RESUMEN
The human colon hosts hundreds of commensal bacterial species, many of which ferment complex dietary carbohydrates. To transform these fibers into metabolically accessible compounds, microbes often express a series of dedicated enzymes homologous to the starch utilization system (Sus) encoded in polysaccharide utilization loci (PULs). The genome of Bacteroides thetaiotaomicron (Bt), a common member of the human gut microbiota, encodes nearly 100 PULs, conferring a strong metabolic versatility. While the structures and functions of individual enzymes within the PULs have been investigated, little is known about how polysaccharide complexity impacts the function of Sus-like systems. We here show that the activity of Sus-like systems depends on polysaccharide size, ultimately impacting bacterial growth. We demonstrate the effect of size-dependent metabolism in the context of dextran metabolism driven by the specific utilization system PUL48. We find that as the molecular weight of dextran increases, Bt growth rate decreases and lag time increases. At the enzymatic level, the dextranase BT3087, a glycoside hydrolase (GH) belonging to the GH family 66, is the main GH for dextran utilization, and BT3087 and BT3088 contribute to Bt dextran metabolism in a size-dependent manner. Finally, we show that the polysaccharide size-dependent metabolism of Bt impacts its metabolic output in a way that modulates the composition of a producer-consumer community it forms with Bacteroides fragilis. Altogether, our results expose an overlooked aspect of Bt metabolism that can impact the composition and diversity of microbiota. IMPORTANCE: Polysaccharides are complex molecules that are commonly found in our diet. While humans lack the ability to degrade many polysaccharides, their intestinal microbiota contain bacterial commensals that are versatile polysaccharide utilizers. The gut commensal Bacteroides thetaiotaomicron dedicates roughly 20% of their genomes to the expression of polysaccharide utilization loci for the broad range utilization of polysaccharides. Although it is known that different polysaccharide utilization loci are dedicated to the degradation of specific polysaccharides with unique glycosidic linkages and monosaccharide compositions, it is often overlooked that specific polysaccharides may also exist in various molecular weights. These different physical attributes may impact their processability by starch utilization system-like systems, leading to differing growth rates and nutrient-sharing properties at the community level. Therefore, understanding how molecular weight impacts utilization by gut microbe may lead to the potential design of novel precision prebiotics.
Asunto(s)
Bacteroides thetaiotaomicron , Humanos , Bacteroides thetaiotaomicron/metabolismo , Peso Molecular , Bacteroides/metabolismo , Dextranos/metabolismo , Tracto Gastrointestinal/microbiología , Polisacáridos/metabolismo , AlmidónRESUMEN
Bacterial gut commensals experience a biologically and physically complex mucosal environment. While many chemical factors mediate the composition and structure of these microbial communities, less is known about the role of mechanics. Here, we demonstrate that fluid flow impacts the spatial organization and composition of gut biofilm communities by shaping how different species interact metabolically. We first demonstrate that a model community composed of Bacteroides thetaiotaomicron (Bt) and Bacteroides fragilis (Bf), two representative human commensals, can form robust biofilms in flow. We identified dextran as a polysaccharide readily metabolized by Bt but not Bf, but whose fermentation generates a public good enabling Bf growth. By combining simulations with experiments, we demonstrate that in flow, Bt biofilms share dextran metabolic by-products, promoting Bf biofilm formation. By transporting this public good, flow structures the spatial organization of the community, positioning the Bf population downstream from Bt. We show that sufficiently strong flows abolish Bf biofilm formation by limiting the effective public good concentration at the surface. Physical factors such as flow may therefore contribute to the composition of intestinal microbial communities, potentially impacting host health.
