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1.
Plant Cell ; 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36454674

RESUMO

The carbon efficiency of storage lipid biosynthesis from imported sucrose in green Brassicaceae seeds is proposed to be enhanced by the PRK/Rubisco shunt, in which ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) acts outside the context of the Calvin-Benson-Bassham cycle to recycle CO2 molecules released during fatty acid synthesis. This pathway utilizes metabolites generated by the nonoxidative steps of the pentose phosphate pathway. Photosynthesis provides energy for reactions such as the phosphorylation of ribulose 5-phosphate by phosphoribulokinase (PRK). Here we show that loss of PRK in Arabidopsis thaliana (Arabidopsis) blocks photoautotrophic growth and is seedling-lethal. However, seeds containing prk embryos develop normally, allowing us to use genetics to assess the importance of the PRK/Rubisco shunt. Compared to nonmutant siblings, prk embryos produce one third less lipids-a greater reduction than expected from simply blocking the proposed PRK/Rubisco shunt. However, developing prk seeds are also chlorotic and have elevated starch contents compared with their siblings, indicative of secondary effects. Overexpressing PRK did not increase embryo lipid content, but metabolite profiling suggested that Rubisco activity becomes limiting. Overall, our findings show that the PRK/Rubisco shunt is tightly integrated into the carbon metabolism of green Arabidopsis seeds, and that its manipulation affects seed glycolysis, starch metabolism, and photosynthesis.

2.
Plant Physiol ; 2022 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-36135791

RESUMO

Leaves of shade-avoiding plants such as Arabidopsis (Arabidopsis thaliana) change their growth pattern and position in response to low red to far-red ratios (LRFRs) encountered in dense plant communities. Under LRFR, transcription factors of the phytochrome interacting factor (PIF) family are de-repressed. PIFs induce auxin production, which is required for promoting leaf hyponasty, thereby favoring access to unfiltered sunlight. Abscisic acid (ABA) has also been implicated in the control of leaf hyponasty, with gene expression patterns suggesting that LRFR regulates the ABA response. Here, we show that LRFR leads to a rapid increase in ABA levels in leaves. Changes in ABA levels depend on PIFs, which regulate the expression of genes encoding isoforms of the enzyme catalyzing a rate-limiting step in ABA biosynthesis. Interestingly, ABA biosynthesis and signaling mutants have more erect leaves than wild-type Arabidopsis under white light but respond less to LRFR. Consistent with this, ABA application decreases leaf angle under white light; however, this response is inhibited under LRFR. Tissue-specific interference with ABA signaling indicates that an ABA response is required in different cell types for LRFR-induced hyponasty. Collectively, our data indicate that LRFR triggers rapid PIF-mediated ABA production. ABA plays a different role in controlling hyponasty under white light than under LRFR. Moreover, ABA exerts its activity in multiple cell types to control leaf position.

3.
BMC Biol ; 20(1): 207, 2022 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-36153520

RESUMO

BACKGROUND: Starch, a vital plant-derived polysaccharide comprised of branched glucans, is essential in nutrition and many industrial applications. Starch is often modified post-extraction to alter its structure and enhance its functionality. Targeted metabolic engineering of crops to produce valuable and versatile starches requires knowledge of the relationships between starch biosynthesis, structure, and properties, but systematic studies to obtain this knowledge are difficult to conduct in plants. Here we used Saccharomyces cerevisiae as a testbed to dissect the functions of plant starch biosynthetic enzymes and create diverse starch-like polymers. RESULTS: We explored yeast promoters and terminators to tune the expression levels of the starch-biosynthesis machinery from Arabidopsis thaliana. We systematically modulated the expression of each starch synthase (SS) together with a branching enzyme (BE) in yeast. Protein quantification by parallel reaction monitoring (targeted proteomics) revealed unexpected effects of glucan biosynthesis on protein abundances but showed that the anticipated broad range of SS/BE enzyme ratios was maintained during the biosynthetic process. The different SS/BE ratios clearly influenced glucan structure and solubility: The higher the SS/BE ratio, the longer the glucan chains and the more glucans were partitioned into the insoluble fraction. This effect was irrespective of the SS isoform, demonstrating that the elongation/branching ratio controls glucan properties separate from enzyme specificity. CONCLUSIONS: Our results provide a quantitative framework for the in silico design of improved starch biosynthetic processes in plants. Our study also exemplifies a workflow for the rational tuning of a complex pathway in yeast, starting from the selection and evaluation of expression modules to multi-gene assembly and targeted protein monitoring during the biosynthetic process.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana , Arabidopsis , Sintase do Amido , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Arabidopsis/metabolismo , Glucanos/química , Plantas/metabolismo , Isoformas de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Amido/metabolismo , Sintase do Amido/química , Sintase do Amido/metabolismo
4.
Curr Biol ; 32(16): R894-R896, 2022 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-35998602

