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1.
Foods ; 12(2)2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36673358

RESUMO

Breeding for less digestible starch in wheat can improve the health impact of bread and other wheat foods. The application of forward genetic approaches has lately opened opportunities for the discovery of new genes that influence the digestibility of starch, without the burden of detrimental effects on yield or on pasta and bread-making quality. In this study we developed a high-throughput in vitro starch digestibility assay (HTA) for use in forward genetic approaches to screen wheat germplasm. The HTA was validated using standard maize and wheat starches. Using the HTA we measured starch digestibility in hydrothermally processed flour samples and found wide variation among 118 wheat landraces from the A. E. Watkins collection and among eight elite UK varieties (23.5 to 39.9% and 31.2 to 43.5% starch digested after 90 min, respectively). We further investigated starch digestibility in fractions of sieved wholemeal flour and purified starch in a subset of the Watkins lines and elite varieties and found that the matrix properties of flour rather than the intrinsic properties of starch granules conferred lower starch digestibility.

3.
J Exp Bot ; 73(18): 6367-6379, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-35716106

RESUMO

Recent work has identified several proteins involved in starch granule initiation, the first step of starch synthesis. However, the degree of conservation in the granule initiation process remains poorly understood, especially among grass species differing in patterns of carbohydrate turnover in leaves, and granule morphology in the endosperm. We therefore compared mutant phenotypes of Hordeum vulgare (barley), Triticum turgidum (durum wheat), and Brachypodium distachyon defective in PROTEIN TARGETING TO STARCH 2 (PTST2), a key granule initiation protein. We report striking differences across species and organs. Loss of PTST2 from leaves resulted in fewer, larger starch granules per chloroplast and normal starch content in wheat, fewer granules per chloroplast and lower starch content in barley, and almost complete loss of starch in Brachypodium. The loss of starch in Brachypodium leaves was accompanied by high levels of ADP-glucose and detrimental effects on growth and physiology. Additionally, we found that loss of PTST2 increased granule initiation in Brachypodium amyloplasts, resulting in abnormal compound granule formation throughout the seed. These findings suggest that the importance of PTST2 varies greatly with the genetic and developmental background and inform the extent to which the gene can be targeted to improve starch in crops.


Assuntos
Brachypodium , Hordeum , Sintase do Amido , Amido/metabolismo , Sintase do Amido/genética , Endosperma/metabolismo , Hordeum/genética , Hordeum/metabolismo , Triticum/genética , Triticum/metabolismo , Glucose/metabolismo , Difosfato de Adenosina/metabolismo
4.
New Phytol ; 230(6): 2371-2386, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33714222

RESUMO

Starch granule initiation is poorly understood at the molecular level. The glucosyltransferase, STARCH SYNTHASE 4 (SS4), plays a central role in granule initiation in Arabidopsis leaves, but its function in cereal endosperms is unknown. We investigated the role of SS4 in wheat, which has a distinct spatiotemporal pattern of granule initiation during grain development. We generated TILLING mutants in tetraploid wheat (Triticum turgidum) that are defective in both SS4 homoeologs. The morphology of endosperm starch was examined in developing and mature grains. SS4 deficiency led to severe alterations in endosperm starch granule morphology. During early grain development, while the wild-type initiated single 'A-type' granules per amyloplast, most amyloplasts in the mutant formed compound granules due to multiple initiations. This phenotype was similar to mutants deficient in B-GRANULE CONTENT 1 (BGC1). SS4 deficiency also reduced starch content in leaves and pollen grains. We propose that SS4 and BGC1 are required for the proper control of granule initiation during early grain development that leads to a single A-type granule per amyloplast. The absence of either protein results in a variable number of initiations per amyloplast and compound granule formation.


Assuntos
Sintase do Amido , Endosperma/genética , Proteínas de Plantas/genética , Plastídeos/genética , Amido , Sintase do Amido/genética , Triticum/genética
5.
Curr Opin Plant Biol ; 60: 102013, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33677239

RESUMO

Genetic approaches to modify starch in crops have been limited by our knowledge of starch biosynthesis. Recent advances in Arabidopsis have revealed key genetic components determining the size, shape and number of granules in a plastid. This has opened the doors to new discoveries on granule initiation in crop species. In parallel, advances in genomic resources and gene editing technologies allow targeted manipulation of starch biosynthesis genes in isogenic crop backgrounds. Such technologies have been successfully deployed to alter starch composition, and can now be used to modify other starch traits. This will allow the complex relationships between starch structure and physicochemical properties to be elucidated, which will facilitate the rational manipulation of starches in crops.


