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1.
Plant Physiol ; 195(3): 1851-1865, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38573555

RESUMO

Starch is the major energy storage compound in plants. Both transient starch and long-lasting storage starch accumulate in the form of insoluble, partly crystalline granules. The structure of these granules is related to the structure of the branched polymer amylopectin: linear chains of glucose units organized in double helices that align to form semicrystalline lamellae, with branching points located in amorphous regions between them. EARLY STARVATION 1 (ESV1) and LIKE EARLY STARVATION 1 (LESV) proteins are involved in the maintenance of starch granule structure and in the phase transition of amylopectin, respectively, in Arabidopsis (Arabidopsis thaliana). These proteins contain a conserved tryptophan-rich C-terminal domain folded into an antiparallel ß-sheet, likely responsible for binding of the proteins to starch, and different N-terminal domains whose structure and function are unknown. In this work, we combined biochemical and biophysical approaches to analyze the structures of LESV and ESV1 and their interactions with the different starch polyglucans. We determined that both proteins interact with amylopectin but not with amylose and that only LESV is capable of interacting with amylopectin during starch biosynthesis. While the C-terminal domain interacts with amylopectin in its semicrystalline form, the N-terminal domain of LESV undergoes induced conformational changes that are probably involved in its specific function of mediating glucan phase transition. These results clarify the specific mechanism of action of these 2 proteins in the biosynthesis of starch granules.


Assuntos
Amilopectina , Proteínas de Arabidopsis , Arabidopsis , Amido , Amilopectina/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Amido/metabolismo , Amido/biossíntese , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Ligação Proteica , Amilose/metabolismo
2.
New Phytol ; 239(1): 132-145, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37010093

RESUMO

The control of starch granule initiation in plant leaves is a complex process that requires active enzymes like Starch Synthase 4 and 3 (SS4 or SS3) and several noncatalytic proteins such as Protein Involved in starch Initiation 1 (PII1). In Arabidopsis leaves, SS4 is the main enzyme that control starch granule initiation, but in its absence, SS3 partly fulfills this function. How these proteins collectively act to control the initiation of starch granules remains elusive. PII1 and SS4 physically interact, and PII1 is required for SS4 to be fully active. However, Arabidopsis mutants lacking SS4 or PII1 still accumulate starch granules. Combining pii1 KO mutation with either ss3 or ss4 KO mutations provide new insights of how the remaining starch granules are synthesized. The ss3 pii1 line still accumulates starch, while the phenotype of ss4 pii1 is stronger than that of ss4. Our results indicate first that SS4 initiates starch granule synthesis in the absence of PII1 albeit being limited to one large lenticular granule per plastid. Second, that if in the absence of SS4, SS3 is able to initiate starch granules with low efficiency, this ability is further reduced with the additional absence of PII1.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Sintase do Amido , Arabidopsis/metabolismo , Amido/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Sintase do Amido/genética , Folhas de Planta/metabolismo , Mutação/genética
3.
New Phytol ; 221(1): 356-370, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30055112

RESUMO

The initiation of starch granule formation is still poorly understood. However, the soluble starch synthase 4 (SS4) appears to be a major component of this process since it is required to synthesize the correct number of starch granules in the chloroplasts of Arabidopsis thaliana plants. A yeast two-hybrid screen allowed the identification of several putative SS4 interacting partners. We identified the product of At4g32190 locus as a chloroplast-targeted PROTEIN INVOLVED IN STARCH INITIATION (named PII1). Arabidopsis mutants devoid of PII1 display an alteration of the starch initiation process and accumulate, on average, one starch granule per plastid instead of the five to seven granules found in plastids of wild-type plants. These granules are larger than in wild-type, and they remain flat and lenticular. pii1 mutants display wild-type growth rates and accumulate standard starch amounts. Moreover, starch characteristics, such as amylopectin chain length distribution, remain unchanged. Our results reveal the involvement of PII1 in the starch priming process in Arabidopsis leaves through interaction with SS4.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Amido/metabolismo , Amilopectina/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Proteínas de Cloroplastos/genética , Cloroplastos/genética , Microscopia Eletrônica de Varredura , Mutação , Cadeias Pesadas de Miosina/genética , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plastídeos/genética , Plastídeos/metabolismo , Amido/genética , Amido/ultraestrutura , Sintase do Amido/genética , Sintase do Amido/metabolismo
4.
J Exp Bot ; 68(18): 5177-5189, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29040651