RESUMO

Starch metabolism is linked to plant growth, yet blocking its biosynthesis has species-specific consequences. In a new study, plastidial phosphoglucomutase is knocked out in aspen trees using CRISPR-Cas9, limiting starch production and altering photosynthesis, but growth, bud break and wood production proceed unaffected.


Assuntos
Fotossíntese , Amido , Metabolismo dos Carboidratos , Folhas de Planta , Plastídeos/metabolismo , Amido/metabolismo , Árvores
5.
Plant Physiol ; 189(4): 1976-2000, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35486376

RESUMO

Many plants, including Arabidopsis (Arabidopsis thaliana), accumulate starch in the light and remobilize it to support maintenance and growth at night. Starch synthesis and degradation are usually viewed as temporally separate processes. Recently, we reported that starch is also degraded in the light. Degradation rates are generally low early in the day but rise with time. Here, we show that the rate of degradation in the light depends on time relative to dawn rather than dusk. We also show that degradation in the light is inhibited by trehalose 6-phosphate, a signal for sucrose availability. The observed responses of degradation in the light can be simulated by a skeletal model in which the rate of degradation is a function of starch content divided by time remaining until dawn. The fit is improved by extension to include feedback inhibition of starch degradation by trehalose 6-phosphate. We also investigate possible functions of simultaneous starch synthesis and degradation in the light, using empirically parameterized models and experimental approaches. The idea that this cycle buffers growth against falling rates of photosynthesis at twilight is supported by data showing that rates of protein and cell wall synthesis remain high during a simulated dusk twilight. Degradation of starch in the light may also counter over-accumulation of starch in long photoperiods and stabilize signaling around dusk. We conclude that starch degradation in the light is regulated by mechanisms similar to those that operate at night and is important for stabilizing carbon availability and signaling, thus optimizing growth in natural light conditions.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Carbono/metabolismo , Fosfatos/metabolismo , Fotossíntese/fisiologia , Amido/metabolismo , Trealose/metabolismo
6.
Curr Biol ; 32(11): 2517-2528.e6, 2022 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-35413240

RESUMO

Recurrent damage by lepidopteran folivores triggers repeated leaf-to-leaf electrical signaling. We found that the ability to propagate electrical signals-called slow wave potentials-was unexpectedly robust and was maintained in plants that had experienced severe damage. We sought genes that maintain tissue excitability during group insect attack. When Arabidopsis thaliana P-Type II Ca2+-ATPase mutants were mechanically wounded, all mutants tested displayed leaf-to-leaf electrical signals. However, when the auto-inhibited Ca2+-ATPase double-mutant aca10 aca12 was attacked by Spodoptera littoralis caterpillars, electrical signaling failed catastrophically, and the insects consumed these plants rapidly. The attacked double mutant displayed petiole base deformation and chlorosis, which spread acropetally into laminas and led to senescence. A phloem-feeding aphid recapitulated these effects, implicating the vasculature in electrical signaling failure. Consistent with this, ACA10 expressed in phloem companion cells in an aca10 aca12 background rescued electrical signaling and defense during protracted S. littoralis attack. When expressed in xylem contact cells, ACA10 partially rescued these phenotypes. Extending our analyses, we found that prolonged darkness also caused wound-response electrical signaling failure in aca10 aca12 mutants. Our results lead to a model in which the plant vasculature acts as a capacitor that discharges temporarily when leaves are subjected to energy-depleting stresses. Under these conditions, ACA10 and ACA12 function allows the restoration of vein cell membrane potentials. In the absence of these gene functions, vascular cell excitability can no longer be restored efficiently. Additionally, this work demonstrates that non-invasive electrophysiology is a powerful tool for probing early events underlying senescence.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Adenosina Trifosfatases/metabolismo , Animais , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Herbivoria , Insetos , Folhas de Planta/fisiologia
7.
Plant Cell Environ ; 45(6): 1779-1795, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35229892

