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1.
Glycobiology ; 34(6)2024 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-38690785

RESUMO

Cellulose is an abundant component of plant cell wall matrices, and this para-crystalline polysaccharide is synthesized at the plasma membrane by motile Cellulose Synthase Complexes (CSCs). However, the factors that control CSC activity and motility are not fully resolved. In a targeted chemical screen, we identified the alkylated nojirimycin analog N-Dodecyl Deoxynojirimycin (ND-DNJ) as a small molecule that severely impacts Arabidopsis seedling growth. Previous work suggests that ND-DNJ-related compounds inhibit the biosynthesis of glucosylceramides (GlcCers), a class of glycosphingolipid associated with plant membranes. Our work uncovered major changes in the sphingolipidome of plants treated with ND-DNJ, including reductions in GlcCer abundance and altered acyl chain length distributions. Crystalline cellulose content was also reduced in ND-DNJ-treated plants as well as plants treated with the known GlcCer biosynthesis inhibitor N-[2-hydroxy-1-(4-morpholinylmethyl)-2-phenyl ethyl]-decanamide (PDMP) or plants containing a genetic disruption in GLUCOSYLCERAMIDE SYNTHASE (GCS), the enzyme responsible for sphingolipid glucosylation that results in GlcCer synthesis. Live-cell imaging revealed that CSC speed distributions were reduced upon treatment with ND-DNJ or PDMP, further suggesting an important relationship between glycosylated sphingolipid composition and CSC motility across the plasma membrane. These results indicate that multiple interventions compromising GlcCer biosynthesis disrupt cellulose deposition and CSC motility, suggesting that GlcCers regulate cellulose biosynthesis in plants.


Assuntos
Arabidopsis , Celulose , Glucosilceramidas , Glucosiltransferases , Arabidopsis/metabolismo , Glucosiltransferases/metabolismo , Glucosiltransferases/genética , Celulose/metabolismo , Celulose/biossíntese , Glucosilceramidas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , 1-Desoxinojirimicina/farmacologia , 1-Desoxinojirimicina/análogos & derivados , Parede Celular/metabolismo
2.
New Phytol ; 239(6): 2212-2224, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37431066

RESUMO

Cellulose is an essential component of plant cell walls and an economically important source of food, paper, textiles, and biofuel. Despite its economic and biological significance, the regulation of cellulose biosynthesis is poorly understood. Phosphorylation and dephosphorylation of cellulose synthases (CESAs) were shown to impact the direction and velocity of cellulose synthase complexes (CSCs). However, the protein kinases that phosphorylate CESAs are largely unknown. We conducted research in Arabidopsis thaliana to reveal protein kinases that phosphorylate CESAs. In this study, we used yeast two-hybrid, protein biochemistry, genetics, and live-cell imaging to reveal the role of calcium-dependent protein kinase32 (CPK32) in the regulation of cellulose biosynthesis in A. thaliana. We identified CPK32 using CESA3 as a bait in a yeast two-hybrid assay. We showed that CPK32 phosphorylates CESA3 while it interacts with both CESA1 and CESA3. Overexpressing functionally defective CPK32 variant and phospho-dead mutation of CESA3 led to decreased motility of CSCs and reduced crystalline cellulose content in etiolated seedlings. Deregulation of CPKs impacted the stability of CSCs. We uncovered a new function of CPKs that regulates cellulose biosynthesis and a novel mechanism by which phosphorylation regulates the stability of CSCs.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cálcio/metabolismo , Parede Celular/metabolismo , Celulose/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional
3.
Plant Physiol ; 188(4): 2115-2130, 2022 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-35022793