Asunto(s)
Bacteroides thetaiotaomicron , Microbioma Gastrointestinal , Microbiota , Humanos , Dextranos , Bacteroides fragilis , BiopelículasRESUMEN
Starch, the most abundant carbohydrate reserve in plants, primarily consists of the branched glucan amylopectin, which forms semi-crystalline granules. Phase transition from a soluble to an insoluble form depends on amylopectin architecture, requiring a compatible distribution of glucan chain lengths and a branch-point distribution. Here, we show that two starch-bound proteins, LIKE EARLY STARVATION 1 (LESV) and EARLY STARVATION 1 (ESV1), which have unusual carbohydrate-binding surfaces, promote the phase transition of amylopectin-like glucans, both in a heterologous yeast system expressing the starch biosynthetic machinery and in Arabidopsis plants. We propose a model wherein LESV serves as a nucleating role, with its carbohydrate-binding surfaces helping align glucan double helices to promote their phase transition into semi-crystalline lamellae, which are then stabilized by ESV1. Because both proteins are widely conserved, we suggest that protein-facilitated glucan crystallization may be a general and previously unrecognized feature of starch biosynthesis.
Asunto(s)
Amilopectina , Arabidopsis , Amilopectina/química , Amilopectina/metabolismo , Almidón/química , Glucanos/química , Glucanos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Plantas/metabolismoRESUMEN
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 with 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.
Asunto(s)
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismo , Carbono/metabolismo , Fotosíntesis/genética , Semillas/genética , Semillas/metabolismo , Almidón/metabolismo , LípidosRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Adenosina Trifosfatasas/metabolismo , Animales , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Herbivoria , Insectos , Hojas de la Planta/fisiologíaRESUMEN
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.
Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Hojas de la Planta/metabolismo , Plastidios/metabolismo , Almidón/metabolismo , beta-Amilasa/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Reguladores del Crecimiento de las Plantas/genética , Hojas de la Planta/genética , Plastidios/genética , Almidón/genética , beta-Amilasa/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Frío , Proteínas de Transporte de Membrana/genética , Fenotipo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte de Membrana/metabolismoRESUMEN
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.
Asunto(s)
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/metabolismo , Fotosíntesis , Filogenia , Plastidios/metabolismoRESUMEN
The plant cell wall is a complex network of polysaccharides and proteins that provides strength and structural integrity to plant cells, as well as playing a vital role in growth, development, and defense response. Cell wall polysaccharides can be broadly grouped into three categories: cellulose, pectins, and hemicelluloses. Dynamic interactions between polysaccharides and cell wall-associated proteins contribute to regions of flexibility and rigidity within the cell wall, allowing for remodeling when necessary during growth, environmental adaptation, or stress response activation. These polysaccharide interactions are vital to plant growth, however they also contribute to the level of difficulty encountered when attempting to analyze cell wall structure and composition. In the past, lengthy protocols to quantify cell wall monosaccharides contributing to cellulose as well as neutral and acidic cell wall polysaccharides have been used. Recently, a streamlined approach for monosaccharide quantification was described. This protocol combines a simplified hydrolysis method followed by several runs of high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Here, we present an updated version of this protocol in which we can analyze all nine cell wall monosaccharides in a single high-performance liquid chromatography HPAEC-PAD gradient profile. The inclusion of an enzymatic starch degradation, as well as alternate internal standards for added quantification accuracy, and a ready-to-use Python script facilitating data analysis adds a broadened scope of utility to this protocol. This protocol was used to analyze Arabidopsis light-grown seedlings and dark-grown hypocotyls, but is suitable for any plant tissues.
RESUMEN
Isoamylases hydrolyse (1-6)-alpha-D-glucosidic linkages in starch and are involved in both starch granule formation and starch degradation. In plants, three isoamylase isoforms with distinct functions in starch synthesis (ISA1 and ISA2) and degradation (ISA3) have been described. Here, we created transgenic potato plants with simultaneously decreased expression of all three isoamylases using a chimeric RNAi construct targeting all three isoforms. Constitutive expression of the hairpin RNA using the 35S CaMV promoter resulted in efficient silencing of all three isoforms in leaves, growing tubers, and sprouting tubers. Neither plant growth nor tuber yield was effected in isoamylase-deficient potato lines. Interestingly, starch metabolism was found to be impaired in a tissue-specific manner. While leaf starch content was unaffected, tuber starch was significantly reduced. The reduction in tuber starch content in the transgenic plants was accompanied by a decrease in starch granules size, an increased sucrose content and decreased hexose levels. Despite the effects on granule size, only little changes in chain length composition of soluble and insoluble glucose polymers were detected. The transgenic tubers displayed an early sprouting phenotype that was accompanied by an increased level of sucrose in parenchyma cells below the outgrowing bud. Since high sucrose levels promote sprouting, we propose that the increased number of small starch granules may cause an accelerated turnover of glucan chains and hence a more rapid synthesis of sucrose. This observation links alterations in starch structure/degradation with developmental processes like meristem activation and sprout outgrowth in potato tubers.
