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1.
Redox Biol ; 69: 103015, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38183796

RESUMO

Redox status of protein cysteinyl residues is mediated via glutathione (GSH)/glutaredoxin (GRX) and thioredoxin (TRX)-dependent redox cascades. An oxidative challenge can induce post-translational protein modifications on thiols, such as protein S-glutathionylation. Class I GRX are small thiol-disulfide oxidoreductases that reversibly catalyse S-glutathionylation and protein disulfide formation. TRX and GSH/GRX redox systems can provide partial backup for each other in several subcellular compartments, but not in the plastid stroma where TRX/light-dependent redox regulation of primary metabolism takes place. While the stromal TRX system has been studied at detail, the role of class I GRX on plastid redox processes is still unknown. We generate knockout lines of GRXC5 as the only chloroplast class I GRX of the moss Physcomitrium patens. While we find that PpGRXC5 has high activities in GSH-dependent oxidoreductase assays using hydroxyethyl disulfide or redox-sensitive GFP2 as substrates in vitro, Δgrxc5 plants show no detectable growth defect or stress sensitivity, in contrast to mutants with a less negative stromal EGSH (Δgr1). Using stroma-targeted roGFP2, we show increased protein Cys steady state oxidation and decreased reduction rates after oxidative challenge in Δgrxc5 plants in vivo, indicating kinetic uncoupling of the protein Cys redox state from EGSH. Compared to wildtype, protein Cys disulfide formation rates and S-glutathionylation levels after H2O2 treatment remained unchanged. Lack of class I GRX function in the stroma did not result in impaired carbon fixation. Our observations suggest specific roles for GRXC5 in the efficient transfer of electrons from GSH to target protein Cys as well as negligible cross-talk with metabolic regulation via the TRX system. We propose a model for stromal class I GRX function in efficient catalysis of protein dithiol/disulfide equilibria upon redox steady state alterations affecting stromal EGSH and highlight the importance of identifying in vivo target proteins of GRXC5.


Assuntos
Glutarredoxinas , Peróxido de Hidrogênio , Peróxido de Hidrogênio/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Oxirredução , Glutationa/metabolismo , Estresse Oxidativo , Cloroplastos/metabolismo , Dissulfetos/química
2.
Acta Crystallogr D Struct Biol ; 78(Pt 10): 1259-1272, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36189745

RESUMO

The folding of newly synthesized polypeptides requires the coordinated action of molecular chaperones. Prokaryotic cells and the chloroplasts of plant cells possess the ribosome-associated chaperone trigger factor, which binds nascent polypeptides at their exit stage from the ribosomal tunnel. The structure of bacterial trigger factor has been well characterized and it has a dragon-shaped conformation, with flexible domains responsible for ribosome binding, peptidyl-prolyl cis-trans isomerization (PPIase) activity and substrate protein binding. Chloroplast trigger-factor sequences have diversified from those of their bacterial orthologs and their molecular mechanism in plant organelles has been little investigated to date. Here, the crystal structure of the plastidic trigger factor from the green alga Chlamydomonas reinhardtii is presented at 2.6 Šresolution. Due to the high intramolecular flexibility of the protein, diffraction to this resolution was only achieved using a protein that lacked the N-terminal ribosome-binding domain. The eukaryotic trigger factor from C. reinhardtii exhibits a comparable dragon-shaped conformation to its bacterial counterpart. However, the C-terminal chaperone domain displays distinct charge distributions, with altered positioning of the helical arms and a specifically altered charge distribution along the surface responsible for substrate binding. While the PPIase domain shows a highly conserved structure compared with other PPIases, its rather weak activity and an unusual orientation towards the C-terminal domain points to specific adaptations of eukaryotic trigger factor for function in chloroplasts.