RESUMO

The common ancestor of seed plants and mosses contained homo-oligomeric cellulose synthesis complexes (CSCs) composed of identical subunits encoded by a single CELLULOSE SYNTHASE (CESA) gene. Seed plants use different CESA isoforms for primary and secondary cell wall deposition. Both primary and secondary CESAs form hetero-oligomeric CSCs that assemble and function in planta only when all the required isoforms are present. The moss Physcomitrium (Physcomitrella) patens has seven CESA genes that can be grouped into two functionally and phylogenetically distinct classes. Previously, we showed that PpCESA3 and/or PpCESA8 (class A) together with PpCESA6 and/or PpCESA7 (class B) form obligate hetero-oligomeric complexes required for normal secondary cell wall deposition. Here, we show that gametophore morphogenesis requires a member of class A, PpCESA5, and is sustained in the absence of other PpCESA isoforms. PpCESA5 also differs from the other class A PpCESAs as it is able to self-interact and does not co-immunoprecipitate with other PpCESA isoforms. These results are consistent with the hypothesis that homo-oligomeric CSCs containing only PpCESA5 subunits synthesize cellulose required for gametophore morphogenesis. Analysis of mutant phenotypes also revealed that, like secondary cell wall deposition, normal protonemal tip growth requires class B isoforms (PpCESA4 or PpCESA10), along with a class A partner (PpCESA3, PpCESA5, or PpCESA8). Thus, P. patens contains both homo-oligomeric and hetero-oligomeric CSCs.


Assuntos
Briófitas , Bryopsida , Bryopsida/genética , Parede Celular , Celulose , Glucosiltransferases/genética , Sementes
4.
Int J Mol Sci ; 22(22)2021 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-34830110

RESUMO

In angiosperms, double fertilization requires pollen tubes to transport non-motile sperm to distant egg cells housed in a specialized female structure known as the pistil, mediating the ultimate fusion between male and female gametes. During this journey, the pollen tube encounters numerous physical barriers that must be mechanically circumvented, including the penetration of the stigmatic papillae, style, transmitting tract, and synergid cells as well as the ultimate fusion of sperm cells to the egg or central cell. Additionally, the pollen tube must maintain structural integrity in these compact environments, while responding to positional guidance cues that lead the pollen tube to its destination. Here, we discuss the nature of these physical barriers as well as efforts to genetically and cellularly identify the factors that allow pollen tubes to successfully, specifically, and quickly circumnavigate them.


Assuntos
Comunicação Celular/fisiologia , Flores/metabolismo , Tubo Polínico/metabolismo , Polinização/fisiologia
5.
Plant J ; 99(5): 1003-1013, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31034103

RESUMO

Post-translational modifications (PTMs) are critical regulators of protein function, and nearly 200 different types of PTM have been identified. Advances in high-resolution mass spectrometry have led to the identification of an unprecedented number of PTM sites in numerous organisms, potentially facilitating a more complete understanding of how PTMs regulate cellular behavior. While databases have been created to house the resulting data, most of these resources focus on individual types of PTM, do not consider quantitative PTM analyses or do not provide tools for the visualization and analysis of PTM data. Here, we describe the Functional Analysis Tools for Post-Translational Modifications (FAT-PTM) database (https://bioinformatics.cse.unr.edu/fat-ptm/), which currently supports eight different types of PTM and over 49 000 PTM sites identified in large-scale proteomic surveys of the model organism Arabidopsis thaliana. The FAT-PTM database currently supports tools to visualize protein-centric PTM networks, quantitative phosphorylation site data from over 10 different quantitative phosphoproteomic studies, PTM information displayed in protein-centric metabolic pathways and groups of proteins that are co-modified by multiple PTMs. Overall, the FAT-PTM database provides users with a robust platform to share and visualize experimentally supported PTM data, develop hypotheses related to target proteins or identify emergent patterns in PTM data for signaling and metabolic pathways.


