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1.
Front Plant Sci ; 13: 833612, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35251104

RESUMO

The molecular machinery orchestrating microautophagy, whereby eukaryotic cells sequester autophagic cargo by direct invagination of the vacuolar/lysosomal membrane, is still largely unknown, especially in plants. Here, we demonstrate microautophagy of storage proteins in the maize aleurone cells of the endosperm and analyzed proteins with potential regulatory roles in this process. Within the cereal endosperm, starchy endosperm cells accumulate storage proteins (mostly prolamins) and starch whereas the peripheral aleurone cells store oils, storage proteins, and specialized metabolites. Although both cell types synthesize prolamins, they employ different pathways for their subcellular trafficking. Starchy endosperm cells accumulate prolamins in protein bodies within the endoplasmic reticulum (ER), whereas aleurone cells deliver prolamins to vacuoles via an autophagic mechanism, which we show is by direct association of ER prolamin bodies with the tonoplast followed by engulfment via microautophagy. To identify candidate proteins regulating this process, we performed RNA-seq transcriptomic comparisons of aleurone and starchy endosperm tissues during seed development and proteomic analysis on tonoplast-enriched fractions of aleurone cells. From these datasets, we identified 10 candidate proteins with potential roles in membrane modification and/or microautophagy, including phospholipase-Dα5 and a possible EUL-like lectin. We found that both proteins increased the frequency of tonoplast invaginations when overexpressed in Arabidopsis leaf protoplasts and are highly enriched at the tonoplast surface surrounding ER protein bodies in maize aleurone cells, thus supporting their potential connections to microautophagy. Collectively, this candidate list now provides useful tools to study microautophagy in plants.

2.
J Exp Bot ; 70(19): 5071-5088, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31145803

RESUMO

Upon imbibition, epidermal cells of Arabidopsis thaliana seeds release a mucilage formed mostly by pectic polysaccharides. The Arabidopsis mucilage is composed mainly of unbranched rhamnogalacturonan-I (RG-I), with low amounts of cellulose, homogalacturonan, and traces of xylan, xyloglucan, galactoglucomannan, and galactan. The pectin-rich composition of the mucilage and their simple extractability makes this structure a good candidate to study the biosynthesis of pectic polysaccharides and their modification. Here, we characterize the mucilage phenotype of a mutant in the UDP-rhamnose/galactose transporter 2 (URGT2), which exhibits a reduction in RG-I and also shows pleiotropic changes, suggesting the existence of compensation mechanisms triggered by the lack of URGT2. To gain an insight into the possible compensation mechanisms activated in the mutant, we performed a transcriptome analysis of developing seeds using RNA sequencing (RNA-seq). The results showed a significant misregulation of 3149 genes, 37 of them (out of the 75 genes described to date) encoding genes proposed to be involved in mucilage biosynthesis and/or its modification. The changes observed in urgt2 included the up-regulation of UAFT2, a UDP-arabinofuranose transporter, and UUAT3, a paralog of the UDP-uronic acid transporter UUAT1, suggesting that they play a role in mucilage biosynthesis. Mutants in both genes showed changes in mucilage composition and structure, confirming their participation in mucilage biosynthesis. Our results suggest that plants lacking a UDP-rhamnose/galactose transporter undergo important changes in gene expression, probably to compensate modifications in the plant cell wall due to the lack of a gene involved in its biosynthesis.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Transporte de Monossacarídeos/genética , Mucilagem Vegetal/biossíntese , Transcriptoma , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutação
3.
Plant Cell ; 29(1): 129-143, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28062750