Assuntos
Proteínas de Escherichia coli , Peptidilprolil Isomerase , Cloroplastos/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Modelos Moleculares , Chaperonas Moleculares/química , Peptídeos/metabolismo , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Dobramento de Proteína
3.
J Exp Bot ; 72(20): 6867-6881, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34244747

RESUMO

The plant vacuole recycles proteins and RNA delivered to it by autophagy. In this study, by isolating intact vacuoles from Arabidopsis plants, followed by subsequent RNA purification, and deep sequencing, we provide a comprehensive characterization of Arabidopsis vacuolar RNAome. In the vacuolar RNAome, we detected ribosomal RNAs, transfer RNAs, including those of chloroplast origin, and in addition small RNA types. As autophagy is a main mechanism for the transport of RNA to the vacuole, atg5-1 mutants deficient in autophagy were included in our analysis. We observed severely reduced amounts of most chloroplast-derived RNA species in these mutants. Comparisons with cellular RNA composition provided an indication of possible up-regulation of alternative RNA breakdown pathways. By contrast, vacuolar RNA processing and composition in plants lacking vacuolar ribonuclease 2, involved in cellular RNA homeostasis, only showed minor alterations, possibly because of the presence of further so far unknown vacuolar RNase species. Among the small RNA types, we detected mature miRNAs in all vacuolar preparations but at much lower frequency in atg5-1, raising the possibility of a biological role for vacuolar miRNAs.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Autofagia/genética , RNA , Vacúolos
4.
Plant Physiol ; 186(1): 315-329, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-33650638

RESUMO

Maltose, the major product of starch breakdown in Arabidopsis (Arabidopsis thaliana) leaves, exits the chloroplast via the maltose exporter1 MEX1. Consequently, mex1 loss-of-function plants exhibit substantial maltose accumulation, a starch-excess phenotype and a specific chlorotic phenotype during leaf development. Here, we investigated whether the introduction of an alternative metabolic route could suppress the marked developmental defects typical for mex1 loss-of-function mutants. To this end, we ectopically expressed in mex1  chloroplasts a functional maltase (MAL) from baker's yeast (Saccharomyces cerevisiae, chloroplastidial MAL [cpMAL] mutants). Remarkably, the stromal MAL activity substantially alleviates most phenotypic peculiarities typical for mex1 plants. However, the cpMAL lines contained only slightly less maltose than parental mex1 plants and their starch levels were, surprisingly, even higher. These findings point to a threshold level of maltose responsible for the marked developmental defects in mex1. While growth and flowering time were only slightly retarded, cpMAL lines exhibited a substantially improved frost tolerance, when compared to wild-types. In summary, these results demonstrate the possibility to bypass the MEX1 transporter, allow us to differentiate between possible starch-excess and maltose-excess responses, and demonstrate that stromal maltose accumulation prevents frost defects. The latter insight may be instrumental for the development of crop plants with improved frost tolerance.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Temperatura Baixa , Proteínas de Membrana Transportadoras/genética , Fenótipo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Membrana Transportadoras/metabolismo
5.
Plant J ; 106(1): 23-40, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33368770

RESUMO

Acclimation is the capacity to adapt to environmental changes within the lifetime of an individual. This ability allows plants to cope with the continuous variation in ambient conditions to which they are exposed as sessile organisms. Because environmental changes and extremes are becoming even more pronounced due to the current period of climate change, enhancing the efficacy of plant acclimation is a promising strategy for mitigating the consequences of global warming on crop yields. At the cellular level, the chloroplast plays a central role in many acclimation responses, acting both as a sensor of environmental change and as a target of cellular acclimation responses. In this Perspective article, we outline the activities of the Green Hub consortium funded by the German Science Foundation. The main aim of this research collaboration is to understand and strategically modify the cellular networks that mediate plant acclimation to adverse environments, employing Arabidopsis, tobacco (Nicotiana tabacum) and Chlamydomonas as model organisms. These efforts will contribute to 'smart breeding' methods designed to create crop plants with improved acclimation properties. To this end, the model oilseed crop Camelina sativa is being used to test modulators of acclimation for their potential to enhance crop yield under adverse environmental conditions. Here we highlight the current state of research on the role of gene expression, metabolism and signalling in acclimation, with a focus on chloroplast-related processes. In addition, further approaches to uncovering acclimation mechanisms derived from systems and computational biology, as well as adaptive laboratory evolution with photosynthetic microbes, are highlighted.