Assuntos
Bases de Dados de Proteínas , Redes e Vias Metabólicas , Proteínas de Plantas/metabolismo , Processamento de Proteína Pós-Traducional , Arabidopsis , Biologia Computacional/métodos , Espectrometria de Massas , Proteômica/métodos
6.
Plant J ; 99(5): 862-876, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31021018

RESUMO

In seed plants, cellulose is synthesized by rosette-shaped cellulose synthesis complexes (CSCs) that are obligate hetero-oligomeric, comprising three non-interchangeable cellulose synthase (CESA) isoforms. The moss Physcomitrella patens has rosette CSCs and seven CESAs, but its common ancestor with seed plants had rosette CSCs and a single CESA gene. Therefore, if P. patens CSCs are hetero-oligomeric, then CSCs of this type evolved convergently in mosses and seed plants. Previous gene knockout and promoter swap experiments showed that PpCESAs from class A (PpCESA3 and PpCESA8) and class B (PpCESA6 and PpCESA7) have non-redundant functions in secondary cell wall cellulose deposition in leaf midribs, whereas the two members of each class are redundant. Based on these observations, we proposed the hypothesis that the secondary class A and class B PpCESAs associate to form hetero-oligomeric CSCs. Here we show that transcription of secondary class A PpCESAs is reduced when secondary class B PpCESAs are knocked out and vice versa, as expected for genes encoding isoforms that occupy distinct positions within the same CSC. The class A and class B isoforms co-accumulate in developing gametophores and co-immunoprecipitate, suggesting that they interact to form a complex in planta. Finally, secondary PpCESAs interact with each other, whereas three of four fail to self-interact when expressed in two different heterologous systems. These results are consistent with the hypothesis that obligate hetero-oligomeric CSCs evolved independently in mosses and seed plants and we propose the constructive neutral evolution hypothesis as a plausible explanation for convergent evolution of hetero-oligomeric CSCs.


Assuntos
Bryopsida/genética , Bryopsida/metabolismo , Celulose/biossíntese , Celulose/química , Sementes/genética , Sementes/metabolismo , Bryopsida/enzimologia , Parede Celular , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Genes de Plantas/genética , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Folhas de Planta , Proteínas de Plantas/genética , Isoformas de Proteínas
7.
Ann Bot ; 126(5): 807-824, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32619216

RESUMO

BACKGROUND: Phytohormones are small molecules that regulate virtually every aspect of plant growth and development, from basic cellular processes, such as cell expansion and division, to whole plant environmental responses. While the phytohormone levels and distribution thus tell the plant how to adjust itself, the corresponding growth alterations are actuated by cell wall modification/synthesis and internal turgor. Plant cell walls are complex polysaccharide-rich extracellular matrixes that surround all plant cells. Among the cell wall components, cellulose is typically the major polysaccharide, and is the load-bearing structure of the walls. Hence, the cell wall distribution of cellulose, which is synthesized by large Cellulose Synthase protein complexes at the cell surface, directs plant growth. SCOPE: Here, we review the relationships between key phytohormone classes and cellulose deposition in plant systems. We present the core signalling pathways associated with each phytohormone and discuss the current understanding of how these signalling pathways impact cellulose biosynthesis with a particular focus on transcriptional and post-translational regulation. Because cortical microtubules underlying the plasma membrane significantly impact the trajectories of Cellulose Synthase Complexes, we also discuss the current understanding of how phytohormone signalling impacts the cortical microtubule array. CONCLUSION: Given the importance of cellulose deposition and phytohormone signalling in plant growth and development, one would expect that there is substantial cross-talk between these processes; however, mechanisms for many of these relationships remain unclear and should be considered as the target of future studies.


Assuntos
Embriófitas , Reguladores de Crescimento de Plantas , Parede Celular , Celulose , Células Vegetais
8.
Proc Natl Acad Sci U S A ; 114(13): 3533-3538, 2017 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-28289192