Asunto(s)
Isoamilasa/metabolismo , Proteínas de Plantas/metabolismo , Interferencia de ARN , Almidón/metabolismo , Hexosas/metabolismo , Isoamilasa/antagonistas & inhibidores , Isoamilasa/genética , Fenotipo , Hojas de la Planta/metabolismo , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/genética , Tubérculos de la Planta/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/metabolismo , Plantones/fisiología , Solanum tuberosum/crecimiento & desarrollo , Solanum tuberosum/metabolismo , Sacarosa/metabolismoRESUMEN
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.
Asunto(s)
Arabidopsis/metabolismo , Almidón Sintasa/metabolismo , Almidón/biosíntesis , alfa-Amilasas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Mutación , Almidón/genética , Almidón Sintasa/genética , alfa-Amilasas/genéticaRESUMEN
Arabidopsis proton gradient regulation (pgr) mutants have high chlorophyll fluorescence and reduced non-photochemical quenching (NPQ) caused by defects in photosynthetic electron transport. Here, we identify PGR6 as the chloroplast lipid droplet (plastoglobule, PG) kinase ABC1K1 (activity of bc1 complex kinase 1). The members of the ABC1/ADCK/UbiB family of atypical kinases regulate ubiquinone synthesis in bacteria and mitochondria, and impact various metabolic pathways in plant chloroplasts. Here, we demonstrate that abc1k1 has a unique photosynthetic and metabolic phenotype that is distinct from that of the abc1k3 homolog. The abc1k1/pgr6 single mutant is specifically deficient in the electron carrier plastoquinone, as well as in ß-carotene and the xanthophyll lutein, and is defective in membrane antioxidant tocopherol metabolism. After 2 days of continuous high light stress, abc1k1/pgr6 plants suffer extensive photosynthetic and metabolic perturbations, strongly affecting carbohydrate metabolism. Remarkably, however, the mutant acclimates to high light after 7 days together with a recovery of carotenoid levels and a drastic alteration in the starch-to-sucrose ratio. Moreover, ABC1K1 behaves as an active kinase and phosphorylates VTE1, a key enzyme of tocopherol (vitamin E) metabolism in vitro. Our results indicate that the ABC1K1 kinase constitutes a new type of regulatory link between photosynthetic activity and chloroplast metabolism.
Asunto(s)
Arabidopsis/fisiología , Cloroplastos/metabolismo , Fotosíntesis , Proteínas Quinasas/metabolismo , Arabidopsis/enzimología , Arabidopsis/metabolismo , Western Blotting , Datos de Secuencia Molecular , Mutación , Fosforilación , Proteínas Quinasas/genéticaRESUMEN
Isoamylase-type debranching enzymes (ISAs) play an important role in determining starch structure. Amylopectin - a branched polymer of glucose - is the major component of starch granules and its architecture underlies the semi-crystalline nature of starch. Mutants of several species lacking the ISA1-subclass of isoamylase are impaired in amylopectin synthesis. Consequently, starch levels are decreased and an aberrant soluble glucan (phytoglycogen) with altered branch lengths and branching pattern accumulates. Here we use TAP (tandem affinity purification) tagging to provide direct evidence in Arabidopsis that ISA1 interacts with its homolog ISA2. No evidence for interaction with other starch biosynthetic enzymes was found. Analysis of the single mutants shows that each protein is destabilised in the absence of the other. Co-expression of both ISA1 and ISA2 Escherichia coli allowed the formation of the active recombinant enzyme and we show using site-directed mutagenesis that ISA1 is the catalytic subunit. The presence of the active isoamylase alters glycogen biosynthesis in E. coli, resulting in colonies that stain more starch-like with iodine. However, analysis of the glucans reveals that rather than producing an amylopectin like substance, cells expressing the active isoamylase still accumulate small amounts of glycogen together with a population of linear oligosaccharides that stain strongly with iodine. We conclude that for isoamylase to promote amylopectin synthesis it needs to act on a specific precursor (pre-amylopectin) generated by the combined actions of plant starch synthase and branching enzyme isoforms and when presented with an unsuitable substrate (i.e. E. coli glycogen) it simply degrades it.