RESUMO

The MEX1 locus of Chlamydomonas reinhardtii was identified in a genetic screen as a factor that affects starch metabolism. Mutation of MEX1 causes a slow-down in the mobilization of storage polysaccharide. Cosegregation and functional complementation analyses were used to assess the involvement of the Mex1 protein in starch degradation. Heterologous expression experiments performed in Escherichia coli and Arabidopsis thaliana allowed us to test the capacity of the algal protein in maltose export. In contrast to the A. thaliana mex1 mutant, the mutation in C. reinhardtii does not lead to maltose accumulation and growth impairment. Although localized in the plastid envelope, the algal protein does not transport maltose efficiently across the envelope, but partly complements the higher plant mutant. Both Mex orthologs restore the growth of the E. coli ptsG mutant strain on glucose-containing medium, revealing the capacity of these proteins to transport this hexose. These findings suggest that Mex1 is essential for starch mobilization in both Chlamydomonas and Arabidopsis, and that this protein family may support several functions and not only be restricted to maltose export across the plastidial envelope.


Assuntos
Chlamydomonas reinhardtii/genética , Maltose/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Amido/metabolismo , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Transporte Biológico , Chlamydomonas reinhardtii/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Filogenia , Plastídeos/metabolismo , Proteínas Recombinantes de Fusão , Plântula/citologia , Plântula/genética , Plântula/metabolismo , Transgenes
5.
Plant Cell Environ ; 39(7): 1432-47, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26715025

RESUMO

Starch synthesis requires several enzymatic activities including branching enzymes (BEs) responsible for the formation of α(1 → 6) linkages. Distribution and number of these linkages are further controlled by debranching enzymes that cleave some of them, rendering the polyglucan water-insoluble and semi-crystalline. Although the activity of BEs and debranching enzymes is mandatory to sustain normal starch synthesis, the relative importance of each in the establishment of the plant storage polyglucan (i.e. water insolubility, crystallinity and presence of amylose) is still debated. Here, we have substituted the activity of BEs in Arabidopsis with that of the Escherichia coli glycogen BE (GlgB). The latter is the BE counterpart in the metabolism of glycogen, a highly branched water-soluble and amorphous storage polyglucan. GlgB was expressed in the be2 be3 double mutant of Arabidopsis, which is devoid of BE activity and consequently free of starch. The synthesis of a water-insoluble, partly crystalline, amylose-containing starch-like polyglucan was restored in GlgB-expressing plants, suggesting that BEs' origin only has a limited impact on establishing essential characteristics of starch. Moreover, the balance between branching and debranching is crucial for the synthesis of starch, as an excess of branching activity results in the formation of highly branched, water-soluble, poorly crystalline polyglucan.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Arabidopsis/metabolismo , Glucanos/biossíntese , Plantas Geneticamente Modificadas/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Arabidopsis/genética , Metabolismo dos Carboidratos , Cloroplastos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Glucanos/ultraestrutura , Plantas Geneticamente Modificadas/genética
6.
Biochim Biophys Acta ; 1840(1): 113-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24016601