RESUMO

The deposition of cellulose is a defining aspect of plant growth and development, but regulation of this process is poorly understood. Here, we demonstrate that the protein kinase BRASSINOSTEROID INSENSITIVE2 (BIN2), a key negative regulator of brassinosteroid (BR) signaling, can phosphorylate Arabidopsis cellulose synthase A1 (CESA1), a subunit of the primary cell wall cellulose synthase complex, and thereby negatively regulate cellulose biosynthesis. Accordingly, point mutations of the BIN2-mediated CESA1 phosphorylation site abolished BIN2-dependent regulation of cellulose synthase activity. Hence, we have uncovered a mechanism for how BR signaling can modulate cellulose synthesis in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Celulose/biossíntese , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/metabolismo , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Glucosiltransferases/química , Glucosiltransferases/genética , Dados de Sequência Molecular , Fosforilação , Mutação Puntual , Proteínas Quinases/genética , Alinhamento de Sequência
9.
Biophys J ; 116(6): 1064-1074, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30824115

RESUMO

Bombolitins, a class of peptides produced by bees of the genus Bombus, target and disrupt cellular membranes, leading to lysis. Antimicrobial peptides exhibit various mechanisms of action resulting from the interplay between peptide structure, lipid composition, and cellular target membrane selectivity. Herein, two bombolitins displaying significant amino-acid-sequence similarity, BII and BL6, were assessed for antimicrobial activity as well as correlated dodecylphosphocholine (DPC) micelle binding and membrane-induced peptide conformational changes. Infrared and circular dichroism spectroscopies were used to assess the structure-function relationship of each bombolitin, and the results indicate that BII forms a rigid and helically ordered secondary structure upon binding to DPC micelles, whereas BL6 largely lacks secondary structural order. Moreover, the binding affinity of each peptide to DPC micelles was determined, revealing that BL6 displayed a difference in binding affinity by over two orders of magnitude. Further investigations into the growth-inhibitory activity of the two bombolitins were performed against Escherichia coli and Saccharomyces cerevisiae. Interestingly, BII specifically targeted S. cerevisiae, whereas BL6 more effectively inhibited E. coli growth. Overall, the antimicrobial selectivity and specificity of BII and BL6 are largely dependent on the primary as well as secondary structural content of the peptides and the membrane composition.


Assuntos
Membrana Celular/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Animais , Abelhas , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Peptídeos/farmacologia , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Especificidade da Espécie
10.
Plant Physiol ; 176(4): 2804-2818, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29467178

RESUMO

During pollen-pistil interactions in angiosperms, the male gametophyte (pollen) germinates to produce a pollen tube. To fertilize ovules located within the female pistil, the pollen tube must physically penetrate specialized tissues. Whereas the process of pollen tube penetration through the pistil has been anatomically well described, the genetic regulation remains poorly understood. In this study, we identify a novel Arabidopsis (Arabidopsis thaliana) gene, O-FUCOSYLTRANSFERASE1 (AtOFT1), which plays a key role in pollen tube penetration through the stigma-style interface. Semi-in vivo growth assays demonstrate that oft1 mutant pollen tubes have a reduced ability to penetrate the stigma-style interface, leading to a nearly 2,000-fold decrease in oft1 pollen transmission efficiency and a 5- to 10-fold decreased seed set. We also demonstrate that AtOFT1 is localized to the Golgi apparatus, indicating its potential role in cellular glycosylation events. Finally, we demonstrate that AtOFT1 and other similar Arabidopsis genes represent a novel clade of sequences related to metazoan protein O-fucosyltransferases and that mutation of residues that are important for O-fucosyltransferase activity compromises AtOFT1 function in vivo. The results of this study elucidate a physiological function for AtOFT1 in pollen tube penetration through the stigma-style interface and highlight the potential importance of protein O-glycosylation events in pollen-pistil interactions.


Assuntos
Proteínas de Arabidopsis/genética , Flores/genética , Fucosiltransferases/genética , Tubo Polínico/genética , Polinização/genética , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fertilização/genética , Flores/metabolismo , Fucosiltransferases/classificação , Fucosiltransferases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Mutação , Filogenia , Plantas Geneticamente Modificadas , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Homologia de Sequência de Aminoácidos
11.
Oecologia ; 191(1): 141-152, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31367913