RESUMO

Despite the importance of storage root (SR) organs for cassava and the other root crops yield, their developmental origin is poorly understood. Here we use multiple approaches to shed light on the initial stages of root development demonstrating that SR and fibrous roots (FR) follow different rhizogenic processes. Transcriptome analysis carried out on roots collected before, during and after root bulking highlighted early and specific activation of a number of functions essential for root swelling and identified root-specific genes able to effectively discriminate emerging FR and SR. Starch and sugars start to accumulate at a higher rate in SR before they swell but only after parenchyma tissue has been produced. Finally, using non-destructive computed tomography measurements, we show that SR (but not FR) contain, since their emergence from the stem, an inner channel structure in continuity with the stem secondary xylem, indicating that SR derive from a distinct rhizogenic process compared with FR.


Assuntos
Manihot , Regulação da Expressão Gênica de Plantas , Manihot/genética , Raízes de Plantas , Amido , Xilema
8.
ACS Appl Mater Interfaces ; 14(6): 8417-8426, 2022 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-35107245

RESUMO

The development of controlled processes for continuous hydrogen generation from solid-state storage chemicals such as ammonia borane is central to integrating renewable hydrogen into a clean energy mix. However, to date, most reported platforms operate in batch mode, posing a challenge for controllable hydrogen release, catalyst reusability, and large-scale operation. To address these issues, we developed flow-through wood-based catalytic microreactors, characterized by inherent natural oriented microchannels. The prepared structured catalysts utilize silver-promoted palladium nanoparticles supported on metal-organic framework (MOF)-coated wood microreactors as the active phase. Catalytic tests demonstrate their highly controllable hydrogen production in continuous mode, and by adjusting the ammonia borane flow and wood species, we reach stable productivities of up to 10.4 cmH23 min-1 cmcat-3. The modular design of the structured catalysts proves readily scalable. Our versatile approach is applicable for other metals and MOF combinations, thus comprising a sustainable and scalable platform for catalytic dehydrogenations and applications in the energy-water nexus.

9.
Plant Physiol ; 188(1): 191-207, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-34662400

RESUMO

ß-Amylases (BAMs) are key enzymes of transitory starch degradation in chloroplasts, a process that buffers the availability of photosynthetically fixed carbon over the diel cycle to maintain energy levels and plant growth at night. However, during vascular plant evolution, the BAM gene family diversified, giving rise to isoforms with different compartmentation and biological activities. Here, we characterized BETA-AMYLASE 9 (BAM9) of Arabidopsis (Arabidopsis thaliana). Among the BAMs, BAM9 is most closely related to BAM4 but is more widely conserved in plants. BAM9 and BAM4 share features including their plastidial localization and lack of measurable α-1,4-glucan hydrolyzing capacity. BAM4 is a regulator of starch degradation, and bam4 mutants display a starch-excess phenotype. Although bam9 single mutants resemble the wild-type (WT), genetic experiments reveal that the loss of BAM9 markedly enhances the starch-excess phenotypes of mutants already impaired in starch degradation. Thus, BAM9 also regulates starch breakdown, but in a different way. Interestingly, BAM9 gene expression is responsive to several environmental changes, while that of BAM4 is not. Furthermore, overexpression of BAM9 in the WT reduced leaf starch content, but overexpression in bam4 failed to complement fully that mutant's starch-excess phenotype, suggesting that BAM9 and BAM4 are not redundant. We propose that BAM9 activates starch degradation, helping to manage carbohydrate availability in response to fluctuations in environmental conditions. As such, BAM9 represents an interesting gene target to explore in crop species.


Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/metabolismo , Plastídeos/metabolismo , Amido/metabolismo , beta-Amilase/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Reguladores de Crescimento de Plantas/genética , Folhas de Planta/genética , Plastídeos/genética , Amido/genética , beta-Amilase/genética
10.
Nat Commun ; 12(1): 6944, 2021 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-34836943

RESUMO

Living cells orchestrate enzyme activities to produce myriads of biopolymers but cell-biological understanding of such processes is scarce. Starch, a plant biopolymer forming discrete, semi-crystalline granules within plastids, plays a central role in glucose storage, which is fundamental to life. Combining complementary imaging techniques and Arabidopsis genetics we reveal that, in chloroplasts, multiple starch granules initiate in stromal pockets between thylakoid membranes. These initials coalesce, then grow anisotropically to form lenticular granules. The major starch polymer, amylopectin, is synthesized at the granule surface, while the minor amylose component is deposited internally. The non-enzymatic domain of STARCH SYNTHASE 4, which controls the protein's localization, is required for anisotropic growth. These results present us with a conceptual framework for understanding the biosynthesis of this key nutrient.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Sintase do Amido/metabolismo , Amido/metabolismo , Anisotropia , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Grânulos Citoplasmáticos/metabolismo , Glucose/metabolismo , Plantas Geneticamente Modificadas , Sintase do Amido/genética
11.
J Exp Bot ; 72(10): 3739-3755, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33684221

RESUMO

Plastid metabolism is critical in both photoautotrophic and heterotrophic plant cells. In chloroplasts, fructose-1,6-bisphosphate aldolase (FBA) catalyses the formation of both fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate within the Calvin-Benson cycle. Three Arabidopsis genes, AtFBA1-AtFBA3, encode plastidial isoforms of FBA, but the contribution of each isoform is unknown. Phylogenetic analysis indicates that FBA1 and FBA2 derive from a recently duplicated gene, while FBA3 is a more ancient paralog. fba1 mutants are phenotypically indistinguishable from the wild type, while both fba2 and fba3 have reduced growth. We show that FBA2 is the major isoform in leaves, contributing most of the measurable activity. Partial redundancy with FBA1 allows both single mutants to survive, but combining both mutations is lethal, indicating a block of photoautotrophy. In contrast, FBA3 is expressed predominantly in heterotrophic tissues, especially the leaf and root vasculature, but not in the leaf mesophyll. We show that the loss of FBA3 affects plastidial glycolytic metabolism of the root, potentially limiting the biosynthesis of essential compounds such as amino acids. However, grafting experiments suggest that fba3 is dysfunctional in leaf phloem transport, and we suggest that a block in photoassimilate export from leaves causes the buildup of high carbohydrate concentrations and retarded growth.


Assuntos
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Fotossíntese , Filogenia , Plastídeos/metabolismo
12.
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
13.
Nat Commun ; 12(1): 1049, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594064

RESUMO

Eukaryotic phytoplankton have a small global biomass but play major roles in primary production and climate. Despite improved understanding of phytoplankton diversity and evolution, we largely ignore the cellular bases of their environmental plasticity. By comparative 3D morphometric analysis across seven distant phytoplankton taxa, we observe constant volume occupancy by the main organelles and preserved volumetric ratios between plastids and mitochondria. We hypothesise that phytoplankton subcellular topology is modulated by energy-management constraints. Consistent with this, shifting the diatom Phaeodactylum from low to high light enhances photosynthesis and respiration, increases cell-volume occupancy by mitochondria and the plastid CO2-fixing pyrenoid, and boosts plastid-mitochondria contacts. Changes in organelle architectures and interactions also accompany Nannochloropsis acclimation to different trophic lifestyles, along with respiratory and photosynthetic responses. By revealing evolutionarily-conserved topologies of energy-managing organelles, and their role in phytoplankton acclimation, this work deciphers phytoplankton responses at subcellular scales.