Assuntos
Arabidopsis , Amido , Arabidopsis/genética , Produtos Agrícolas , Edição de Genes , Plastídeos/genética
6.
New Phytol ; 228(5): 1490-1504, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32767769

RESUMO

Starch granules are composed of two distinct glucose polymers - amylose and amylopectin. Amylose constitutes 5-35% of most natural starches and has a major influence over starch properties in foods. Its synthesis and storage occurs within the semicrystalline amylopectin matrix of starch granules, this poses a great challenge for biochemical and structural analyses. However, the last two decades have seen vast progress in understanding amylose synthesis, including new insights into the action of GRANULE BOUND STARCH SYNTHASE (GBSS), the major glucosyltransferase that synthesises amylose, and the discovery of PROTEIN TARGETING TO STARCH1 (PTST1) that targets GBSS to starch granules. Advances in analytical techniques have resolved the fine structure of amylose, raising new questions on how structure is determined during biosynthesis. Furthermore, the discovery of wild plants that do not produce amylose revives a long-standing question of why starch granules contain amylose, rather than amylopectin alone. Overall, these findings contribute towards a full understanding of amylose biosynthesis, structure and function that will be essential for future approaches to improve starch quality in crops.


Assuntos
Amilose , Sintase do Amido , Amilopectina , Glucanos , Amido , Sintase do Amido/genética
7.
Plant Cell ; 32(8): 2543-2565, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32471861

RESUMO

What determines the number of starch granules in plastids is an enigmatic aspect of starch metabolism. Several structurally and functionally diverse proteins have been implicated in the granule initiation process in Arabidopsis (Arabidopsis thaliana), with each protein exerting a varying degree of influence. Here, we show that a conserved starch synthase-like protein, STARCH SYNTHASE5 (SS5), regulates the number of starch granules that form in Arabidopsis chloroplasts. Among the starch synthases, SS5 is most closely related to SS4, a major determinant of granule initiation and morphology. However, unlike SS4 and the other starch synthases, SS5 is a noncanonical isoform that lacks catalytic glycosyltransferase activity. Nevertheless, loss of SS5 reduces starch granule numbers that form per chloroplast in Arabidopsis, and ss5 mutant starch granules are larger than wild-type granules. Like SS4, SS5 has a conserved putative surface binding site for glucans and also interacts with MYOSIN-RESEMBLING CHLOROPLAST PROTEIN, a proposed structural protein influential in starch granule initiation. Phenotypic analysis of a suite of double mutants lacking both SS5 and other proteins implicated in starch granule initiation allows us to propose how SS5 may act in this process.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Cloroplastos/metabolismo , Glicosiltransferases/metabolismo , Sintase do Amido/metabolismo , Amido/metabolismo , Proteínas de Arabidopsis/química , Sítios de Ligação , Proteínas de Cloroplastos/química , Cloroplastos/metabolismo , Sequência Conservada , Glucanos/metabolismo , Glicosiltransferases/química , Modelos Moleculares , Mutação/genética , Fenótipo , Folhas de Planta/enzimologia , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Sintase do Amido/química
8.
Plant Physiol ; 182(2): 870-881, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31694903