RESUMO

Herbivorous insects can defend themselves against pathogens via an immune response, which is influenced by the nutritional quality and phytochemistry of the host plant. However, it is unclear how these aspects of diet interact to influence the insect immune response and what role is played by ingested foliar microbes. We examined dietary protein, phytochemistry, and the caterpillar microbiome to understand variation in immune response of the Melissa blue butterfly, Lycaeides melissa. We also asked if these factors have host plant-specific effects by measuring L. melissa immune response when reared on a recently colonized exotic host plant (Medicago sativa) as compared to the immune response on an ancestral, native host (Astragalus canadensis). L. melissa did not experience immunological benefits directly related to consumption of the novel plant M. sativa. However, we did find negative, direct effects of phytochemical diversity and negative, direct effects of diet-derived microbial diversity on constitutive immune response for caterpillars fed M. sativa, as measured by phenoloxidase activity. Foliar protein did not directly influence the immune response, but did do so indirectly by increasing weight gain. Our results highlight the important effects of host diet on caterpillar physiology and raise the possibility that foliar microbiota, despite being rapidly passed through the gut, can affect the caterpillar immune response.


Assuntos
Borboletas , Microbiota , Animais , Herbivoria , Larva , Plantas
12.
Proc Natl Acad Sci U S A ; 109(4): 1329-34, 2012 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-22232683

RESUMO

Polysaccharide-rich cell walls are a defining feature of plants that influence cell division and growth, but many details of cell-wall organization and dynamics are unknown because of a lack of suitable chemical probes. Metabolic labeling using sugar analogs compatible with click chemistry has the potential to provide new insights into cell-wall structure and dynamics. Using this approach, we found that an alkynylated fucose analog (FucAl) is metabolically incorporated into the cell walls of Arabidopsis thaliana roots and that a significant fraction of the incorporated FucAl is present in pectic rhamnogalacturonan-I (RG-I). Time-course experiments revealed that FucAl-containing RG-I first localizes in cell walls as uniformly distributed punctae that likely mark the sites of vesicle-mediated delivery of new polysaccharides to growing cell walls. In addition, we found that the pattern of incorporated FucAl differs markedly along the developmental gradient of the root. Using pulse-chase experiments, we also discovered that the pectin network is reoriented in elongating root epidermal cells. These results reveal previously undescribed details of polysaccharide delivery, organization, and dynamics in cell walls.


Assuntos
Arabidopsis/fisiologia , Parede Celular/metabolismo , Parede Celular/fisiologia , Química Click/métodos , Pectinas/metabolismo , Raízes de Plantas/citologia , Alcinos/metabolismo , Epiderme/metabolismo , Fucose/metabolismo , Hidrazinas , Microscopia de Fluorescência , Pectinas/química , Raízes de Plantas/fisiologia
13.
bioRxiv ; 2024 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-38464008

RESUMO

Rhamnose is an essential component of the plant cell wall and is synthesized from uridine diphosphate (UDP)-glucose by the RHAMNOSE1 (RHM1) enzyme. RHM1 localizes to biomolecular condensates in plants, but their identity, formation, and function remain elusive. Combining live imaging, genetics, and biochemical approaches in Arabidopsis and heterologous systems, we show that RHM1 alone is sufficient to form enzymatically active condensates, which we name rhamnosomes. Rhamnosome formation is required for UDP-rhamnose synthesis and organ development. Overall, our study demonstrates a novel role for biomolecular condensation in metabolism and organismal development, and provides further support for how organisms have harnessed this biophysical process to regulate small molecule metabolism.