RESUMO

BACKGROUND: Native starch accumulates as granules containing two glucose polymers: amylose and amylopectin. Phosphate (0.2-0.5%) and proteins (0.1-0.7%) are also present in some starches. Phosphate groups play a major role in starch metabolism while granule-bound starch synthase 1 (GBSS1) which represents up to 95% of the proteins bound to the granule is responsible for amylose biosynthesis. METHODS: Synchrotron micro-X-ray fluorescence (µXRF) was used for the first time for high-resolution mapping of GBSS1 and phosphate groups based on the XRF signal of sulfur (S) and phosphorus (P), respectively. Wild-type starches were studied as well as their related mutants lacking GBSS1 or starch-phosphorylating enzyme. RESULTS: Wild-type potato and maize starch exhibited high level of phosphorylation and high content of sulfur respectively when compared to mutant potato starch lacking glucan water dikinase (GWD) and mutant maize starch lacking GBSS1. Phosphate groups are mostly present at the periphery of wild-type potato starch granules, and spread all over the granule in the amylose-free mutant. P and S XRF were also measured within single small starch granules from Arabidopsis or Chlamydomonas not exceeding 3-5µm in diameter. CONCLUSIONS: Imaging GBSS1 (by S mapping) in potato starch sections showed that the antisense technique suppresses the expression of GBSS1 during biosynthesis. P mapping confirmed that amylose is mostly present in the center of the granule, which had been suggested before. GENERAL SIGNIFICANCE: µXRF is a potentially powerful technique to analyze the minor constituents of starch and understand starch structure/properties or biosynthesis by the use of selected genetic backgrounds.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Fósforo/metabolismo , Solanum tuberosum/metabolismo , Espectrometria por Raios X/métodos , Sintase do Amido/metabolismo , Amido/metabolismo , Enxofre/metabolismo , Síncrotrons , Triticum/metabolismo , Solanum tuberosum/crescimento & desenvolvimento , Espectrometria por Raios X/instrumentação , Triticum/crescimento & desenvolvimento
7.
Biochim Biophys Acta ; 1830(1): 2167-77, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23041072

RESUMO

BACKGROUND: Glycogen and starch branching enzymes catalyze the formation of α(1→6) linkages in storage polysaccharides by rearrangement of preexisting α-glucans. This reaction occurs through the cleavage of α(1→4) linkage and transfer in α(1→6) of the fragment in non-reducing position. These enzymes define major elements that control the structure of both glycogen and starch. METHODS: The kinetic parameters of the branching enzyme of Rhodothermus obamensis (RoBE) were established after in vitro incubation with different branched or unbranched α-glucans of controlled structure. RESULTS: A minimal chain length of ten glucosyl units was required for the donor substrate to be recognized by RoBE that essentially produces branches of DP 3-8. We show that RoBE preferentially creates new branches by intermolecular mechanism. Branched glucans define better substrates for the enzyme leading to the formation of hyper-branched particles of 30-70nm in diameter (dextrins). Interestingly, RoBE catalyzes an additional α-4-glucanotransferase activity not described so far for a member of the GH13 family. CONCLUSIONS: RoBE is able to transfer α(1→4)-linked-glucan in C4 position (instead of C6 position for the branching activity) of a glucan to create new α(1→4) linkages yielding to the elongation of linear chains subsequently used for further branching. This result is a novel case for the thin border that exists between enzymes of the GH13 family. GENERAL SIGNIFICANCE: This work reveals the original catalytic properties of the thermostable branching enzyme of R. obamensis. It defines new approach to produce highly branched α-glucan particles in vitro.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Rhodothermus/enzimologia , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Proteínas de Bactérias/metabolismo , Catálise , Estabilidade Enzimática , Especificidade por Substrato/fisiologia
8.
Plant Physiol ; 163(3): 1363-75, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24027240