RESUMO

UDP-glucuronic acid (UDP-GlcA) is the precursor of many plant cell wall polysaccharides and is required for production of seed mucilage. Following synthesis in the cytosol, it is transported into the lumen of the Golgi apparatus, where it is converted to UDP-galacturonic acid (UDP-GalA), UDP-arabinose, and UDP-xylose. To identify the Golgi-localized UDP-GlcA transporter, we screened Arabidopsis thaliana mutants in genes coding for putative nucleotide sugar transporters for altered seed mucilage, a structure rich in the GalA-containing polysaccharide rhamnogalacturonan I. As a result, we identified UUAT1, which encodes a Golgi-localized protein that transports UDP-GlcA and UDP-GalA in vitro. The seed coat of uuat1 mutants had less GalA, rhamnose, and xylose in the soluble mucilage, and the distal cell walls had decreased arabinan content. Cell walls of other organs and cells had lower arabinose levels in roots and pollen tubes, but no differences were observed in GalA or xylose contents. Furthermore, the GlcA content of glucuronoxylan in the stem was not affected in the mutant. Interestingly, the degree of homogalacturonan methylation increased in uuat1 These results suggest that this UDP-GlcA transporter plays a key role defining the seed mucilage sugar composition and that its absence produces pleiotropic effects in this component of the plant extracellular matrix.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Polissacarídeos/metabolismo , Sementes/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Parede Celular/genética , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Immunoblotting , Microscopia Confocal , Mutação , Proteínas de Transporte de Nucleotídeos/genética , Pectinas/metabolismo , Plantas Geneticamente Modificadas , Sementes/genética , Açúcares de Uridina Difosfato/metabolismo
4.
Glycobiology ; 26(9): 913-925, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27507902

RESUMO

The cell wall is a complex extracellular matrix composed primarily of polysaccharides. Noncellulosic polysaccharides, glycoproteins and proteoglycans are synthesized in the Golgi apparatus by glycosyltransferases (GTs), which use nucleotide sugars as donors to glycosylate nascent glycan and glycoprotein acceptors that are subsequently exported to the extracellular space. Many nucleotide sugars are synthesized in the cytosol, leading to a topological issue because the active sites of most GTs are located in the Golgi lumen. Nucleotide sugar transporters (NSTs) overcome this problem by translocating nucleoside diphosphate sugars from the cytosol into the lumen of the organelle. The structures of the cell wall components synthesized in the Golgi are diverse and complex; therefore, transporter activities are necessary so that the nucleotide sugars can provide substrates for the GTs. In this review, we describe the topology of reactions involved in polysaccharide biosynthesis in the Golgi and focus on the roles of NSTs as well as their impacts on cell wall structure when they are altered.


Assuntos
Parede Celular/genética , Células Vegetais/metabolismo , Polissacarídeos/biossíntese , Açúcares/metabolismo , Transporte Biológico/genética , Parede Celular/química , Parede Celular/metabolismo , Glicosilação , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Proteínas de Transporte de Monossacarídeos , Nucleotídeos/química , Nucleotídeos/metabolismo , Polissacarídeos/genética
5.
Plant Physiol ; 166(2): 889-902, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25149602

RESUMO

The molecular mechanisms by which vascular tissues acquire their identities are largely unknown. Here, we report on the identification and characterization of VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC), a member of a 15-member, plant-specific gene family in Arabidopsis (Arabidopsis thaliana) that encodes proteins of unknown function with four predicted transmembrane domains. Homozygous vcc mutants displayed cotyledon vein networks of reduced complexity and disconnected veins. Similar disconnections or gaps were observed in the provasculature of vcc embryos, indicating that defects in vein connectivity appear early in mutant embryo development. Consistently, the overexpression of VCC leads to an unusually high proportion of cotyledons with high-complexity vein networks. Neither auxin distribution nor the polar localization of the auxin efflux carrier were affected in vcc mutant embryos. Expression of VCC was detected in developing embryos and procambial, cambial, and vascular cells of cotyledons, leaves, roots, hypocotyls, and anthers. To evaluate possible genetic interactions with other genes that control vasculature patterning in embryos, we generated a double mutant for VCC and OCTOPUS (OPS). The vcc ops double mutant embryos showed a complete loss of high-complexity vascular networks in cotyledons and a drastic increase in both provascular and vascular disconnections. In addition, VCC and OPS interact physically, suggesting that VCC and OPS are part of a complex that controls cotyledon vascular complexity.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Genes de Plantas , Sementes/metabolismo , Sequência de Aminoácidos , Arabidopsis/embriologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/fisiologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos
6.
Methods Mol Biol ; 1209: 143-8, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25117281