14.
Biochim Biophys Acta ; 1818(3): 627-35, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22166843

RESUMO

Annotation of the recently sequenced genome of the pea aphid (Acyrthosiphon pisum) identified a gene ApAQP2 (ACYPI009194, Gene ID: 100168499) with homology to the Major Intrinsic Protein/aquaporin superfamily of membrane channel proteins. Phylogenetic analysis suggests that ApAQP2 is a member of an insect-specific clade of this superfamily. Homology model structures of ApAQP2 showed a novel array of amino acids comprising the substrate selectivity-determining "aromatic/arginine" region of the putative transport pore. Subsequent characterization of the transport properties of ApAQP2 upon expression in Xenopus oocytes supports an unusual substrate selectivity profile. Water permeability analyses show that the ApAQP2 protein exhibits a robust mercury-insensitive aquaporin activity. However unlike the water-specific ApAQP1 protein, ApAQP2 forms a multifunctional transport channel that shows a wide permeability profile to a range of linear polyols, including the potentially biologically relevant substrates glycerol, mannitol and sorbitol. Gene expression analysis indicates that ApAQP2 is highly expressed in the insect bacteriocytes (cells bearing the symbiotic bacteria Buchnera) and the fat body. Overall the results demonstrate that ApAQP2 is a novel insect aquaglyceroporin which may be involved in water and polyol transport in support of the Buchnera symbiosis and aphid osmoregulation.


Assuntos
Afídeos/metabolismo , Aquaporina 2/metabolismo , Proteínas de Insetos/metabolismo , Água/metabolismo , Sequência de Aminoácidos , Animais , Afídeos/genética , Afídeos/microbiologia , Aquaporina 1/genética , Aquaporina 1/metabolismo , Aquaporina 2/genética , Transporte Biológico/fisiologia , Buchnera/fisiologia , Permeabilidade da Membrana Celular , Proteínas de Insetos/genética , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Simbiose/fisiologia , Xenopus laevis
15.
Plant Reprod ; 36(3): 263-272, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37222783

RESUMO

During angiosperm sexual reproduction, pollen tubes must penetrate through multiple cell types in the pistil to mediate successful fertilization. Although this process is highly choreographed and requires complex chemical and mechanical signaling to guide the pollen tube to its destination, aspects of our understanding of pollen tube penetration through the pistil are incomplete. Our previous work demonstrated that disruption of the Arabidopsis thaliana O-FUCOSYLTRANSFERASE1 (OFT1) gene resulted in decreased pollen tube penetration through the stigma-style interface. Here, we demonstrate that second site mutations of Arabidopsis GALACTURONOSYLTRANSFERASE 14 (GAUT14) effectively suppress the phenotype of oft1 mutants, partially restoring silique length, seed set, pollen transmission, and pollen tube penetration deficiencies in navigating the female reproductive tract. These results suggest that disruption of pectic homogalacturonan (HG) synthesis can alleviate the penetrative defects associated with the oft1 mutant and may implicate pectic HG deposition in the process of pollen tube penetration through the stigma-style interface in Arabidopsis. These results also support a model in which OFT1 function directly or indirectly modifies structural features associated with the cell wall, with the loss of oft1 leading to an imbalance in the wall composition that can be compensated for by a reduction in pectic HG deposition.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Tubo Polínico/genética , Tubo Polínico/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pólen/genética
16.
Methods Mol Biol ; 2499: 145-154, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35696079

RESUMO

Posttranslational modifications (PTMs) are critical regulators of protein behavior, and over 200 different types of PTMs have been identified. Recent developments in mass spectrometry technology and sample enrichment approaches have led to a massive expansion in the number of identified PTM types and sites within eukaryotic proteins. As these types of data become increasingly available, it is important to develop additional analysis tools and data repositories to investigate PTM cross talk and larger networks of PTMs. Recently, we developed the Functional Analysis Tools for Post-translational Modifications (FAT-PTM) database, which supports data from publicly available proteomic analyses encompassing eight different types of PTMs and over 49,000 PTM sites. In this chapter, we describe the utility of FAT-PTM for analysis of posttranslationally modified proteins in three different contexts. First, a simple protein search tool is available that allows users to investigate proteins in the Arabidopsis proteome to identify types of PTMs that are associated with the query protein as well as quantitative phosphorylation site changes associated with ten different experimental conditions. Second, FAT-PTM contains a metabolic pathway analysis tool to investigate PTMs in the broader context of over 600 different metabolic pathways compiled from the Plant Metabolic Network. Finally, FAT-PTM contains a comodification tool that can be used to identify groups of proteins that are subject to two or more user-defined PTMs. Overall, FAT-PTM provides a user-friendly platform to visualize posttranslationally modified proteins at the individual, metabolic pathway, and PTM cross-talk levels.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Bases de Dados de Proteínas , Redes e Vias Metabólicas , Processamento de Proteína Pós-Traducional , Proteômica/métodos
17.
Biochemistry ; 50(31): 6633-41, 2011 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-21710975

