocarotenoid glycosides that accumulate in the vacuole to a level up to 10% of the stigma dry weight. Previously, we characterized the first dedicated enzyme in the crocin biosynthetic pathway, carotenoid cleavage dioxygenase2 (CsCCD2), which cleaves zeaxanthin to yield crocetin dialdehyde. In this work, we identified six putative aldehyde dehydrogenase (ALDH) genes expressed in C. sativus stigmas. Heterologous expression in Escherichia coli showed that only one of corresponding proteins (CsALDH3I1) was able to convert crocetin dialdehyde into the crocin precursor crocetin. CsALDH3I1 carries a carboxyl-terminal hydrophobic domain, similar to that of the Neurospora crassa membrane-associated apocarotenoid dehydrogenase YLO-1. We also characterized the UDP-glycosyltransferase CsUGT74AD1, which converts crocetin to crocins 1 and 2'. In vitro assays revealed high specificity of CsALDH3I1 for crocetin dialdehyde and long-chain apocarotenals and of CsUGT74AD1 for crocetin. Following extract fractionation, CsCCD2, CsALDH3I1, and CsUGT74AD1 were found in the insoluble fraction, suggesting their association with membranes or large insoluble complexes. Analysis of protein localization in both C. sativus stigmas and following transgene expression in Nicotiana benthamiana leaves revealed that CsCCD2, CsALDH3I, and CsUGT74AD1 were localized to the plastids, the endoplasmic reticulum, and the cytoplasm, respectively, in association with cytoskeleton-like structures. Based on these findings and current literature, we propose that the endoplasmic reticulum and cytoplasm function as transit centers for metabolites whose biosynthesis starts in the plastid and are accumulated in the vacuole.
Asunto(s)
Aldehído Deshidrogenasa/metabolismo , Carotenoides/biosíntesis , Crocus/metabolismo , Glicosiltransferasas/metabolismo , Proteínas de Plantas/metabolismo , Aldehído Deshidrogenasa/genética , Carotenoides/metabolismo , Crocus/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glicosilación , Glicosiltransferasas/genética , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica/métodos , Microscopía Confocal , Especificidad de Órganos , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Nicotiana/genética , Vitamina A/análogos & derivadosRESUMEN
Aquaporins influence water flow in plants, yet little is known of their involvement in the water-driven process of seed germination. We therefore investigated their role in seeds in the laboratory and under field and global warming conditions. We mapped the expression of tonoplast intrinsic proteins (TIPs) during dormancy cycling and during germination under normal and water stress conditions. We found that the two key tonoplast aquaporins, TIP3;1 and TIP3;2, which have previously been implicated in water or solute transport, respectively, act antagonistically to modulate the response to abscisic acid, with TIP3;1 being a positive and TIP3;2 a negative regulator. A third isoform, TIP4;1, which is normally expressed upon completion of germination, was found to play an earlier role during water stress. Seed TIPs also contribute to the regulation of depth of primary dormancy and differences in the induction of secondary dormancy during dormancy cycling. Protein and gene expression during annual cycling under field conditions and a global warming scenario further illustrate this role. We propose that the different responses of the seed TIP contribute to mechanisms that influence dormancy status and the timing of germination under variable soil conditions.