RESUMO

ESCRT (Endosomal Sorting Complex Required for Transport) proteins are required for the sorting of biosynthetic and endocytic proteins at multivesicular bodies (MVBs). Here, I describe an assay to evaluate conservation of endosomal sorting functions of plant ESCRT proteins by trans-species complementation analysis. The assay is based on the imaging of a fluorescent biosynthetic MVB cargo, the carboxypeptidase S (CPS) fused to the green fluorescent protein GFP, in yeast ESCRT mutants expressing putative plant orthologues of the missing yeast ESCRT components.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Biologia Molecular/métodos , Sequência de Aminoácidos , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Proteínas de Fluorescência Verde , Transporte Proteico/genética , Proteínas de Transporte Vesicular/metabolismo
7.
J Biol Chem ; 289(8): 4980-8, 2014 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-24385429

RESUMO

ESCRT proteins mediate membrane remodeling and scission events and are essential for endosomal sorting of plasma membrane proteins for degradation. We have identified a novel, plant-specific ESCRT component called PROS (POSITIVE REGULATOR OF SKD1) in Arabidopsis thaliana. PROS has a strong positive effect on the in vitro ATPase activity of SKD1 (also known as Vacuolar Protein Sorting 4 or VPS4), a critical component required for ESCRT-III disassembly and endosomal vesiculation. PROS interacts with both SKD1 and the SKD1-positive regulator LIP5/VTA1. We have identified a putative MIM domain within PROS that mediate the interaction with the MIT domain of SKD1. Interestingly, whereas MIM domains are commonly found at the C terminus of ESCRT-III subunits, the PROS MIM domain is internal. The heterologous expression of PROS in yeast mutant cells lacking Vta1p partially rescues endosomal sorting defects. PROS is expressed in most tissues and cells types in Arabidopsis thaliana. Silencing of PROS leads to reduced cell expansion and abnormal organ growth.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Desenvolvimento Vegetal , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/química , Proteínas de Transporte/química , Proliferação de Células , Técnicas de Silenciamento de Genes , Inativação Gênica , Dados de Sequência Molecular , Corpos Multivesiculares/metabolismo , Mutação/genética , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie
8.
Curr Opin Plant Biol ; 14(6): 666-73, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21821464

RESUMO

Endosomes regulate both the recycling and degradation of plasma membrane (PM) proteins, thereby modulating many cellular responses triggered at the cell surface. Endosomes also play a role in the biosynthetic pathway by taking proteins to the vacuole and recycling vacuolar cargo receptors. In plants, the trans-Golgi network (TGN) acts as an early/recycling endosome whereas prevacuolar compartments/multivesicular bodies (MVBs) take PM proteins to the vacuole for degradation. Recent studies have demonstrated that some of the molecular complexes that mediate endosomal trafficking, such as the retromer, the ADP-ribosylation factor (ARF) machinery, and the Endosomal Sorting Complexes Required for Transport (ESCRTs) have both conserved and specialized functions in plants. Whereas there is disagreement on the subcellular localization of the plant retromer, its function in recycling vacuolar sorting receptors (VSRs) and modulating the trafficking of PM proteins has been well established. Studies on Arabidopsis ESCRT components highlight the essential role of this complex in cytokinesis, plant development, and vacuolar organization. In addition, post-translational modifications of plant PM proteins, such as phosphorylation and ubiquitination, have been demonstrated to act as sorting signals for endosomal trafficking.