RESUMO

Isoamylase-type starch debranching enzymes (ISA) play important roles in starch biosynthesis in chloroplast-containing organisms, as shown by the strict conservation of both catalytically active ISA1 and the noncatalytic homolog ISA2. Functional distinctions exist between species, although they are not understood yet. Numerous plant tissues require both ISA1 and ISA2 for normal starch biosynthesis, whereas monocot endosperm and leaf exhibit nearly normal starch metabolism without ISA2. This study took in vivo and in vitro approaches to determine whether organism-specific physiology or evolutionary divergence between monocots and dicots is responsible for distinctions in ISA function. Maize (Zea mays) ISA1 was expressed in Arabidopsis (Arabidopsis thaliana) lacking endogenous ISA1 or lacking both native ISA1 and ISA2. The maize protein functioned in Arabidopsis leaves to support nearly normal starch metabolism in the absence of any native ISA1 or ISA2. Analysis of recombinant enzymes showed that Arabidopsis ISA1 requires ISA2 as a partner for enzymatic function, whereas maize ISA1 was active by itself. The electrophoretic mobility of recombinant and native maize ISA differed, suggestive of posttranslational modifications in vivo. Sedimentation equilibrium measurements showed recombinant maize ISA1 to be a dimer, in contrast to previous gel permeation data that estimated the molecular mass as a tetramer. These data demonstrate that evolutionary divergence between monocots and dicots is responsible for the distinctions in ISA1 function.


Assuntos
Arabidopsis/enzimologia , Isoamilase/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Zea mays/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Western Blotting , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Isoamilase/química , Isoamilase/genética , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Mutação , Folhas de Planta/genética , Folhas de Planta/ultraestrutura , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Multimerização Proteica , Proteínas Recombinantes/metabolismo , Amido/metabolismo , Espectrometria de Massas em Tandem , Zea mays/genética
9.
Anal Bioanal Chem ; 406(6): 1607-18, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24220756

RESUMO

Asymmetrical flow field flow fractionation (AF4) has proven to be a very powerful and quantitative method for the determination of the macromolecular structure of high molar mass branched biopolymers, when coupled with multi-angle laser light scattering (MALLS). This work describes a detailed investigation of the macromolecular structure of native glycogens and hyperbranched α-glucans (HBPs), with average molar mass ranging from 2 × 10(6) to 4.3 × 10(7) g mol(-1), which are not well fractionated by means of classical size-exclusion chromatography. HBPs were enzymatically produced from sucrose by the tandem action of an amylosucrase and a branching enzyme mimicking in vitro the elongation and branching steps involved in glycogen biosynthesis. Size and molar mass distributions were studied by AF4, coupled with online quasi-elastic light scattering (QELS) and transmission electron microscopy. AF4-MALLS-QELS has shown a remarkable agreement between hydrodynamic radii obtained by online QELS and by AF4 theory in normal mode with constant cross flow. Molar mass, size, and dispersity were shown to significantly increase with initial sucrose concentration, and to decrease when the branching enzyme activity increases. Several populations with different size range were observed: the amount of small size molecules decreasing with increasing sucrose concentration. The spherical and dense global conformation thus highlighted was partly similar to native glycogens. A more detailed study of HBPs synthesized from low and high initial sucrose concentrations was performed using complementary enzymatic hydrolysis of external chains and chromatography. It emphasized a more homogeneous branching pattern than native glycogens with a denser core and shorter external chains.


Assuntos
Fracionamento por Campo e Fluxo , Glucanos/química , Glicogênio/química , Amilases/metabolismo , Bactérias/enzimologia , Fracionamento por Campo e Fluxo/métodos , Glucanos/isolamento & purificação , Glucanos/metabolismo , Glucosiltransferases/metabolismo , Glicogênio/isolamento & purificação , Glicogênio/metabolismo , Luz , Estrutura Molecular , Peso Molecular , Espalhamento de Radiação , Sacarose/metabolismo
10.
New Phytol ; 200(4): 1009-21, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23952574