RESUMO

Starch granules contain two Glc polymers, amylopectin and amylose. Amylose makes up approximately 10% to 30% (w/w) of all natural starches thus far examined, but mutants of crop and model plants that produce amylose-free starch are generally indistinguishable from their wild-type counterparts with respect to growth, starch content, and granule morphology. Since the function and adaptive significance of amylose are unknown, we asked whether there is natural genetic variation in amylose synthesis within a wild, uncultivated species. We examined polymorphisms among the 1,135 sequenced accessions of Arabidopsis (Arabidopsis thaliana) in GRANULE-BOUND STARCH SYNTHASE (GBSS), encoding the enzyme responsible for amylose synthesis. We identified 18 accessions that are predicted to have polymorphisms in GBSS that affect protein function, and five of these accessions produced starch with no or extremely low amylose (< 0.5% [w/w]). Eight further accessions had amylose contents that were significantly lower or higher than that of Col-0 (9% [w/w]), ranging from 5% to 12% (w/w). We examined the effect of the polymorphisms on GBSS function and uncovered three mechanisms by which GBSS sequence variation led to different amylose contents: (1) altered GBSS abundance, (2) altered GBSS activity, and (3) altered affinity of GBSS for binding PROTEIN TARGETING TO STARCH1-a protein that targets GBSS to starch granules. These findings demonstrate that amylose in leaves is not essential for the viability of some naturally occurring Arabidopsis genotypes, at least over short timescales and under some environmental conditions and open an opportunity to explore the adaptive significance of amylose.


Assuntos
Amilose/biossíntese , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Sintase do Amido/genética , Sintase do Amido/metabolismo , Amido/análise , Amilopectina/análise , Amilopectina/genética , Amilopectina/metabolismo , Amilose/análise , Amilose/genética , Amilose/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Grânulos Citoplasmáticos/enzimologia , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Variação Genética , Genótipo , Fenótipo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Polimorfismo de Nucleotídeo Único , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Amido/metabolismo
9.
J Exp Bot ; 71(1): 105-115, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31633795

RESUMO

In Triticeae endosperm (e.g. wheat and barley), starch granules have a bimodal size distribution (with A- and B-type granules) whereas in other grasses the endosperm contains starch granules with a unimodal size distribution. Here, we identify the gene, BGC1 (B-GRANULE CONTENT 1), responsible for B-type starch granule content in Aegilops and wheat. Orthologues of this gene are known to influence starch synthesis in diploids such as rice, Arabidopsis, and barley. However, using polyploid Triticeae species, we uncovered a more complex biological role for BGC1 in starch granule initiation: BGC1 represses the initiation of A-granules in early grain development but promotes the initiation of B-granules in mid grain development. We provide evidence that the influence of BGC1 on starch synthesis is dose dependent and show that three very different starch phenotypes are conditioned by the gene dose of BGC1 in polyploid wheat: normal bimodal starch granule morphology; A-granules with few or no B-granules; or polymorphous starch with few normal A- or B-granules. We conclude from this work that BGC1 participates in controlling B-type starch granule initiation in Triticeae endosperm and that its precise effect on granule size and number varies with gene dose and stage of development.


Assuntos
Grão Comestível/crescimento & desenvolvimento , Dosagem de Genes , Proteínas de Plantas/genética , Receptores de Superfície Celular/genética , Amido/metabolismo , Triticum/genética , Grão Comestível/genética , Proteínas de Plantas/metabolismo , Poliploidia , Receptores de Superfície Celular/metabolismo , Triticum/crescimento & desenvolvimento
10.
Front Plant Sci ; 10: 993, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31417599

RESUMO

Reactive oxygen species (ROS) are produced in cells as normal cellular metabolic by-products. ROS concentration is normally low, but it increases under stress conditions. To stand ROS exposure, organisms evolved series of responsive mechanisms. One such mechanism is protein S-glutathionylation. S-glutathionylation is a post-translational modification typically occurring in response to oxidative stress, in which a glutathione reacts with cysteinyl residues, protecting them from overoxidation. α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. The Arabidopsis genome contains three genes encoding α-amylases. The sole chloroplastic member, AtAMY3, is involved in osmotic stress response and stomatal opening and is redox-regulated by thioredoxins. Here we show that AtAMY3 activity was sensitive to ROS, such as H2O2. Treatments with H2O2 inhibited enzyme activity and part of the inhibition was irreversible. However, in the presence of glutathione this irreversible inhibition was prevented through S-glutathionylation. The activity of oxidized AtAMY3 was completely restored by simultaneous reduction by both glutaredoxin (specific for the removal of glutathione-mixed disulfide) and thioredoxin (specific for the reduction of protein disulfide), supporting a possible liaison between both redox modifications. By comparing free cysteine residues between reduced and GSSG-treated AtAMY3 and performing oxidation experiments of Cys-to-Ser variants of AtAMY3 using biotin-conjugated GSSG, we could demonstrate that at least three distinct cysteinyl residues can be oxidized/glutathionylated, among those the two previously identified catalytic cysteines, Cys499 and Cys587. Measuring the pK a values of the catalytic cysteines by alkylation at different pHs and enzyme activity measurement (pK a1 = 5.70 ± 0.28; pK a2 = 7.83 ± 0.12) showed the tendency of one of the two catalytic cysteines to deprotonation, even at physiological pHs, supporting its propensity to undergo redox post-translational modifications. Taking into account previous and present findings, a functional model for redox regulation of AtAMY3 is proposed.