Assuntos
Endossomos/metabolismo , Plantas/metabolismo , Transporte Biológico , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteólise , Rede trans-Golgi/metabolismo
9.
Plant Cell ; 23(2): 769-84, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21343414

RESUMO

Zeins, the prolamin storage proteins found in maize (Zea mays), accumulate in accretions called protein bodies inside the endoplasmic reticulum (ER) of starchy endosperm cells. We found that genes encoding zeins, α-globulin, and legumin-1 are transcribed not only in the starchy endosperm but also in aleurone cells. Unlike the starchy endosperm, aleurone cells accumulate these storage proteins inside protein storage vacuoles (PSVs) instead of the ER. Aleurone PSVs contain zein-rich protein inclusions, a matrix, and a large system of intravacuolar membranes. After being assembled in the ER, zeins are delivered to the aleurone PSVs in atypical prevacuolar compartments that seem to arise at least partially by autophagy and consist of multilayered membranes and engulfed cytoplasmic material. The zein-containing prevacuolar compartments are neither surrounded by a double membrane nor decorated by AUTOPHAGY RELATED8 protein, suggesting that they are not typical autophagosomes. The PSV matrix contains glycoproteins that are trafficked through a Golgi-multivesicular body (MVB) pathway. MVBs likely fuse with the multilayered, autophagic compartments before merging with the PSV. The presence of similar PSVs also containing prolamins and large systems of intravacuolar membranes in wheat (Triticum aestivum) and barley (Hordeum vulgare) starchy endosperm suggests that this trafficking mechanism may be common among cereals.


Assuntos
Endosperma/metabolismo , Prolaminas/metabolismo , Vacúolos/metabolismo , Zea mays/metabolismo , Tomografia com Microscopia Eletrônica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Complexo de Golgi/metabolismo , Microscopia Confocal , Prolaminas/genética , Transporte Proteico , Sementes/metabolismo , Zea mays/genética , Zeína/genética , Zeína/metabolismo
10.
Plant Physiol ; 153(2): 624-31, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20357137

RESUMO

Developing maize (Zea mays) endosperms can be excised from the maternal tissues and undergo tissue/cell-type differentiation under in vitro conditions. We have developed a method to transform in vitro-grown endosperms using Agrobacterium tumefaciens and standard binary vectors. We show that both aleurone and starchy endosperm cells can be successfully transformed using a short cocultivation with A. tumefaciens cells. The highest transformation rates were obtained with the A. tumefaciens EHA101 strain and the pTF101.1 binary vector. The percentage of aleurone cells transformed following this method varied between 10% and 22% whereas up to the eighth layer of starchy endosperm cells underneath the aleurone layer showed transformed cells. Cultured endosperms undergo normal cell type (aleurone and starchy endosperm) differentiation and storage protein accumulation, making them suitable for cell biology and biochemical studies. In addition, transgenic cultured endosperms are able to express and accumulate epitope-tagged storage proteins that can be isolated for biochemical assays or used for immunolabeling techniques.


Assuntos
Agrobacterium tumefaciens/genética , Endosperma/genética , Transformação Genética , Zea mays/genética , Endosperma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/genética , Plasmídeos , Proteínas de Armazenamento de Sementes/metabolismo
11.
Plant Cell ; 21(3): 749-66, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19304934

RESUMO

Plasma membrane proteins internalized by endocytosis and targeted for degradation are sorted into lumenal vesicles of multivesicular bodies (MVBs) by the endosomal sorting complexes required for transport (ESCRT) machinery. Here, we show that the Arabidopsis thaliana ESCRT-related CHARGED MULTIVESICULAR BODY PROTEIN/CHROMATIN MODIFYING PROTEIN1A (CHMP1A) and CHMP1B proteins are essential for embryo and seedling development. Double homozygous chmp1a chmp1b mutant embryos showed limited polar differentiation and failed to establish bilateral symmetry. Mutant seedlings show disorganized apical meristems and rudimentary true leaves with clustered stomata and abnormal vein patterns. Mutant embryos failed to establish normal auxin gradients. Three proteins involved in auxin transport, PINFORMED1 (PIN1), PIN2, and AUXIN-RESISTANT1 (AUX1) mislocalized to the vacuolar membrane of the mutant. PIN1 was detected in MVB lumenal vesicles of control cells but remained in the limiting membrane of chmp1a chmp1b MVBs. The chmp1a chmp1b mutant forms significantly fewer MVB lumenal vesicles than the wild type. Furthermore, CHMP1A interacts in vitro with the ESCRT-related proteins At SKD1 and At LIP5. Thus, Arabidopsis CHMP1A and B are ESCRT-related proteins with conserved endosomal functions, and the auxin carriers PIN1, PIN2, and AUX1 are ESCRT cargo proteins in the MVB sorting pathway.