RESUMO

Conserved isoamylase-type starch debranching enzymes (ISAs), including the catalytic ISA1 and noncatalytic ISA2, are major starch biosynthesis determinants. Arabidopsis thaliana leaves require ISA1 and ISA2 for physiological function, whereas endosperm starch is near normal with only ISA1. ISA functions were characterized in maize (Zea mays) leaves to determine whether species-specific distinctions in ISA1 primary structure, or metabolic differences in tissues, are responsible for the differing ISA2 requirement. Genetic methods provided lines lacking ISA1 or ISA2. Biochemical analyses characterized ISA activities in mutant tissues. Starch content, granule morphology, and amylopectin fine structure were determined. Three ISA activity forms were observed in leaves, two ISA1/ISA2 heteromultimers and one ISA1 homomultimer. ISA1 homomultimer activity existed in mutants lacking ISA2. Mutants without ISA2 differed in leaf starch content, granule morphology, and amylopectin structure compared with nonmutants or lines lacking both ISA1 and ISA2. The data imply that both the ISA1 homomultimer and ISA1/ISA2 heteromultimer function in the maize leaf. The ISA1 homomultimer is present and functions in the maize leaf. Evolutionary divergence between monocots and dicots probably explains the ability of ISA1 to function as a homomultimer in maize leaves, in contrast to other species where the ISA1/ISA2 heteromultimer is the only active form.


Assuntos
Isoamilase/metabolismo , Folhas de Planta/enzimologia , Proteínas de Plantas/metabolismo , Amido/metabolismo , Zea mays/enzimologia , Sequência de Aminoácidos , Cromatografia em Gel , Sequência Conservada , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Isoamilase/química , Isoamilase/genética , Dados de Sequência Molecular , Extratos Vegetais , Folhas de Planta/genética , Folhas de Planta/ultraestrutura , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plastídeos/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Amido/ultraestrutura , Zea mays/ultraestrutura
11.
Biomacromolecules ; 14(2): 438-47, 2013 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-23311582

RESUMO

Glycogen biosynthesis requires the coordinated action of elongating and branching enzymes, of which the synergetic action is still not clearly understood. We have designed an experimental plan to develop and fully exploit a biomimetic system reproducing in vitro the activities involved in the formation of α(1,4) and α(1,6) glycosidic linkages during glycogen biosynthesis. This method is based on the use of two bacterial transglucosidases, the amylosucrase from Neisseria polysaccharea and the branching enzyme from Rhodothermus obamensis . The α-glucans synthesized from sucrose, a low cost agroresource, by the tandem action of the two enzymes, have been characterized by using complementary enzymatic, chromatographic, and imaging techniques. In a single step, linear and branched α-glucans were obtained, whose proportions, morphology, molar mass, and branching degree depended on both the initial sucrose concentration and the ratio between elongating and branching enzymes. In particular, spherical hyperbranched α-glucans with a controlled mean diameter (ranging from 10 to 150 nm), branching degree (from 10 to 13%), and weight-average molar mass (3.7 × 10(6) to 4.4 × 10(7) g.mol(-1)) were synthesized. Despite their structure, which is similar to that of natural glycogens, the mechanisms involved in their in vitro synthesis appeared to be different from those involved in the biosynthesis of native hyperbranched α-glucans.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Glucanos/síntese química , Glucosiltransferases/metabolismo , Neisseria/enzimologia , Rhodothermus/enzimologia , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Biomimética , Glucanos/química , Glucanos/ultraestrutura , Glucosiltransferases/genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Amido/metabolismo
12.
J Exp Bot ; 62(13): 4547-59, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21624979

RESUMO

This study assessed the impact on starch metabolism in Arabidopsis leaves of simultaneously eliminating multiple soluble starch synthases (SS) from among SS1, SS2, and SS3. Double mutant ss1- ss2- or ss1- ss3- lines were generated using confirmed null mutations. These were compared to the wild type, each single mutant, and ss1- ss2- ss3- triple mutant lines grown in standardized environments. Double mutant plants developed similarly to the wild type, although they accumulated less leaf starch in both short-day and long-day diurnal cycles. Despite the reduced levels in the double mutants, lines containing only SS2 and SS4, or SS3 and SS4, are able to produce substantial amounts of starch granules. In both double mutants the residual starch was structurally modified including higher ratios of amylose:amylopectin, altered glucan chain length distribution within amylopectin, abnormal granule morphology, and altered placement of α(1→6) branch linkages relative to the reducing end of each linear chain. The data demonstrate that SS activity affects not only chain elongation but also the net result of branch placement accomplished by the balanced activities of starch branching enzymes and starch debranching enzymes. SS3 was shown partially to overlap in function with SS1 for the generation of short glucan chains within amylopectin. Compensatory functions that, in some instances, allow continued residual starch production in the absence of specific SS classes were identified, probaby accomplished by the granule bound starch synthase GBSS1.