11.
Plant Cell ; 31(9): 2169-2186, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31266901

RESUMO

In Arabidopsis (Arabidopsis thaliana) leaves, starch is synthesized during the day and degraded at night to fuel growth and metabolism. Starch is degraded primarily by ß-amylases, liberating maltose, but this activity is preceded by glucan phosphorylation and is accompanied by dephosphorylation. A glucan phosphatase family member, LIKE SEX4 1 (LSF1), binds starch and is required for normal starch degradation, but its exact role is unclear. Here, we show that LSF1 does not dephosphorylate glucans. The recombinant dual specificity phosphatase (DSP) domain of LSF1 had no detectable phosphatase activity. Furthermore, a variant of LSF1 mutated in the catalytic cysteine of the DSP domain complemented the starch-excess phenotype of the lsf1 mutant. By contrast, a variant of LSF1 with mutations in the carbohydrate binding module did not complement lsf1 Thus, glucan binding, but not phosphatase activity, is required for the function of LSF1 in starch degradation. LSF1 interacts with the ß-amylases BAM1 and BAM3, and the BAM1-LSF1 complex shows amylolytic but not glucan phosphatase activity. Nighttime maltose levels are reduced in lsf1, and genetic analysis indicated that the starch-excess phenotype of lsf1 is dependent on bam1 and bam3 We propose that LSF1 binds ß-amylases at the starch granule surface, thereby promoting starch degradation.


Assuntos
Arabidopsis/metabolismo , Metabolismo dos Carboidratos/fisiologia , Fosfatases de Especificidade Dupla/metabolismo , Amido/metabolismo , beta-Amilase/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Metabolismo dos Carboidratos/genética , Proteínas de Transporte , Clonagem Molecular , Fosfatases de Especificidade Dupla/genética , Regulação da Expressão Gênica de Plantas , Glucanos/metabolismo , Fosforilação , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes , Alinhamento de Sequência , Tabaco/genética , Tabaco/metabolismo , beta-Amilase/genética
12.
J Exp Bot ; 70(3): 771-784, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30452691

RESUMO

Starch, the major storage carbohydrate in plants, is synthesized in plastids as semi-crystalline, insoluble granules. Many organs and cell types accumulate starch at some point during their development and maturation. The biosynthesis of the starch polymers, amylopectin and amylose, is relatively well understood and mostly conserved between organs and species. However, we are only beginning to understand the mechanism by which starch granules are initiated, and the factors that control the number of granules per plastid and the size/shape of granules. Here, we review recent progress in understanding starch granule initiation and morphogenesis. In Arabidopsis, granule initiation requires several newly discovered proteins with specific locations within the chloroplast, and also on the availability of maltooligosaccharides which act as primers for initiation. We also describe progress in understanding granule biogenesis in the endosperm of cereal grains-within which there is large interspecies variation in granule initiation patterns and morphology. Investigating whether this diversity results from differences between species in the functions of known proteins, and/or from the presence of novel, unidentified proteins, is a promising area of future research. Expanding our knowledge in these areas will lead to new strategies for improving the quality of cereal crops by modifying starch granule size and shape in vivo.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Produtos Agrícolas/crescimento & desenvolvimento , Desenvolvimento Vegetal , Poaceae/genética , Amido/metabolismo , Arabidopsis/metabolismo , Produtos Agrícolas/metabolismo , Grânulos Citoplasmáticos/metabolismo , Grão Comestível/crescimento & desenvolvimento , Grão Comestível/metabolismo , Poaceae/metabolismo
13.
Sci Adv ; 4(9): eaat6086, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30191180