Assuntos
Arabidopsis/fisiologia , Proteínas de Transporte/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Ácidos Indolacéticos/metabolismo , Sequência de Aminoácidos , Animais , Arabidopsis/citologia , Arabidopsis/embriologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/classificação , Proteínas de Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte , Teste de Complementação Genética , Humanos , Imuno-Histoquímica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Fenótipo , Filogenia , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Plântula/citologia , Plântula/fisiologia , Alinhamento de Sequência , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
12.
Plant Physiol ; 129(4): 1820-8, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12177495

RESUMO

The Golgi apparatus behaves as a bona fide Ca(2+) store in animal cells and yeast (Saccharomyces cerevisiae); however, it is not known whether this organelle plays a similar role in plant cells. In this work, we investigated the presence of an active Ca(2+) accumulation mechanism in the plant cell Golgi apparatus. Toward this end, we measured Ca(2+) uptake in subcellular fractions isolated from the elongating zone of etiolated pea (Pisum sativum) epicotyls. Separation of organelles using sucrose gradients showed a strong correlation between the distribution of an ATP-dependent Ca(2+) uptake activity and the Golgi apparatus marker enzyme, xyloglucan-fucosyltransferase. The kinetic parameters obtained for this activity were: the rate of maximum Ca(2+) uptake of 2.5 nmol mg min(-1) and an apparent K(m) for Ca(2+) of 209 nM. The ATP-dependent Ca(2+) uptake was strongly inhibited by vanadate (inhibitor concentration causing 50% inhibition [I(50)] = 126 microM) and cyclopiazonic acid (I(50) = 0.36 nmol mg protein(-1)) and was not stimulated by calmodulin (1 microM). Addition of Cd(2+) and Cu(2+) at nanomolar concentration inhibited the Ca(2+) uptake, whereas Mn(2+), Fe(2+), and Co(2+) had no significant effect. Interestingly, the active calcium uptake was inhibited by thapsigargin (apparent I(50) = 88 nM), a well-known inhibitor of the endoplasmic reticulum and Golgi sarco-endoplasmic reticulum Ca(2+) ATPase from mammalian cells. A thapsigargin-sensitive Ca(2+) uptake activity was also detected in a cauliflower (Brassica oleracea) Golgi-enriched fraction, suggesting that other plants may also possess thapsigargin-sensitive Golgi Ca(2+) pumps. To our knowledge, this is the first report of a plant Ca(2+) pump activity that shows sensitivity to low concentrations of thapsigargin.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Complexo de Golgi/enzimologia , Membranas Intracelulares/enzimologia , Pisum sativum/enzimologia , Tapsigargina/farmacologia , Trifosfato de Adenosina/metabolismo , Brassica/efeitos dos fármacos , Brassica/fisiologia , Cádmio/farmacologia , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/efeitos dos fármacos , Calmodulina/farmacologia , Cobalto/metabolismo , Cobre/farmacologia , Fucosiltransferases/metabolismo , Complexo de Golgi/efeitos dos fármacos , Indóis/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Ferro/metabolismo , Cinética , Manganês/farmacologia , Pisum sativum/efeitos dos fármacos , Vanadatos/farmacologia
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