Asunto(s)
Acuaporinas/fisiología , Proteínas de Arabidopsis/fisiología , Arabidopsis/fisiología , Estrés Fisiológico , Ácido Abscísico/metabolismo , Acuaporinas/genética , Acuaporinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ambiente , Regulación del Desarrollo de la Expresión Génica , Germinación , Calentamiento Global , Proteínas de la Membrana/metabolismo , Latencia en las Plantas , Reguladores del Crecimiento de las Plantas/metabolismo , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Semillas/genética , Semillas/metabolismo , Semillas/fisiología , Temperatura , Agua/metabolismoRESUMEN
Reticulons (RTNs) are a class of endoplasmic reticulum (ER) membrane proteins that are capable of maintaining high membrane curvature, thus helping shape the ER membrane into tubules. The mechanism of action of RTNs is hypothesized to be a combination of wedging, resulting from the transmembrane topology of their conserved reticulon homology domain, and scaffolding, arising from the ability of RTNs to form low-mobility homo-oligomers within the membrane. We studied the plant RTN isoform RTN13, which has previously been shown to locate to ER tubules and the edges of ER cisternae and to induce constrictions in ER tubules when overexpressed, and identified a region in the C terminus containing a putative amphipathic helix (APH). Here we show that deletion of this region or disruption of the hydrophobic face of the predicted helix abolishes the ability of RTN13 to induce constrictions of ER tubules in vivo. These mutants, however, still retain their ability to interact and form low-mobility oligomers in the ER membrane. Hence, our evidence indicates that the conserved APH is a key structural feature for RTN13 function in vivo, and we propose that RTN, like other membrane morphogens, rely on APHs for their function.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Retículo Endoplásmico/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada , Transferencia Resonante de Energía de Fluorescencia , Interacciones Hidrofóbicas e Hidrofílicas , Membranas Intracelulares/metabolismo , Mutación/genética , Epidermis de la Planta/citología , Estructura Secundaria de Proteína , Eliminación de Secuencia , Relación Estructura-Actividad , Nicotiana/citologíaRESUMEN
The plant endoplasmic reticulum (ER) is crucial to the maintenance of cellular homeostasis. The ER consists of a dynamic and continuously remodelling network of tubules and cisternae. Several conserved membrane proteins have been implicated in formation and maintenance of the ER network in plants, such as RHD3 and the reticulon proteins. Despite the recent work in mammalian and yeast cells, the detailed molecular mechanisms of ER network organization in plants remain largely unknown. Recently, novel ER network-shaping proteins called Lunapark (LNP) have been identified in yeast and mammalian cells. Here we identify two Arabidopsis LNP homologues and investigate their subcellular localization via confocal microscopy and potential function in shaping the ER network using protein-protein interaction assays and mutant analysis. We show that AtLNP1 overexpression in tobacco leaf epidermal cells mainly labels cisternae in the ER network, whereas AtLNP2 labels the whole ER. Overexpression of LNP proteins results in an increased abundance of ER cisternae and lnp1 and lnp1lnp2 amiRNA lines display a reduction in cisternae and larger polygonal areas. Thus, we hypothesize that AtLNP1 and AtLNP2 are involved in determining the network morphology of the plant ER, possibly by regulating the formation of ER cisternae.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Retículo Endoplásmico/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Mutación con Pérdida de Función/genética , MicroARNs/genética , MicroARNs/metabolismo , Unión ProteicaRESUMEN
Reticulons are a large family of integral membrane proteins that are ubiquitous in eukaryotes and play a key role in functional remodelling of the endoplasmic reticulum membrane. The reticulon family is especially large in plants, with the Arabidopsis thaliana genome containing twenty-one isoforms. Reticulons vary in length but all contain a conserved C-terminal reticulon homology domain (RHD) that associates with membranes. An understanding of the structure and membrane interactions of RHDs is key to unlocking their mechanism of function, however no three-dimensional structure has been solved. We believe that this is, in part, due to difficulties in obtaining reticulon proteins in yields sufficient for structural study. To address this, we report here the first bacterial overexpression, purification, and biophysical investigation of a reticulon protein from plants, the RTNLB13 protein from A. thaliana. RTNLB13 is the smallest plant reticulon and is made up of a single RHD. We used circular dichroism, SDS-PAGE and analytical ultracentrifugation to reveal that RTNLB13 is 45% α-helical in a number of detergent environments, monomeric at low concentrations, and capable of self-association at higher concentrations. We used solution-state NMR to screen the effect of detergent type on the fold of isotopically-enriched RTNLB13, and found that â¼60% of the expected protein peaks were broadened due to slow dynamics. This broadening points toward a large network of protein-membrane interactions throughout the sequence. We have interpreted our results in light of current literature and suggest a preliminary description of RTNLB13 structure and topology.