Assuntos
Folhas de Planta/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Camellia/genética , Camellia/metabolismo , Camellia/fisiologia , Chlamydomonas/genética , Chlamydomonas/metabolismo , Chlamydomonas/fisiologia , Folhas de Planta/genética , Biologia de Sistemas/métodos , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/fisiologia
6.
Plant Physiol ; 182(3): 1239-1255, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31932409

RESUMO

The ability of plants to withstand cold temperatures relies on their photosynthetic activity. Thus, the chloroplast is of utmost importance for cold acclimation and acquisition of freezing tolerance. During cold acclimation, the properties of the chloroplast change markedly. To provide the most comprehensive view of the protein repertoire of the chloroplast envelope, we analyzed this membrane system in Arabidopsis (Arabidopsis thaliana) using mass spectrometry-based proteomics. Profiling chloroplast envelope membranes was achieved by a cross comparison of protein intensities across the plastid and the enriched membrane fraction under both normal and cold conditions. We used multivariable logistic regression to model the probabilities for the classification of an envelope localization. In total, we identified 38 envelope membrane intrinsic or associated proteins exhibiting altered abundance after cold acclimation. These proteins comprise several solute carriers, such as the ATP/ADP antiporter nucleotide transporter2 (NTT2; substantially increased abundance) or the maltose exporter MEX1 (substantially decreased abundance). Remarkably, analysis of the frost recovery of ntt loss-of-function and mex1 overexpressor mutants confirmed that the comparative proteome is well suited to identify key factors involved in cold acclimation and acquisition of freezing tolerance. Moreover, for proteins with known physiological function, we propose scenarios explaining their possible roles in cold acclimation. Furthermore, spatial proteomics introduces an additional layer of complexity and enables the identification of proteins differentially localized at the envelope membrane under the changing environmental regime.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Arabidopsis/metabolismo , Temperatura Baixa , Espectrometria de Massas , Proteínas de Membrana Transportadoras/metabolismo , Proteômica
7.
Plant Physiol ; 179(2): 569-587, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30482788

RESUMO

Sucrose (Suc) is one of the most important types of sugars in plants, serving inter alia as a long-distance transport molecule, a carbon and energy storage compound, an osmotically active solute, and fuel for many anabolic reactions. Suc biosynthesis and degradation pathways are well known; however, the regulation of Suc intracellular distribution is poorly understood. In particular, the cellular function of chloroplast Suc reserves and the transporters involved in accumulating these substantial Suc levels remain uncharacterized. Here, we characterize the plastidic sugar transporter (pSuT) in Arabidopsis (Arabidopsis thaliana), which belongs to a subfamily of the monosaccharide transporter-like family. Transport analyses with yeast cells expressing a truncated, vacuole-targeted version of pSuT indicate that both glucose and Suc act as substrates, and nonaqueous fractionation supports a role for pSuT in Suc export from the chloroplast. The latter process is required for a correct transition from vegetative to reproductive growth and influences inflorescence architecture. Moreover, pSuT activity affects freezing-induced electrolyte release. These data further underline the central function of the chloroplast for plant development and the modulation of stress tolerance.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Resposta ao Choque Frio/fisiologia , Flores/fisiologia , Simportadores/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutação , Plantas Geneticamente Modificadas , Plastídeos/metabolismo , Domínios Proteicos , Saccharomyces cerevisiae/genética , Sacarose/metabolismo , Simportadores/química , Simportadores/genética
8.
Plant Cell Physiol ; 59(7): 1290-1299, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29444312