Asunto(s)
Arabidopsis/enzimología , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Isoamilasa/metabolismo , Complejos Multiproteicos/metabolismo , Secuencia de Bases , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cartilla de ADN/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Prueba de Complementación Genética , Glucógeno/biosíntesis , Glucógeno/metabolismo , Isoamilasa/genética , Isoamilasa/aislamiento & purificación , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Estabilidad Proteica , Subunidades de Proteína/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Almidón/biosíntesis , Especificidad por SustratoRESUMEN
A putative phosphatase, LSF1 (for LIKE SEX4; previously PTPKIS2), is closely related in sequence and structure to STARCH-EXCESS4 (SEX4), an enzyme necessary for the removal of phosphate groups from starch polymers during starch degradation in Arabidopsis (Arabidopsis thaliana) leaves at night. We show that LSF1 is also required for starch degradation: lsf1 mutants, like sex4 mutants, have substantially more starch in their leaves than wild-type plants throughout the diurnal cycle. LSF1 is chloroplastic and is located on the surface of starch granules. lsf1 and sex4 mutants show similar, extensive changes relative to wild-type plants in the expression of sugar-sensitive genes. However, although LSF1 and SEX4 are probably both involved in the early stages of starch degradation, we show that LSF1 neither catalyzes the same reaction as SEX4 nor mediates a sequential step in the pathway. Evidence includes the contents and metabolism of phosphorylated glucans in the single mutants. The sex4 mutant accumulates soluble phospho-oligosaccharides undetectable in wild-type plants and is deficient in a starch granule-dephosphorylating activity present in wild-type plants. The lsf1 mutant displays neither of these phenotypes. The phenotype of the lsf1/sex4 double mutant also differs from that of both single mutants in several respects. We discuss the possible role of the LSF1 protein in starch degradation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Hojas de la Planta/metabolismo , Almidón/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cloroplastos/enzimología , ADN Bacteriano/genética , Glucanos/metabolismo , Mutagénesis Insercional , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Hojas de la Planta/genética , ARN de Planta/genéticaRESUMEN
In most plants, a large fraction of photo-assimilated carbon is stored in the chloroplasts during the day as starch and remobilized during the subsequent night to support metabolism. Mutations blocking either starch synthesis or starch breakdown in Arabidopsis thaliana reduce plant growth. Maltose is the major product of starch breakdown exported from the chloroplast at night. The maltose excess 1 mutant (mex1), which lacks the chloroplast envelope maltose transporter, accumulates high levels of maltose and starch in chloroplasts and develops a distinctive but previously unexplained chlorotic phenotype as leaves mature. The introduction of additional mutations that prevent starch synthesis, or that block maltose production from starch, also prevent chlorosis of mex1. In contrast, introduction of mutations in disproportionating enzyme (DPE1) results in the accumulation of maltotriose in addition to maltose, and greatly increases chlorosis. These data suggest a link between maltose accumulation and chloroplast homeostasis. Microscopic analyses show that the mesophyll cells in chlorotic mex1 leaves have fewer than half the number of chloroplasts than wild-type cells. Transmission electron microscopy reveals autophagy-like chloroplast degradation in both mex1 and the dpe1/mex1 double mutant. Microarray analyses reveal substantial reprogramming of metabolic and cellular processes, suggesting that organellar protein turnover is increased in mex1, though leaf senescence and senescence-related chlorophyll catabolism are not induced. We propose that the accumulation of maltose and malto-oligosaccharides causes chloroplast dysfunction, which may by signaled via a form of retrograde signaling and trigger chloroplast degradation.