RESUMO

Plant nodulin-26 intrinsic proteins (NIPs) are members of the aquaporin superfamily that serve as multifunctional transporters of uncharged metabolites. In Arabidopsis thaliana, a specific NIP pore subclass, known as the NIP II proteins, is represented by AtNIP5;1 and AtNIP6;1, which encode channel proteins expressed in roots and leaf nodes, respectively, that participate in the transport of the critical cell wall nutrient boric acid. Modeling of the protein encoded by the AtNIP7;1 gene shows that it is a third member of the NIP II pore subclass in Arabidopsis. However, unlike AtNIP5;1 and AtNIP6;1 proteins, which form constitutive boric acid channels, AtNIP7;1 forms a channel with an extremely low intrinsic boric acid transport activity. Molecular modeling and molecular dynamics simulations of AtNIP7;1 suggest that a conserved tyrosine residue (Tyr81) located in transmembrane helix 2 adjacent to the aromatic arginine (ar/R) pore selectivity region stabilizes a closed pore conformation through interaction with the canonical Arg220 in ar/R region. Substitution of Tyr81 with a Cys residue, characteristic of established NIP boric acid channels, results in opening of the AtNIP7;1 pore that acquires a robust, transport activity for boric acid as well as other NIP II test solutes (glycerol and urea). Substitution of a Phe for Tyr81 also opens the channel, supporting the prediction from MD simulations that hydrogen bond interaction between the Tyr81 phenol group and the ar/R Arg may contribute to the stabilization of a closed pore state. Expression analyses show that AtNIP7;1 is selectively expressed in developing anther tissues of young floral buds of A. thaliana, principally in developing pollen grains of stage 9-11 anthers. Because boric acid is both an essential nutrient as well as a toxic compound at high concentrations, it is proposed that Tyr81 modulates transport and may provide an additional level of regulation for this transporter in male gametophyte development.


Assuntos
Aquaporinas/química , Proteínas de Arabidopsis/química , Arabidopsis/química , Ácidos Bóricos/química , Proteínas de Transporte/química , Regulação da Expressão Gênica de Plantas , Pólen/química , Tirosina/química , Substituição de Aminoácidos/genética , Aquaporinas/biossíntese , Aquaporinas/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/genética , Ácidos Bóricos/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Sequência Conservada , Flores/química , Flores/genética , Flores/crescimento & desenvolvimento , Família Multigênica , Especificidade de Órgãos/genética , Fenilalanina/genética , Pólen/crescimento & desenvolvimento , Pólen/metabolismo , Estrutura Secundária de Proteína/genética , Tirosina/genética
18.
J Biol Chem ; 285(31): 23880-8, 2010 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-20504761

RESUMO

Nodulin 26 (nod26) is a major intrinsic protein that constitutes the major protein component on the symbiosome membrane (SM) of N(2)-fixing soybean nodules. Functionally, nod26 forms a low energy transport pathway for water, osmolytes, and NH(3) across the SM. Besides their transport functions, emerging evidence suggests that high concentrations of major intrinsic proteins on membranes provide interaction and docking targets for various cytosolic proteins. Here it is shown that the C-terminal domain peptide of nod26 interacts with a 40-kDa protein from soybean nodule extracts, which was identified as soybean cytosolic glutamine synthetase GS(1)beta1 by mass spectrometry. Fluorescence spectroscopy assays show that recombinant soybean GS(1)beta1 binds the nod26 C-terminal domain with a 1:1 stoichiometry (K(d) = 266 nm). GS(1)beta1 also binds to isolated SMs, and this binding can be blocked by preincubation with the C-terminal peptide of nod26. In vivo experiments using either a split ubiquitin yeast two-hybrid system or bimolecular fluorescence complementation show that the four cytosolic GS isoforms expressed in soybean nodules interact with full-length nod26. The binding of GS, the principal ammonia assimilatory enzyme, to the conserved C-terminal domain of nod26, a transporter of NH(3), is proposed to promote efficient assimilation of fixed nitrogen, as well as prevent potential ammonia toxicity, by localizing the enzyme to the cytosolic side of the symbiosome membrane.