Assuntos
Amilopectina/química , Amilopectina/metabolismo , Arabidopsis/enzimologia , Folhas de Planta/metabolismo , Sintase do Amido/metabolismo , Amilopectina/ultraestrutura , Amilose/metabolismo , Fracionamento Químico , Cromatografia em Gel , Mutação/genética , Extratos Vegetais/metabolismo , Solubilidade
13.
New Phytol ; 188(1): 13-21, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20618917

RESUMO

Starch is the main polymer in which carbon and energy are stored in land plants, algae and some cyanobacteria. It plays a crucial role in the physiology of these organisms and also represents an important polymer for humans, in terms of both diet and nonfood industry uses. Recent efforts have elucidated most of the steps involved in the synthesis of starch. However, the process that initiates the synthesis of the starch granule remains unclear. Here, we outline the similarities between the synthesis of starch and the synthesis of glycogen, the other widespread and abundant glucose-based polymer in living cells. We place special emphasis on the mechanisms of initiation of the glycogen granule and current knowledge concerning the initiation of the starch granule. We also discuss recent discoveries regarding the function of starch synthases in the priming of the starch granule and possible interactions with other elements of the starch synthesis machinery.


Assuntos
Bactérias/metabolismo , Glicogênio/biossíntese , Plantas/metabolismo , Amido/biossíntese , Glicogênio/química , Glicogênio/metabolismo , Proteínas de Plantas/metabolismo , Plantas/enzimologia , Amido/química , Amido/metabolismo , Sintase do Amido/metabolismo
14.
Trends Plant Sci ; 13(11): 574-82, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18824400

RESUMO

Plastid endosymbiosis was accompanied by the appearance of a novel type of semi-cristalline storage polysaccharide (starch). Interestingly, starch is found in the cytoplasm of Rhodophyceae and Glaucophyta but is localized to the chloroplast stroma of Chloroplastida. The pathway is presumed to have been cytosolic in the common ancestor of the three Archaeplastida lineages. The means by which in green plants and algae an entire suite of nuclear-encoded starch-metabolism genes could have had their protein products rewired simultaneously to plastids are unclear. This opinion article reviews the timing and the possible reasons underlying this rewiring and proposes a hypothesis that explains its mechanism. The consequences of this mechanism on the complexity of starch metabolism in Chloroplastida are discussed.


Assuntos
Cloroplastos/metabolismo , Rodófitas/metabolismo , Amido/metabolismo , Evolução Biológica , Cianobactérias/metabolismo , Glicogênio/biossíntese , Glicogênio/metabolismo , Oligossacarídeos/biossíntese , Oligossacarídeos/metabolismo , Plastídeos/genética , Polissacarídeos/biossíntese , Polissacarídeos/metabolismo
15.
Mol Biol Evol ; 25(3): 536-48, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18093994

RESUMO

Eukaryotic cells are composed of a variety of membrane-bound organelles that are thought to derive from symbiotic associations involving bacteria, archaea, or other eukaryotes. In addition to acquiring the plastid, all Archaeplastida and some of their endosymbiotic derivatives can be distinguished from other organisms by the fact that they accumulate starch, a semicrystalline-storage polysaccharide distantly related to glycogen and never found elsewhere. We now provide the first evidence for the existence of starch in a particular species of single-cell diazotrophic cyanobacterium. We provide evidence for the existence in the eukaryotic host cell at the time of primary endosymbiosis of an uridine diphosphoglucose (UDP-glucose)-based pathway similar to that characterized in amoebas. Because of the monophyletic origin of plants, we can define the genetic makeup of the Archaeplastida ancestor with respect to storage polysaccharide metabolism. The most likely enzyme-partitioning scenario between the plastid's ancestor and its eukaryotic host immediately suggests the precise nature of the ancient metabolic symbiotic relationship. The latter consisted in the export of adenosine diphosphoglucose (ADP-glucose) from the cyanobiont in exchange for the import of reduced nitrogen from the host. We further speculate that the monophyletic origin of plastids may lie in an organism with close relatedness to present-day group V cyanobacteria.