RESUMO

Crop diversification required to meet demands for food security and industrial use is often challenged by breeding time and amenability of varieties to genome modification. Cassava is one such crop. Grown for its large starch-rich storage roots, it serves as a staple food and a commodity in the multibillion-dollar starch industry. Starch is composed of the glucose polymers amylopectin and amylose, with the latter strongly influencing the physicochemical properties of starch during cooking and processing. We demonstrate that CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated targeted mutagenesis of two genes involved in amylose biosynthesis, PROTEIN TARGETING TO STARCH (PTST1) or GRANULE BOUND STARCH SYNTHASE (GBSS), can reduce or eliminate amylose content in root starch. Integration of the Arabidopsis FLOWERING LOCUS T gene in the genome-editing cassette allowed us to accelerate flowering-an event seldom seen under glasshouse conditions. Germinated seeds yielded S1, a transgene-free progeny that inherited edited genes. This attractive new plant breeding technique for modified cassava could be extended to other crops to provide a suite of novel varieties with useful traits for food and industrial applications.


Assuntos
Manihot/genética , Melhoramento Vegetal/métodos , Proteínas de Plantas/genética , Sintase do Amido/genética , Amido/genética , Proteínas de Arabidopsis/genética , Sistemas CRISPR-Cas , Produtos Agrícolas/genética , Edição de Genes , Germinação , Manihot/química , Mutagênese , Plantas Geneticamente Modificadas/genética , Amido/química
14.
Plant Cell ; 30(7): 1523-1542, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29866647

RESUMO

The mechanism of starch granule initiation in chloroplasts is not fully understood. Here, we aimed to build on our recent discovery that PROTEIN TARGETING TO STARCH (PTST) family members, PTST2 and PTST3, are key players in starch granule initiation, by identifying and characterizing additional proteins involved in the process in Arabidopsis thaliana chloroplasts. Using immunoprecipitation and mass spectrometry, we demonstrate that PTST2 interacts with two plastidial coiled-coil proteins. Surprisingly, one of the proteins is the thylakoid-associated MAR BINDING FILAMENT-LIKE PROTEIN1 (MFP1), which was proposed to bind plastid nucleoids. The other protein, MYOSIN-RESEMBLING CHLOROPLAST PROTEIN (MRC), contains long coiled coils and no known domains. Whereas wild-type chloroplasts contained multiple starch granules, only one large granule was observed in most chloroplasts of the mfp1 and mrc mutants. The mfp1 mrc double mutant had a higher proportion of chloroplasts containing no visible granule than either single mutant and accumulated ADP-glucose, the substrate for starch synthesis. PTST2 was partially associated with the thylakoid membranes in wild-type plants, and fluorescently tagged PTST2 was located in numerous discrete patches within the chloroplast in which MFP1 was also located. In the mfp1 mutant, PTST2 was not associated with the thylakoids and formed discrete puncta, suggesting that MFP1 is necessary for normal PTST2 localization. Overall, we reveal that proper granule initiation requires the presence of MFP1 and MRC, and the correct location of PTST2.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Mutação/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Amido/metabolismo , Sintase do Amido/genética , Sintase do Amido/metabolismo , Tilacoides/genética , Tilacoides/metabolismo
15.
Dermatol Surg ; 44(9): 1170-1173, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29933297

RESUMO

BACKGROUND: Perineural invasion (PNI) is a high-risk feature of cutaneous squamous cell carcinoma (CSCC). Depths at which PNI occurs are unknown. OBJECTIVE: To determine the most superficial depth at which PNI occurs in CSCC and stratify by tumor clinical diameter and body location. METHODS AND MATERIALS: Single-institution retrospective review of CSCC specimens reporting PNI on pathology reports between January 2004 and August 2014. Depth was defined as distance from top of granular layer to middle of nerve invaded by CSCC or distance from erosion to middle of nerve affected by CSCC. RESULTS: Of 66 specimens identified with PNI, 45 specimens were included. Mean histopathologic depth to PNI was 2.7 mm (SD = 1.8 mm, median depth = 2.2 mm, range 0.5-12 mm). Perineural invasion depth varied by anatomic location, with the head associated with most superficial average PNI depth (2.2 mm) and trunk with greatest average PNI depth (4.3 mm). Perineural invasion depth correlated with clinical tumor diameter. The largest percentage of specimens with PNI were of clinical diameter of at least 2 cm (20/45 = 44%). CONCLUSION: Clinicians encountering lesions suspicious for CSCC have the greatest chance of detecting PNI using biopsy techniques that reach at least 3 to 4 mm deep.