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/química , Proteínas de la Membrana/química , Proteínas Recombinantes de Fusión/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/biosíntesis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Cromatografía en Gel/métodos , Clonación Molecular , Detergentes/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Histidina/genética , Histidina/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Micelas , Oligopéptidos/genética , Oligopéptidos/metabolismo , Dominios Proteicos , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Aquaporins (AQPs) are water channels allowing fast and passive diffusion of water across cell membranes. It was hypothesized that AQPs contribute to cell elongation processes by allowing water influx across the plasma membrane and the tonoplast to maintain adequate turgor pressure. Here, we report that, in Arabidopsis (Arabidopsis thaliana), the highly abundant tonoplast AQP isoforms AtTIP1;1, AtTIP1;2, and AtTIP2;1 facilitate the emergence of new lateral root primordia (LRPs). The number of lateral roots was strongly reduced in the triple tip mutant, whereas the single, double, and triple tip mutants showed no or minor reduction in growth of the main root. This phenotype was due to the retardation of LRP emergence. Live cell imaging revealed that tight spatiotemporal control of TIP abundance in the tonoplast of the different LRP cells is pivotal to mediating this developmental process. While lateral root emergence is correlated to a reduction of AtTIP1;1 and AtTIP1;2 protein levels in LRPs, expression of AtTIP2;1 is specifically needed in a restricted cell population at the base, then later at the flanks, of developing LRPs. Interestingly, the LRP emergence phenotype of the triple tip mutants could be fully rescued by expressing AtTIP2;1 under its native promoter. We conclude that TIP isoforms allow the spatial and temporal fine-tuning of cellular water transport, which is critically required during the highly regulated process of LRP morphogenesis and emergence.
Asunto(s)
Acuaporinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Raíces de Plantas/metabolismo , Vacuolas/metabolismo , Acuaporinas/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Transporte Biológico/genética , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Meristema/genética , Meristema/crecimiento & desarrollo , Meristema/metabolismo , Microscopía Confocal , Mutación , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vacuolas/genética , Agua/metabolismoRESUMEN
Natural rubber (polyisoprene) from the rubber tree Hevea brasiliensis is synthesized by specialized cells called laticifers. It is not clear how rubber particles arise, although one hypothesis is that they derive from the endoplasmic reticulum (ER) membrane. Here we cloned the genes encoding four key proteins found in association with rubber particles and studied their intracellular localization by transient expression in Nicotiana benthamiana leaves. We show that, while the cis-prenyltransferase (CPT), responsible for the synthesis of long polyisoprene chains, is a soluble, cytosolic protein, other rubber particle proteins such as rubber elongation factor (REF), small rubber particle protein (SRPP) and Hevea rubber transferase 1-REF bridging protein (HRBP) are associated with the endoplasmic reticulum (ER). We also show that SRPP can recruit CPT to the ER and that interaction of CPT with HRBP leads to both proteins relocating to the plasma membrane. We discuss these results in the context of the biogenesis of rubber particles.
Asunto(s)
Antígenos de Plantas/metabolismo , Hevea/enzimología , Proteínas de Plantas/metabolismo , Goma/metabolismo , Transferasas/metabolismo , Secuencia de Aminoácidos , Antígenos de Plantas/genética , Citosol/enzimología , Retículo Endoplásmico/metabolismo , Genes Reporteros , Hevea/citología , Hevea/genética , Modelos Biológicos , Hojas de la Planta/citología , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Proteínas de Plantas/genética , Alineación de Secuencia , Nicotiana/citología , Nicotiana/genética , Nicotiana/metabolismo , Transferasas/genéticaRESUMEN
X-Intrinsic Proteins (XIP) were recently identified in a narrow range of plants as a full clade within the aquaporins. These channels reportedly facilitate the transport of a wide range of hydrophobic solutes. The functional roles of XIP in planta remain poorly identified. In this study, we found three XIP genes (HbXIP1;1, HbXIP2;1 and HbXIP3;1) in the Hevea brasiliensis genome. Comprehensive bioinformatics, biochemical and structural analyses were used to acquire a better understanding of this AQP subfamily. Phylogenetic analysis revealed that HbXIPs clustered into two major groups, each distributed in a specific lineage of the order Malpighiales. Tissue-specific expression profiles showed that only HbXIP2;1 was expressed in all the vegetative tissues tested (leaves, stem, bark, xylem and latex), suggesting that HbXIP2;1 could take part in a wide range of cellular processes. This is particularly relevant to the rubber-producing laticiferous system, where this isoform was found to be up-regulated during tapping and ethylene treatments. Furthermore, the XIP transcriptional pattern is significantly correlated to latex production level. Structural comparison with SoPIP2;1 from Spinacia oleracea species provides new insights into the possible role of structural checkpoints by which HbXIP2;1 ensures glycerol transfer across the membrane. From these results, we discuss the physiological involvement of glycerol and HbXIP2;1 in water homeostasis and carbon stream of challenged laticifers. The characterization of HbXIP2;1 during rubber tree tapping lends new insights into molecular and physiological response processes of laticifer metabolism in the context of latex exploitation.