RESUMO

The sessile lifestyle of higher plants is accompanied by their remarkable ability to tolerate unfavorable environmental conditions. This is because, during evolution, plants developed a sophisticated repertoire of molecular and metabolic reactions to cope with changing biotic and abiotic challenges. In particular, the abiotic factors light intensity and ambient temperature are characterized by altering their amplitude within comparably short periods of time and are causative for onset of dynamic plant responses. These rapid responses in plants are also classified as 'acclimation reactions' which differ, due to their reversibility and duration, from non-reversible 'adaptation reactions'. In this review, we demonstrate the remarkable importance of stress-induced changes in carbohydrate homeostasis of plants exposed to high light or low temperatures. These changes represent a co-ordinated process comprising modifications of (i) the concentrations of selected sugars; (ii) starch turnover; (iii) intracellular sugar compartmentation; and (iv) corresponding gene expression patterns. The critical importance of these individual processes has been underlined in the recent past by the analyses of a large number of mutant plants. The outcome of these analyses raised our understanding of acclimation processes in plants per se but might even become instrumental to develop new concepts for directed breeding approaches with the aim to increase abiotic stress tolerance of crop species, which in most cases have high stress sensitivity. The latter direction of plant research is of special importance since abiotic stress stimuli strongly impact on crop productivity and are expected to become even more pronounced because of human activities which alter environmental conditions rapidly.


Assuntos
Metabolismo dos Carboidratos/fisiologia , Plantas/metabolismo , Estresse Fisiológico/fisiologia , Transporte Biológico , Resposta ao Choque Frio/fisiologia , Frutanos/metabolismo , Açúcares/metabolismo , Temperatura
9.
Front Plant Sci ; 7: 411, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27148277

RESUMO

Measurements of protein abundance changes are important for biological conclusions on protein-related processes such as activity or complex formation. Proteomic analyses in general are almost routine tasks in many laboratories, but a precise and quantitative description of (absolute) protein abundance changes require careful experimental design and precise data quality. Today, a vast choice of metabolic labeling and label-free quantitation protocols are available, but the trade-off between quantitative precision and proteome coverage of quantified proteins including missing value problems remain. Here, we provide an example of a targeted proteomic approach using artificial standard proteins consisting of concatenated peptides of interest (QconCAT) to specifically quantify abiotic stress-induced abundance changes in low abundant vacuolar transporters. An advantage of this approach is the reliable quantitation of alimited set of low-abundant target proteins throughout different conditions. We show that vacuolar ATPase AVP1 and sugar transporters of the ERDL (early responsive to dehydration-like) family and TMT2 (tonoplast monosaccharide transporter 2) showed increased abundance upon salt stress.

10.
Front Plant Sci ; 5: 460, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25309559

RESUMO

In plants, the vacuole builds up the cellular turgor and represents an important component in cellular responses to diverse stress stimuli. Rapid volume changes of cells, particularly of motor cells, like guard cells, are caused by variation of osmolytes and consequently of the water contents in the vacuole. Moreover, directed solute uptake into or release out of the large central vacuole allows adaptation of cytosolic metabolite levels according to the current physiological requirements and specific cellular demands. Therefore, solute passage across the vacuolar membrane, the tonoplast, has to be tightly regulated. Important principles in vacuolar transport regulation are changes of tonoplast transport protein abundances by differential expression of genes or changes of their activities, e.g., due to post-translational modification or by interacting proteins. Because vacuolar transport is in most cases driven by an electro-chemical gradient altered activities of tonoplast proton pumps significantly influence vacuolar transport capacities. Intense studies on individual tonoplast proteins but also unbiased system biological approaches have provided important insights into the regulation of vacuolar transport. This short review refers to selected examples of tonoplast proteins and their regulation, with special focus on protein phosphorylation.

11.
New Phytol ; 202(1): 188-197, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24329902

RESUMO

Arabidopsis vacuoles harbor, besides sugar transporter of the TMT-type, an early response to dehydration like 6 (ERDL6) protein involved in glucose export into the cytosol. However, the mode of transport of ERDL6 and the plant's feedback to overexpression of its activity on essential properties such as, for example, seed germination or freezing tolerance, remain unexplored. Using patch-clamp studies on vacuoles expressing AtERDL6 we demonstrated directly that this carrier operates as a proton-driven glucose exporter. Overexpression of BvIMP, the closest sugar beet (Beta vulgaris) homolog to AtERDL6, in Arabidopsis leads surprisingly to impaired seed germination under both conditions, sugar application and low environmental temperatures, but not under standard conditions. Upon cold treatment, BvIMP overexpressor plants accumulated lower quantities of monosaccharides than the wild-type, a response in line with the reduced frost tolerance of the transgenic Arabidopsis plants, and the fact that cold temperatures inhibits BvIMP transcription in sugar beet leaves. With these findings we show that the tight control of vacuolar sugar import and export is a key requisite for cold tolerance and seed germination of plants.