Assuntos
Carcinoma de Células Escamosas/patologia , Nervos Periféricos/patologia , Neoplasias Cutâneas/patologia , Biópsia/métodos , Humanos , Invasividade Neoplásica , Estadiamento de Neoplasias , Estudos Retrospectivos
16.
Plant Cell ; 29(7): 1657-1677, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28684429

RESUMO

The molecular mechanism that initiates the synthesis of starch granules is poorly understood. Here, we discovered two plastidial proteins involved in granule initiation in Arabidopsis thaliana leaves. Both contain coiled coils and a family-48 carbohydrate binding module (CBM48) and are homologs of the PROTEIN TARGETING TO STARCH (PTST) protein; thus, we named them PTST2 and PTST3. Chloroplasts in mesophyll cells typically contain five to seven granules, but remarkably, most chloroplasts in ptst2 mutants contained zero or one large granule. Chloroplasts in ptst3 had a slight reduction in granule number compared with the wild type, while those of the ptst2 ptst3 double mutant contained even fewer granules than ptst2 The ptst2 granules were larger but similar in morphology to wild-type granules, but those of the double mutant had an aberrant morphology. Immunoprecipitation showed that PTST2 interacts with STARCH SYNTHASE4 (SS4), which influences granule initiation and morphology. Overexpression of PTST2 resulted in chloroplasts containing many small granules, an effect that was dependent on the presence of SS4. Furthermore, isothermal titration calorimetry revealed that the CBM48 domain of PTST2, which is essential for its function, interacts with long maltooligosaccharides. We propose that PTST2 and PTST3 are critical during granule initiation, as they bind and deliver suitable maltooligosaccharide primers to SS4.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Folhas de Planta/metabolismo , Amido/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Glucanos/metabolismo , Isoamilase/metabolismo , Mutação , Filogenia , Plantas Geneticamente Modificadas , Amido/genética , Sintase do Amido/genética , Sintase do Amido/metabolismo
17.
Metab Eng ; 40: 23-32, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28216105

RESUMO

Global demand for higher crop yields and for more efficient utilization of agricultural products will grow over the next decades. Here, we present a new concept for boosting the carbohydrate content of plants, by channeling photosynthetically fixed carbon into a newly engineered glucose polymer pool. We transiently expressed the starch/glycogen synthases from either Saccharomyces cerevisiae or Cyanidioschyzon merolae, together with the starch branching enzyme from C. merolae, in the cytosol of Nicotiana benthamiana leaves. This effectively built a UDP-glucose-dependent glycogen biosynthesis pathway. Glycogen synthesis was observed with Transmission Electron Microscopy, and the polymer structure was further analyzed. Within three days of enzyme expression, glycogen content of the leaf was 5-10 times higher than the starch levels of the control. Further, the leaves produced less starch and sucrose, which are normally the carbohydrate end-products of photosynthesis. We conclude that after enzyme expression, the newly fixed carbohydrates were routed into the new glycogen sink and trapped. Our approach allows carbohydrates to be efficiently stored in a new subcellular compartment, thus increasing the value of vegetative crop tissues for biofuel production or animal feed. The method also opens new potential for increasing the sink strength of heterotrophic tissues.


Assuntos
Metabolismo dos Carboidratos/fisiologia , Melhoramento Genético/métodos , Glicogênio/metabolismo , Folhas de Planta/fisiologia , Plantas Geneticamente Modificadas/metabolismo , Amido/metabolismo , Tabaco/fisiologia , Citosol/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Glucose/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Plantas Geneticamente Modificadas/genética , Amido/genética , Regulação para Cima/fisiologia
18.
Plant Cell ; 28(8): 1860-78, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27436713