Asunto(s)
Acuaporinas/química , Acuaporinas/genética , Genoma de Planta , Hevea/genética , Látex/biosíntesis , Proteínas de Plantas/genética , Acuaporinas/aislamiento & purificación , Biología Computacional , Regulación de la Expresión Génica de las Plantas , Modelos Moleculares , Familia de Multigenes , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología Estructural de Proteína , Fracciones Subcelulares/metabolismoRESUMEN
Primary plasmodesmata (PD) arise at cytokinesis when the new cell plate forms. During this process, fine strands of endoplasmic reticulum (ER) are laid down between enlarging Golgi-derived vesicles to form nascent PD, each pore containing a desmotubule, a membranous rod derived from the cortical ER. Little is known about the forces that model the ER during cell plate formation. Here, we show that members of the reticulon (RTNLB) family of ER-tubulating proteins in Arabidopsis (Arabidopsis thaliana) may play a role in the formation of the desmotubule. RTNLB3 and RTNLB6, two RTNLBs present in the PD proteome, are recruited to the cell plate at late telophase, when primary PD are formed, and remain associated with primary PD in the mature cell wall. Both RTNLBs showed significant colocalization at PD with the viral movement protein of Tobacco mosaic virus, while superresolution imaging (three-dimensional structured illumination microscopy) of primary PD revealed the central desmotubule to be labeled by RTNLB6. Fluorescence recovery after photobleaching studies showed that these RTNLBs are mobile at the edge of the developing cell plate, where new wall materials are being delivered, but significantly less mobile at its center, where PD are forming. A truncated RTNLB3, unable to constrict the ER, was not recruited to the cell plate at cytokinesis. We discuss the potential roles of RTNLBs in desmotubule formation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Pared Celular/metabolismo , Citocinesis , Retículo Endoplásmico/metabolismo , Plasmodesmos/metabolismo , Proteínas de Arabidopsis/genética , Línea Celular , Pared Celular/genética , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Proteínas de Movimiento Viral en Plantas/genética , Proteínas de Movimiento Viral en Plantas/metabolismo , Plantas Modificadas Genéticamente , Plasmodesmos/genética , Transporte de Proteínas , Nicotiana/citología , Nicotiana/genética , Nicotiana/metabolismo , Virus del Mosaico del Tabaco/genética , Virus del Mosaico del Tabaco/metabolismoRESUMEN
The endoplasmic reticulum (ER) is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three-way junctions, and back to tubules. Plant reticulon family proteins (RTNLB) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmata (PD) proteome and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD. Here, we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis in tobacco (Nicotiana tabacum). Coimmunoprecipitation of green fluorescent protein-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localized proteins as well as plasma membrane proteins with putative membrane-anchoring roles. Förster resonance energy transfer by fluorescence lifetime imaging microscopy assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modeling, may play important roles in linking the cortical ER to the plasma membrane.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Nicotiana/metabolismo , Plasmodesmos/metabolismo , Proteínas de Arabidopsis/genética , Transporte Biológico , Membrana Celular/ultraestructura , Retículo Endoplásmico/ultraestructura , Expresión Génica , Proteínas Fluorescentes Verdes , Inmunoprecipitación , Proteínas de la Membrana/genética , Plasmodesmos/ultraestructura , Mapeo de Interacción de Proteínas , Proteómica , Nicotiana/genética , Nicotiana/ultraestructuraRESUMEN
Transient gene expression, in plant protoplasts or specific plant tissues, is a key technique in plant molecular cell biology, aimed at exploring gene products and their modifications to examine functional subdomains, their interactions with other biomolecules, and their subcellular localization. Here, we highlight some of the major advantages and potential pitfalls of the most commonly used transient gene expression models and illustrate how ectopic expression and the use of dominant mutants can provide insights into protein function.