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Arabidopsis/metabolismo , Cloroplastos/metabolismo , Almidón/antagonistas & inhibidores , Almidón/metabolismo , Arabidopsis/genética , Cloroplastos/genética , Cloroplastos/ultraestructura , Mutación , Fenotipo , Fotosíntesis/genética , Hojas de la Planta/crecimiento & desarrolloRESUMEN
This work investigated the roles of beta-amylases in the breakdown of leaf starch. Of the nine beta-amylase (BAM)-like proteins encoded in the Arabidopsis thaliana genome, at least four (BAM1, -2, -3, and -4) are chloroplastic. When expressed as recombinant proteins in Escherichia coli, BAM1, BAM2, and BAM3 had measurable beta-amylase activity but BAM4 did not. BAM4 has multiple amino acid substitutions relative to characterized beta-amylases, including one of the two catalytic residues. Modeling predicts major differences between the glucan binding site of BAM4 and those of active beta-amylases. Thus, BAM4 probably lost its catalytic capacity during evolution. Total beta-amylase activity was reduced in leaves of bam1 and bam3 mutants but not in bam2 and bam4 mutants. The bam3 mutant had elevated starch levels and lower nighttime maltose levels than the wild type, whereas bam1 did not. However, the bam1 bam3 double mutant had a more severe phenotype than bam3, suggesting functional overlap between the two proteins. Surprisingly, bam4 mutants had elevated starch levels. Introduction of the bam4 mutation into the bam3 and bam1 bam3 backgrounds further elevated the starch levels in both cases. These data suggest that BAM4 facilitates or regulates starch breakdown and operates independently of BAM1 and BAM3. Together, our findings are consistent with the proposal that beta-amylase is a major enzyme of starch breakdown in leaves, but they reveal unexpected complexity in terms of the specialization of protein function.
Asunto(s)
Arabidopsis/enzimología , Cloroplastos/enzimología , Almidón/metabolismo , beta-Amilasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Catálisis , Cartilla de ADN , Escherichia coli/genética , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , beta-Amilasa/química , beta-Amilasa/genéticaRESUMEN
The aim of this article is to provide an overview of current models of starch breakdown in leaves. We summarise the results of our recent work focusing on Arabidopsis, relating them to other work in the field. Early biochemical studies of starch containing tissues identified numerous enzymes capable of participating in starch degradation. In the non-living endosperms of germinated cereal seeds, starch breakdown proceeds by the combined actions of α-amylase, limit dextrinase (debranching enzyme), ß-amylase and α-glucosidase. The activities of these enzymes and the regulation of some of the respective genes on germination have been extensively studied. In living plant cells, additional enzymes are present, such as α-glucan phosphorylase and disproportionating enzyme, and the major pathway of starch breakdown appears to differ from that in the cereal endosperm in some important aspects. For example, reverse-genetic studies of Arabidopsis show that α-amylase and limit-dextrinase play minor roles and are dispensable for starch breakdown in leaves. Current data also casts doubt on the involvement of α-glucosidase. In contrast, several lines of evidence point towards a major role for ß-amylase in leaves, which functions together with disproportionating enzyme and isoamylase (debranching enzyme) to produce maltose and glucose. Furthermore, the characterisation of Arabidopsis mutants with elevated leaf starch has contributed to the discovery of previously unknown proteins and metabolic steps in the pathway. In particular, it is now apparent that glucan phosphorylation is required for normal rates of starch mobilisation to occur, although a detailed understanding of this step is still lacking. We use this review to give a background to some of the classical genetic mutants that have contributed to our current knowledge.