Assuntos
Adaptação Fisiológica , Arabidopsis/fisiologia , Germinação , Glucose/metabolismo , Proteínas de Plantas/metabolismo , Prótons , Sementes/crescimento & desenvolvimento , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Beta vulgaris , Biocatálise , Transporte Biológico , Metabolismo dos Carboidratos , Condutividade Elétrica , Congelamento , Regulação da Expressão Gênica de Plantas , Complexos de Proteínas Captadores de Luz/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sementes/genética , Transdução de Sinais , Amido/metabolismo , Vacúolos/metabolismo
12.
Front Plant Sci ; 4: 34, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23459586

RESUMO

Vacuoles of plants fulfill various biologically important functions, like turgor generation and maintenance, detoxification, solute sequestration, or protein storage. Different types of plant vacuoles (lytic versus protein storage) are characterized by different functional properties apparently caused by a different composition/abundance and regulation of transport proteins in the surrounding membrane, the tonoplast. Proteome analyses allow the identification of vacuolar proteins and provide an informative basis for assigning observed transport processes to specific carriers or channels. This review summarizes techniques required for vacuolar proteome analyses, like e.g., isolation of the large central vacuole or tonoplast membrane purification. Moreover, an overview about diverse published vacuolar proteome studies is provided. It becomes evident that qualitative proteomes from different plant species represent just the tip of the iceberg. During the past few years, mass spectrometry achieved immense improvement concerning its accuracy, sensitivity, and application. As a consequence, modern tonoplast proteome approaches are suited for detecting alterations in membrane protein abundance in response to changing environmental/physiological conditions and help to clarify the regulation of tonoplast transport processes.

13.
Plant J ; 69(3): 529-41, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21988472

RESUMO

Because they are immotile organisms, higher plants have developed efficient strategies for adaptation to temperature changes. During cold acclimation, plants accumulate specific types of solutes to enhance freezing tolerance. The vacuole is a major solute storage organelle, but until now the role of tonoplast proteins in cold acclimation has not been investigated. In a comparative tonoplast proteome analysis, we identified several membrane proteins with altered abundance upon cold acclimation. We found an increased protein abundance of the tonoplast pyrophosphatase and subunits of the vacuolar V-ATPase and a significantly increased V-ATPase activity. This was accompanied by increased vacuolar concentrations of dicarbonic acids and soluble sugars. Consistently, the abundance of the tonoplast dicarbonic acid transporter was also higher in cold-acclimatized plants. However, no change in the protein abundance of tonoplast monosaccharide transporters was detectable. However, a generally higher cold-induced phosphorylation of members of this sugar transporter sub-group was observed. Our results indicate that cold-induced solute accumulation in the vacuole is mediated by increased acidification of this organelle. Thus solute transport activity is either modulated by increased protein amounts or by modification of proteins via phosphorylation.


Assuntos
Aclimatação , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Temperatura Baixa , Proteínas de Transporte de Monossacarídeos/metabolismo , Vacúolos/metabolismo , Arabidopsis/genética , Carboidratos/análise , Ácido Carbônico/análise , Regulação da Expressão Gênica de Plantas , Fosforilação , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Proteoma/análise , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vacúolos/enzimologia
14.
Plant J ; 68(5): 890-900, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21838775