RESUMO

Starch serves functions that range over a timescale of minutes to years, according to the cell type from which it is derived. In guard cells, starch is rapidly mobilized by the synergistic action of ß-AMYLASE1 (BAM1) and α-AMYLASE3 (AMY3) to promote stomatal opening. In the leaves, starch typically accumulates gradually during the day and is degraded at night by BAM3 to support heterotrophic metabolism. During osmotic stress, starch is degraded in the light by stress-activated BAM1 to release sugar and sugar-derived osmolytes. Here, we report that AMY3 is also involved in stress-induced starch degradation. Recently isolated Arabidopsis thaliana amy3 bam1 double mutants are hypersensitive to osmotic stress, showing impaired root growth. amy3 bam1 plants close their stomata under osmotic stress at similar rates as the wild type but fail to mobilize starch in the leaves. (14)C labeling showed that amy3 bam1 plants have reduced carbon export to the root, affecting osmolyte accumulation and root growth during stress. Using genetic approaches, we further demonstrate that abscisic acid controls the activity of BAM1 and AMY3 in leaves under osmotic stress through the AREB/ABF-SnRK2 kinase-signaling pathway. We propose that differential regulation and isoform subfunctionalization define starch-adaptive plasticity, ensuring an optimal carbon supply for continued growth under an ever-changing environment.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Folhas de Planta/metabolismo , Amido/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Pressão Osmótica/fisiologia , Folhas de Planta/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
19.
J Biol Chem ; 291(39): 20718-28, 2016 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-27458017

RESUMO

Arabidopsis leaf chloroplasts typically contain five to seven semicrystalline starch granules. It is not understood how the synthesis of each granule is initiated or how starch granule number is determined within each chloroplast. An Arabidopsis mutant lacking the glucosyl-transferase, STARCH SYNTHASE 4 (SS4) is impaired in its ability to initiate starch granules; its chloroplasts rarely contain more than one large granule, and the plants have a pale appearance and reduced growth. Here we report that the chloroplastic α-amylase AMY3, a starch-degrading enzyme, interferes with granule initiation in the ss4 mutant background. The amy3 single mutant is similar in phenotype to the wild type under normal growth conditions, with comparable numbers of starch granules per chloroplast. Interestingly, the ss4 mutant displays a pleiotropic reduction in the activity of AMY3. Remarkably, complete abolition of AMY3 (in the amy3 ss4 double mutant) increases the number of starch granules produced in each chloroplast, suppresses the pale phenotype of ss4, and nearly restores normal growth. The amy3 mutation also restores starch synthesis in the ss3 ss4 double mutant, which lacks STARCH SYNTHASE 3 (SS3) in addition to SS4. The ss3 ss4 line is unable to initiate any starch granules and is thus starchless. We suggest that SS4 plays a key role in granule initiation, allowing it to proceed in a way that avoids premature degradation of primers by starch hydrolases, such as AMY3.


Assuntos
Arabidopsis/metabolismo , Sintase do Amido/metabolismo , Amido/biossíntese , alfa-Amilases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Mutação , Amido/genética , Sintase do Amido/genética , alfa-Amilases/genética
20.
Plant Cell ; 28(6): 1472-89, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27207856

RESUMO

To uncover components of the mechanism that adjusts the rate of leaf starch degradation to the length of the night, we devised a screen for mutant Arabidopsis thaliana plants in which starch reserves are prematurely exhausted. The mutation in one such mutant, named early starvation1 (esv1), eliminates a previously uncharacterized protein. Starch in mutant leaves is degraded rapidly and in a nonlinear fashion, so that reserves are exhausted 2 h prior to dawn. The ESV1 protein and a similar uncharacterized Arabidopsis protein (named Like ESV1 [LESV]) are located in the chloroplast stroma and are also bound into starch granules. The region of highest similarity between the two proteins contains a series of near-repeated motifs rich in tryptophan. Both proteins are conserved throughout starch-synthesizing organisms, from angiosperms and monocots to green algae. Analysis of transgenic plants lacking or overexpressing ESV1 or LESV, and of double mutants lacking ESV1 and another protein necessary for starch degradation, leads us to propose that these proteins function in the organization of the starch granule matrix. We argue that their misexpression affects starch degradation indirectly, by altering matrix organization and, thus, accessibility of starch polymers to starch-degrading enzymes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Folhas de Planta/metabolismo , Amido/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Mutação , Folhas de Planta/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo
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