Assuntos
Aquagliceroporinas/química , Citosol/metabolismo , Glutamato-Amônia Ligase/química , Glycine max/enzimologia , Glycine max/metabolismo , Proteínas de Membrana/química , Proteínas de Plantas/química , Raízes de Plantas/enzimologia , Regulação da Expressão Gênica , Teste de Complementação Genética , Cinética , Espectrometria de Massas/métodos , Nitrogênio/química , Mapeamento de Interação de Proteínas , Isoformas de Proteínas , Estrutura Terciária de Proteína , Espectrometria de Fluorescência/métodos
19.
Methods Mol Biol ; 2160: 129-147, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32529433

RESUMO

Double-fertilization in angiosperms requires precise communication between the male gametophyte (pollen), the female tissues, and the associated female gametophyte (embryo sac) to facilitate efficient fertilization. Numerous small molecules, proteins, and peptides have been shown to impact double-fertilization through the disruption of pollen germination, pollen tube growth, pollen tube guidance, or pollen tube penetration of the female tissues. The genetic basis of signaling events that lead to successful double-fertilization has been greatly facilitated by studies in the model organism Arabidopsis thaliana, which possesses a relatively simple reproductive physiology and a widely available T-DNA mutant seed collection. In this chapter, we detail methods for determining the effects of single gene loss-of-function mutations on pollen behavior through the creation of an internally controlled fluorescent hemizygous complement line. By transforming a single copy of the disrupted gene back into the homozygous mutant background, a precise endogenous control is generated due to the fact that pollen containing equal ratios of mutant and complemented pollen can be collected from a single flower. Using this experimental design, we describe multiple assays that can be performed in series to assess mutant pollen defects in germination, pollen tube elongation rate, and pistil penetration, which can be easily quantified alongside a "near-wildtype" complemented counterpart.


Assuntos
Técnicas Genéticas , Infertilidade das Plantas , Tubo Polínico/fisiologia , Arabidopsis , Mutação com Perda de Função , Óvulo Vegetal/citologia , Óvulo Vegetal/genética , Óvulo Vegetal/fisiologia , Melhoramento Vegetal/métodos , Tubo Polínico/citologia , Tubo Polínico/genética
20.
Plants (Basel) ; 7(3)2018 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-29966291

RESUMO

Cellulose, the most abundant biopolymer on the planet, is synthesized at the plasma membrane of plant cells by the cellulose synthase complex (CSC). Cellulose is the primary load-bearing polysaccharide of plant cell walls and enables cell walls to maintain cellular shape and rigidity. The CSC is comprised of functionally distinct cellulose synthase A (CESA) proteins, which are responsible for synthesizing cellulose, and additional accessory proteins. Moreover, CESA-like (CSL) proteins are proposed to synthesize other essential non-cellulosic polysaccharides that comprise plant cell walls. The deposition of cell-wall polysaccharides is dynamically regulated in response to a variety of developmental and environmental stimuli, and post-translational phosphorylation has been proposed as one mechanism to mediate this dynamic regulation. In this review, we discuss CSC composition, the dynamics of CSCs in vivo, critical studies that highlight the post-translational control of CESAs and CSLs, and the receptor kinases implicated in plant cell-wall biosynthesis. Furthermore, we highlight the emerging importance of post-translational phosphorylation-based regulation of CSCs on the basis of current knowledge in the field.

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