Asunto(s)
Investigación , Vías Secretoras , Transporte Biológico , Colorantes Fluorescentes/metabolismo , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Protoplastos/metabolismo , Protoplastos/ultraestructuraRESUMEN
Embryogenesis in flowering plants is controlled by a complex interplay of genetic, biochemical, and physiological regulators. LEAFY COTYLEDON2 (LEC2) is among a small number of key transcriptional regulators that are known to play important roles in controlling major events during the maturation stage of embryogenesis, notably, the synthesis and accumulation of storage reserves. LEC2 overexpression causes vegetative tissues to change their developmental fate to an embryonic state; however, little information exists about the cellular changes that take place. We show that LEC2 alters leaf morphology and anatomy and causes embryogenic structures to form subcellularly in leaves of Arabidopsis (Arabidopsis thaliana). Chloroplasts accumulate more starch, the cytoplasm fills with oil bodies, and lytic vacuoles (LVs) appear smaller in size and accumulate protein deposits. Because LEC2 is responsible for activating the synthesis of seed storage proteins (SSPs) during seed development, SSP accumulation was investigated in leaves. The major Arabidopsis SSP families were shown to accumulate within small leaf vacuoles. By exploiting the developmental and tissue-specific localization of two tonoplast intrinsic protein isoforms, the small leaf vacuoles were identified as protein storage vacuoles (PSVs). Confocal analyses of leaf vacuoles expressing fluorescently labeled tonoplast intrinsic protein isoforms reveal an altered tonoplast morphology resembling an amalgamation of a LV and PSV. Results suggest that as the LV transitions to a PSV, the tonoplast remodels before the large vacuole lumen is replaced by smaller PSVs. Finally, using vegetative and seed markers to monitor the transition, we show that LEC2 induces a reprogramming of leaf development.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Hojas de la Planta/genética , Factores de Transcripción/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Cotiledón/fisiología , Citoplasma/metabolismo , Regulación de la Expresión Génica de las Plantas , Hojas de la Planta/anatomía & histología , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Proteínas de Almacenamiento de Semillas/genética , Proteínas de Almacenamiento de Semillas/metabolismo , Semillas/genética , Semillas/fisiología , Almidón/metabolismo , Almidón/ultraestructura , Factores de Transcripción/metabolismo , Vacuolas/genética , Vacuolas/metabolismoRESUMEN
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.
Asunto(s)
Arabidopsis/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/metabolismo , Cuerpos Multivesiculares/metabolismo , Red trans-Golgi/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Endocitosis , Cuerpos Multivesiculares/ultraestructura , Raíces de Plantas/metabolismo , Transporte de Proteínas , Vacuolas/metabolismo , Vacuolas/ultraestructura , Red trans-Golgi/ultraestructuraRESUMEN
Imaging plant embryos at the cellular level over time is technically challenging, since the embryo, once its protective seed coat is removed, must be kept viable and unstressed on a microscope slide for the duration of the experiment. Here we describe a procedure and suitable apparatus for the visualization, over several days, of changes in endoplasmic reticulum (ER) morphology associated with the process of germination in Arabidopsis thaliana seeds. Moreover, we also present a user-friendly image analysis tool, which enables subtle perturbations in the ER network to be measured.