Assuntos
Cianobactérias/genética , Filogenia , Plantas/metabolismo , Amido/metabolismo , Simbiose/fisiologia , Adenosina Difosfato Glucose/metabolismo , Evolução Biológica , Compartimento Celular/genética , Compartimento Celular/fisiologia , Cianobactérias/metabolismo , Glucose/metabolismo , Nitrogênio/metabolismo , Plantas/genética , Simbiose/genética , Uridina Difosfato Glucose/metabolismo
16.
Eukaryot Cell ; 7(2): 247-57, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18055913

RESUMO

The nature of the cytoplasmic pathway of starch biosynthesis was investigated in the model glaucophyte Cyanophora paradoxa. The storage polysaccharide granules are shown to be composed of both amylose and amylopectin fractions, with a chain length distribution and crystalline organization similar to those of green algae and land plant starch. A preliminary characterization of the starch pathway demonstrates that Cyanophora paradoxa contains several UDP-glucose-utilizing soluble starch synthase activities related to those of the Rhodophyceae. In addition, Cyanophora paradoxa synthesizes amylose with a granule-bound starch synthase displaying a preference for UDP-glucose. A debranching enzyme of isoamylase specificity and multiple starch phosphorylases also are evidenced in the model glaucophyte. The picture emerging from our biochemical and molecular characterizations consists of the presence of a UDP-glucose-based pathway similar to that recently proposed for the red algae, the cryptophytes, and the alveolates. The correlative presence of isoamylase and starch among photosynthetic eukaryotes is discussed.


Assuntos
Cyanophora/metabolismo , Citosol/metabolismo , Modelos Biológicos , Amido Fosforilase/metabolismo , Sintase do Amido/metabolismo , Amido/metabolismo , Uridina Difosfato Glucose/metabolismo , Amilopectina/metabolismo , Clonagem Molecular , Cyanophora/ultraestrutura , DNA Complementar/genética , Isoamilase/metabolismo , Filogenia , Amido/química , Amido Fosforilase/química , Sintase do Amido/química
17.
Plants (Basel) ; 8(9)2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31487879

RESUMO

Starch granule morphology is highly variable depending on the botanical origin. Moreover, all investigated plant species display intra-tissular variability of granule size. In potato tubers, the size distribution of starch granules follows a unimodal pattern with diameters ranging from 5 to 100 µm. Several evidences indicate that granule morphology in plants is related to the complex starch metabolic pathway. However, the intra-sample variability of starch-binding metabolic proteins remains unknown. Here, we report on the molecular characterization of size-fractionated potato starch granules with average diameters of 14.2 ± 3.7 µm, 24.5 ± 6.5 µm, 47.7 ± 12.8 µm, and 61.8 ± 17.4 µm. In addition to changes in the phosphate contents as well as small differences in the amylopectin structure, we found that the starch-binding protein stoichiometry varies significantly according to granule size. Label-free quantitative proteomics of each granule fraction revealed that individual proteins can be grouped according to four distinct abundance patterns. This study corroborates that the starch proteome may influence starch granule growth and architecture and opens up new perspectives in understanding the dynamics of starch biosynthesis.