RESUMO

Vacuolar solute accumulation is an important process during plant development, growth and stress responses. Although several vacuolar carriers have been identified recently, knowledge regarding the regulation of transport is still limited. Solute accumulation may be controlled by various factors, such as alterations in carrier abundance or activity. Phosphorylation via kinases is a well-known principle for activation or deactivation of proteins. Several phosphorylated proteins have been identified in the tonoplast proteome; however, kinases that catalyse the phosphorylation of tonoplast proteins are currently unknown. The tonoplast monosaccaride transporter from Arabidopsis (AtTMT1) and its homologue from barley have multiple phosphorylation sites in their extremely large loops. Here we demonstrate that the loop of AtTMT1 interacts with a mitogen-activated triple kinase-like protein kinase (VIK), that an aspartate-rich loop domain is required for effective interaction, and that the presence of VIK stimulates glucose import into isolated vacuoles. Furthermore, the phenotype of VIK loss-of-function plants strikingly resembles that of plants lacking AtTMT1/2. These data suggest that VIK-mediated phosphorylation of the AtTMT1 loop enhances carrier activity and consequently vacuolar sugar accumulation. As many phosphorylated proteins have been identified in the tonoplast, differential phosphorylation may be a general mechanism regulating vacuolar solute import.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glucose/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Vacúolos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Transporte Biológico , Respiração Celular , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas , MAP Quinase Quinase Quinases/genética , Células do Mesofilo/metabolismo , Microscopia de Fluorescência/métodos , Proteínas de Transporte de Monossacarídeos/genética , Mutagênese Sítio-Dirigida , Fosforilação , Células Vegetais/metabolismo , Folhas de Planta/metabolismo , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Redobramento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
15.
J Exp Bot ; 62(13): 4627-37, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21642237

RESUMO

ENT1 of Arabidopsis thaliana was the first member of the equilibrative nucleoside transporter (ENT) family to be identified in plants and characterized as a cellular, high-affinity nucleoside importer. Evidence is presented here for a tonoplast localization of ENT1 based on proteome data and Western blot analyses. Increased export of adenosine from reconstituted tonoplast preparations from 35S:ENT1 mutants compared with those from the wild type and ENT1-RNAi mutants support this view. Furthermore, increased vacuolar adenosine and vacuolar 2'3'-cAMP (an intermediate of RNA catabolism) contents in ENT1-RNAi mutants, but decreased contents of these metabolites in 35S:ENT1 over-expresser mutants, were observed. An up-regulation of the salvage pathway was detected in the latter mutants, leading to the conclusion that draining the vacuolar adenosine storage by ENT1 over-expression interferes with cellular nucleotide metabolism. As a consequence of the observed metabolic alterations 35S:ENT1 over-expresser mutants exhibited a smaller phenotypic appearance compared with wild-type plants. In addition, ENT1:RNAi mutants exhibited significantly lower in vitro germination of pollen and contained reduced internal and external ATP levels. This indicates that ENT1-mediated nucleosides, especially adenosine transport, is important for nucleotide metabolism, thus influencing growth and pollen germination.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Germinação/fisiologia , Pólen/crescimento & desenvolvimento , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , AMP Cíclico/metabolismo , Proteínas de Transporte de Nucleosídeo Equilibrativas/genética , Transportador Equilibrativo 1 de Nucleosídeo/genética , Espaço Extracelular/metabolismo , Regulação da Expressão Gênica de Plantas , Espaço Intracelular/metabolismo , Modelos Biológicos , Mutação/genética , Especificidade de Órgãos/genética , Pólen/anatomia & histologia , Pólen/genética , Pólen/fisiologia , Transporte Proteico , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo , Vacúolos/metabolismo
16.
New Phytol ; 192(2): 393-404, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21692805