Assuntos
Metabolismo Energético , Imageamento Tridimensional , Fitoplâncton/citologia , Fitoplâncton/fisiologia , Aclimatação/efeitos da radiação , Metabolismo Energético/efeitos da radiação , Luz , Microalgas/metabolismo , Microalgas/efeitos da radiação , Microalgas/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Mitocôndrias/ultraestrutura , Fitoplâncton/efeitos da radiação , Fitoplâncton/ultraestrutura , Plastídeos/metabolismo , Frações Subcelulares/metabolismo
14.
Elife ; 102021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33629953

RESUMO

Light triggers chloroplast differentiation whereby the etioplast transforms into a photosynthesizing chloroplast and the thylakoid rapidly emerges. However, the sequence of events during chloroplast differentiation remains poorly understood. Using Serial Block Face Scanning Electron Microscopy (SBF-SEM), we generated a series of chloroplast 3D reconstructions during differentiation, revealing chloroplast number and volume and the extent of envelope and thylakoid membrane surfaces. Furthermore, we used quantitative lipid and whole proteome data to complement the (ultra)structural data, providing a time-resolved, multi-dimensional description of chloroplast differentiation. This showed two distinct phases of chloroplast biogenesis: an initial photosynthesis-enabling 'Structure Establishment Phase' followed by a 'Chloroplast Proliferation Phase' during cell expansion. Moreover, these data detail thylakoid membrane expansion during de-etiolation at the seedling level and the relative contribution and differential regulation of proteins and lipids at each developmental stage. Altogether, we establish a roadmap for chloroplast differentiation, a critical process for plant photoautotrophic growth and survival.


Assuntos
Arabidopsis/fisiologia , Cloroplastos/fisiologia , Estiolamento , Biogênese de Organelas
15.
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
16.
Annu Rev Plant Biol ; 71: 217-245, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32075407

RESUMO

Research in the past decade has uncovered new and surprising information about the pathways of starch synthesis and degradation. This includes the discovery of previously unsuspected protein families required both for processes and for the long-sought mechanism of initiation of starch granules. There is also growing recognition of the central role of leaf starch turnover in making carbon available for growth across the day-night cycle. Sophisticated systems-level control mechanisms involving the circadian clock set rates of nighttime starch mobilization that maintain a steady supply of carbon until dawn and modulate partitioning of photosynthate into starch in the light, optimizing the fraction of assimilated carbon that can be used for growth. These discoveries also uncover complexities: Results from experiments with Arabidopsis leaves in conventional controlled environments are not necessarily applicable to other organs or species or to growth in natural, fluctuating environments.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Relógios Circadianos , Carbono , Folhas de Planta , Amido
17.
Photosynth Res ; 145(1): 55-70, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31955343

RESUMO

Starch, a plant-derived insoluble carbohydrate composed of glucose polymers, is the principal carbohydrate in our diet and a valuable raw material for industry. The properties of starch depend on the arrangement of glucose units within the constituent polymers. However, key aspects of starch structure and the underlying biosynthetic processes are not well understood, limiting progress towards targeted improvement of our starch crops. In particular, the major component of starch, amylopectin, has a complex three-dimensional, branched architecture. This architecture stems from the combined actions of a multitude of enzymes, each having broad specificities that are difficult to capture experimentally. In this review, we reflect on experimental approaches and limitations to decipher the enzymes' specificities and explore possibilities for in silico simulations of these activities. We believe that the synergy between experimentation and simulation is needed for the correct interpretation of experimental data and holds the potential to greatly advance our understanding of the overall starch biosynthetic process. We furthermore propose that the formation of glucan secondary structures, concomitant with its synthesis, is a previously overlooked factor that directly affects amylopectin architecture through its impact on enzyme function.


Assuntos
Amilopectina/biossíntese , Arabidopsis/metabolismo , Amido/biossíntese , Glucanos/metabolismo , Folhas de Planta/metabolismo
18.
Plant J ; 102(6): 1202-1219, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31950549

RESUMO

Cassava is an important staple crop in sub-Saharan Africa, due to its high productivity even on nutrient poor soils. The metabolic characteristics underlying this high productivity are poorly understood including the mode of photosynthesis, reasons for the high rate of photosynthesis, the extent of source/sink limitation, the impact of environment, and the extent of variation between cultivars. Six commercial African cassava cultivars were grown in a greenhouse in Erlangen, Germany, and in the field in Ibadan, Nigeria. Source leaves, sink leaves, stems and storage roots were harvested during storage root bulking and analyzed for sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, protein, activities of enzymes in central metabolism and yield traits. High ratios of RuBisCO:phosphoenolpyruvate carboxylase activity support a C3 mode of photosynthesis. The high rate of photosynthesis is likely to be attributed to high activities of enzymes in the Calvin-Benson cycle and pathways for sucrose and starch synthesis. Nevertheless, source limitation is indicated because root yield traits correlated with metabolic traits in leaves rather than in the stem or storage roots. This situation was especially so in greenhouse-grown plants, where irradiance will have been low. In the field, plants produced more storage roots. This was associated with higher AGPase activity and lower sucrose in the roots, indicating that feedforward loops enhanced sink capacity in the high light and low nitrogen environment in the field. Overall, these results indicated that carbon assimilation rate, the K battery, root starch synthesis, trehalose, and chlorogenic acid accumulation are potential target traits for genetic improvement.