18.
Front Plant Sci ; 10: 1075, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31552073

RESUMO

Starch granules that accumulate in the plastids of plants vary in size, shape, phosphate, or protein content according to their botanical origin. Depending on their size, the applications in food and nonfood industries differ. Being able to master starch granule size for a specific plant, without alteration of other characteristics (phosphate content, protein content, etc.), is challenging. The development of a simple and effective screening method to determine the size and shape of starch granules in a plant population is therefore of prime interest. In this study, we propose a new method, NegFluo, that combines negative confocal autofluorescence imaging in leaf and machine learning (ML)-based image analysis. It provides a fast, automated, and easy-to-use pipeline for both in situ starch granule imaging and its morphological analysis. NegFluo was applied to Arabidopsis leaves of wild-type and ss4 mutant plants. We validated its accuracy by comparing morphological quantifications using NegFluo and state-of-the-art methods relying either on starch granule purification or on preparation-intensive electron microscopy combined with manual image analysis. NegFluo thus opens the way to fast in situ analysis of starch granules.

19.
Sci Rep ; 9(1): 1990, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760823

RESUMO

Chlamydomonas reinhardtii represents an ideal model microbial system to decipher starch metabolism. In this green algae, in cells growing in photosynthetic conditions, starch mainly accumulates as a sheath surrounding the pyrenoid while in cells subjected to a nutrient starvation, numerous starch granules are filling up the plastid stroma. The mechanisms underlying and regulating this switch from photosynthetic to storage starch metabolisms are not known. In this work, we have isolated a Chlamydomonas mutant strain containing a deletion in chromosome 2 which displays abnormal starch granule distribution. Under nitrogen starvation, this strain contains an additional starch granules population. These granules are twice as big as the wild-type granules and display characteristics of photosynthetic starch. Genetic and functional complementation analyses allowed us to identify the gene responsible for this original phenotype which was called BSG1 for "Bimodal Starch Granule". Possible roles of BSG1 in starch metabolism modifications during the transition from photosynthetic to starved growth conditions are discussed.


Assuntos
Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Grânulos Citoplasmáticos/genética , Amido/metabolismo , Deleção Cromossômica , Grânulos Citoplasmáticos/química , Fotossíntese/fisiologia , Inanição/patologia
20.
BMC Plant Biol ; 8: 96, 2008 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-18811962

RESUMO

BACKGROUND: The biochemical mechanisms that determine the molecular architecture of amylopectin are central in plant biology because they allow long-term storage of reduced carbon. Amylopectin structure imparts the ability to form semi-crystalline starch granules, which in turn provides its glucose storage function. The enzymatic steps of amylopectin biosynthesis resemble those of the soluble polymer glycogen, however, the reasons for amylopectin's architectural distinctions are not clearly understood. The multiplicity of starch biosynthetic enzymes conserved in plants likely is involved. For example, amylopectin chain elongation in plants involves five conserved classes of starch synthase (SS), whereas glycogen biosynthesis typically requires only one class of glycogen synthase. RESULTS: Null mutations were characterized in AtSS2, which codes for SSII, and mutant lines were compared to lines lacking SSIII and to an Atss2, Atss3 double mutant. Loss of SSII did not affect growth rate or starch quantity, but caused increased amylose/amylopectin ratio, increased total amylose, and deficiency in amylopectin chains with degree of polymerization (DP) 12 to DP28. In contrast, loss of both SSII and SSIII caused slower plant growth and dramatically reduced starch content. Extreme deficiency in DP12 to DP28 chains occurred in the double mutant, far more severe than the summed changes in SSII- or SSIII-deficient plants lacking only one of the two enzymes. CONCLUSION: SSII and SSIII have partially redundant functions in determination of amylopectin structure, and these roles cannot be substituted by any other conserved SS, specifically SSI, GBSSI, or SSIV. Even though SSIII is not required for the normal abundance of glucan chains of DP12 to DP18, the enzyme clearly is capable of functioning in production such chains. The role of SSIII in producing these chains cannot be detected simply by analysis of an individual mutation. Competition between different SSs for binding to substrate could in part explain the specific distribution of glucan chains within amylopectin.


Assuntos
Amilopectina/biossíntese , Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Glucosiltransferases/genética , Proteínas de Plantas/genética , Sintase do Amido/genética , Amilose/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cruzamentos Genéticos , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Glucosiltransferases/metabolismo , Mutagênese Insercional , Mutação , Fenótipo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/metabolismo , RNA de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sintase do Amido/metabolismo
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