RESUMO

Copper is an essential micronutrient for all organisms because it serves as a cofactor of several proteins involved in electron transfer. Elevated copper concentrations can cause toxic effects and organisms have established suitable mechanisms to regulate the uptake and internal distribution of copper to balance the content at an optimal concentration. In recent studies, a family of copper transporters (COPT) with high homology to other eukaryotic copper transporters (Ctr) has been identified in Arabidopsis thaliana. In this study we clarified the physiological function of COPT5. This carrier is located in the tonoplast and functions as a vacuolar copper exporter. Mutants lacking this transporter have altered copper contents in different organs when compared with wild-type plants. We were able to detect copper accumulation in the root and a decreased copper content in siliques and seeds when the COPT5 gene is mutated by T-DNA insertion. Vacuoles purified from copt5 T-DNA-insertion mutants show remarkably increased copper concentrations compared with wild-type organelles. We assume that on the cellular level COPT5 is important for copper export from the vacuole and on the level of the whole plant it is involved in the interorgan reallocation of copper ions from the root to reproductive organs.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico , Proteínas de Transporte de Cátions/genética , Transporte de Elétrons , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Proteínas de Fluorescência Verde/metabolismo , Homeostase , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas SLC31 , Vacúolos/metabolismo
17.
Biotechnol J ; 6(4): 452-62, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21259439

RESUMO

Secretory production of a single-chain antibody fragment (scFv) by recombinant Pichia pastoris using the methanol inducible AOX1 promoter is limited biochemically by retarded secretion, and economically by the high demand for pure oxygen. To address the problem, the adaptation phase with growth-limiting feeding of glycerol before the production phase was optimized. In a standard procedure with a short glycerol-feeding phase before induction, scFv accumulated in the supernatant only after 15 h. Conversely, scFv started to appear immediately in the medium upon methanol induction when the glycerol-feeding phase was extended to 18 h. Interestingly, despite a significantly lower cell density in the cultivation with extended glycerol feeding, the same amount of functional product of 300 mg/L was obtained about 30 h after the start of glycerol feeding with both methods. mRNA analysis revealed that the higher and faster production of the product was related to longer lasting induction of the scFv mRNA. Additional effects of a better adaptation of the secretion machinery may be suggested by higher expression of unfolded protein response-related genes KAR2 and PDI. A clear benefit of the longer glycerol-feeding phase was a 75% reduction of the consumption of both pure oxygen and methanol, and a significantly lower cell density, which would be beneficial for down-stream purification of the product.


Assuntos
Reatores Biológicos/microbiologia , Biotecnologia/métodos , Glicerol/metabolismo , Pichia/metabolismo , Anticorpos de Cadeia Única/biossíntese
18.
Plant Physiol ; 154(2): 665-77, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20709831

RESUMO

The extent to which vacuolar sugar transport activity affects molecular, cellular, and developmental processes in Arabidopsis (Arabidopsis thaliana) is unknown. Electrophysiological analysis revealed that overexpression of the tonoplast monosaccharide transporter TMT1 in a tmt1-2::tDNA mutant led to increased proton-coupled monosaccharide import into isolated mesophyll vacuoles in comparison with wild-type vacuoles. TMT1 overexpressor mutants grew faster than wild-type plants on soil and in high-glucose (Glc)-containing liquid medium. These effects were correlated with increased vacuolar monosaccharide compartmentation, as revealed by nonaqueous fractionation and by chlorophyll(ab)-binding protein1 and nitrate reductase1 gene expression studies. Soil-grown TMT1 overexpressor plants respired less Glc than wild-type plants and only about half the amount of Glc respired by tmt1-2::tDNA mutants. In sum, these data show that TMT activity in wild-type plants limits vacuolar monosaccharide loading. Remarkably, TMT1 overexpressor mutants produced larger seeds and greater total seed yield, which was associated with increased lipid and protein content. These changes in seed properties were correlated with slightly decreased nocturnal CO(2) release and increased sugar export rates from detached source leaves. The SUC2 gene, which codes for a sucrose transporter that may be critical for phloem loading in leaves, has been identified as Glc repressed. Thus, the observation that SUC2 mRNA increased slightly in TMT1 overexpressor leaves, characterized by lowered cytosolic Glc levels than wild-type leaves, provided further evidence of a stimulated source capacity. In summary, increased TMT activity in Arabidopsis induced modified subcellular sugar compartmentation, altered cellular sugar sensing, affected assimilate allocation, increased the biomass of Arabidopsis seeds, and accelerated early plant development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Metabolismo dos Carboidratos , Proteínas de Transporte de Monossacarídeos/metabolismo , Sementes/crescimento & desenvolvimento , Vacúolos/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Transporte Biológico/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Técnicas de Patch-Clamp , Fotossíntese , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Sementes/metabolismo
19.
J Am Chem Soc ; 131(41): 14868-78, 2009 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-19778046