Assuntos
Manihot/metabolismo , Raízes de Plantas/metabolismo , Metabolismo dos Carboidratos , Produção Agrícola , Manihot/crescimento & desenvolvimento , Redes e Vias Metabólicas , Fotossíntese , Folhas de Planta/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Caules de Planta/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo
19.
Curr Protoc Plant Biol ; 4(4): e20102, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31834991

RESUMO

Cassava plays an important role as a staple food for more than 800 million people in the world due to its ability to maintain relatively high productivity even in nutrient-depleted soils. Even though cassava has been the focus of several breeding programs and has become a strong focus of research in the last few years, relatively little is currently known about its metabolism and metabolic composition in different tissues. In this article, the absolute content of sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, carotenoids, chlorophylls, tocopherols, and total protein as well as starch quality is described based on multiple analytical techniques, with protocols specifically adjusted for material from different cassava tissues. Moreover, quantification of secondary metabolites relative to internal standards is presented using both non-targeted and targeted metabolomics approaches. The protocols have also been adjusted to apply to freeze-dried material in order to allow processing of field harvest samples that typically will require long-distance transport. © 2019 The Authors. Basic Protocol 1: Metabolic profiling by gas chromatography-mass spectrometry (GC-MS) Support Protocol 1: Preparation of freeze-dried cassava material Support Protocol 2: Preparation of standard compound mixtures for absolute quantification of metabolites by GC-MS Support Protocol 3: Preparation of retention-time standard mixture Basic Protocol 2: Determination of organic acids and phosphorylated intermediates by ion chromatography-mass spectrometry (IC-MS) Support Protocol 4: Preparation of standards and recovery experimental procedure Basic Protocol 3: Determination of soluble sugars, starch, and free amino acids Alternate Protocol: Determination of soluble sugars and starch Basic Protocol 4: Determination of anions Basic Protocol 5: Determination of elements Basic Protocol 6: Determination of total protein Basic Protocol 7: Determination of non-targeted and targeted secondary metabolites Basic Protocol 8: Determination of carotenoids, chlorophylls, and tocopherol Basic Protocol 9: Determination of starch quality.


Assuntos
Manihot , Aminoácidos , Cromatografia Gasosa-Espectrometria de Massas , Metabolômica , Amido
20.
Plant Cell Physiol ; 60(12): 2692-2706, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31397873

RESUMO

Abiotic environmental stresses have a negative impact on the yield and quality of crops. Understanding these stresses is an essential enabler for mitigating breeding strategies and it becomes more important as the frequency of extreme weather conditions increases due to climate change. This study analyses the response of barley (Hordeum vulgare L.) to a heat wave during grain filling in three distinct stages: the heat wave itself, the return to a normal temperature regime, and the process of maturation and desiccation. The properties and structure of the starch produced were followed throughout the maturational stages. Furthermore, the key enzymes involved in the carbohydrate supply to the grain were monitored. We observed differences in starch structure with well-separated effects because of heat stress and during senescence. Heat stress produced marked effects on sucrolytic enzymes in source and sink tissues. Early cessation of plant development as an indirect consequence of the heat wave was identified as the major contributor to final yield loss from the stress, highlighting the importance for functional stay-green traits for the development of heat-resistant cereals.


Assuntos
Amilopectina/metabolismo , Parede Celular/enzimologia , Parede Celular/metabolismo , Hordeum/enzimologia , Hordeum/metabolismo , beta-Frutofuranosidase/metabolismo , Amilopectina/genética , Parede Celular/fisiologia , Resposta ao Choque Térmico/fisiologia , Hordeum/fisiologia , beta-Frutofuranosidase/genética
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