RESUMO

Essential for the biological function of the light-driven proton pump, bacteriorhodopsin (BR), and the light sensor, sensory rhodopsin II (SRII), is the coupling of the activated retinal chromophore to the hosting protein moiety. In order to explore the dynamics of this process we have performed ultrafast transient mid-infrared spectroscopy on isotopically labeled BR and SRII samples. These include SRII in D(2)O buffer, BR in H(2)(18)O medium, SRII with (15)N-labeled protein, and BR with (13)C(14)(13)C(15)-labeled retinal chromophore. Via observed shifts of infrared difference bands after photoexcitation and their kinetics we provide evidence for nonchromophore bands in the amide I and the amide II region of BR and SRII. A band around 1550 cm(-1) is very likely due to an amide II vibration. In the amide I region, contributions of modes involving exchangeable protons and modes not involving exchangeable protons can be discerned. Observed bands in the amide I region of BR are not due to bending vibrations of protein-bound water molecules. The observed protein bands appear in the amide I region within the system response of ca. 0.3 ps and in the amide II region within 3 ps, and decay partially in both regions on a slower time scale of 9-18 ps. Similar observations have been presented earlier for BR5.12, containing a nonisomerizable chromophore (R. Gross et al. J. Phys. Chem. B 2009, 113, 7851-7860). Thus, the results suggest a common mechanism for ultrafast protein response in the artificial and the native system besides isomerization, which could be induced by initial chromophore polarization.


Assuntos
Bacteriorodopsinas/metabolismo , Luz , Rodopsinas Sensoriais/metabolismo , Absorção/efeitos da radiação , Amidas/metabolismo , Bacteriorodopsinas/química , Soluções Tampão , Isomerismo , Marcação por Isótopo , Cinética , Rodopsinas Sensoriais/química , Espectrofotometria Infravermelho , Água/metabolismo
20.
J Biol Chem ; 283(52): 36486-93, 2008 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-19001371

RESUMO

Chlamydiales and Rickettsiales as metabolically impaired, intracellular pathogenic bacteria essentially rely on "energy parasitism" by the help of nucleotide transporters (NTTs). Also in plant plastids NTT-type carriers catalyze ATP/ADP exchange to fuel metabolic processes. The uptake of ATP4-, followed by energy consumption and the release of ADP3-, would lead to a metabolically disadvantageous accumulation of negative charges in form of inorganic phosphate (Pi) in the bacterium or organelle if no interacting Pi export system exists. We identified that Pi is a third substrate of several NTT-type ATP/ADP transporters. During adenine nucleotide hetero-exchange, Pi is cotransported with ADP in a one-to-one stoichiometry. Additionally, Pi can be transported in exchange with solely Pi. This Pi homo-exchange depends on the presence of ADP and provides a first indication for only one binding center involved in import and export. Furthermore, analyses of mutant proteins revealed that Pi interacts with the same amino acid residue as the gamma-phosphate of ATP. Import of ATP in exchange with ADP plus Pi is obviously an efficient way to couple energy provision with the export of the two metabolic products (ADP plus Pi) and to maintain cellular phosphate homeostasis in intracellular living "energy parasites" and plant plastids. The additional Pi transport capacity of NTT-type ATP/ADP transporters makes the existence of an interacting Pi exporter dispensable and might explain why a corresponding protein so far has not been identified.


Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Fosfatos/química , Adenina/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Aminoácidos/química , Catálise , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Cinética , Lipossomos/química , Mutação , Plastídeos/química , Ligação Proteica , Fatores de Tempo
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