RESUMO
Starch metabolism in guard cells plays a central role in regulating stomatal movement in response to light, elevated ambient CO2 and potentially other abiotic and biotic factors. Here, we discuss how various guard cell signal transduction pathways converge to promote rearrangements in guard cell starch metabolism for efficient stomatal responses, an essential physiological process that sustains plant productivity and stress tolerance. We suggest manipulation of guard cell starch dynamics as a previously overlooked strategy to improve stomatal behaviour under changing environmental conditions.
RESUMO
Acclimation of root growth is vital for plants to survive salt stress. Halophytes are great examples of plants that thrive even under severe salinity, but their salt tolerance mechanisms, especially those mediated by root responses, are still largely unknown. We compared root growth responses of the halophyte Schrenkiella parvula with its glycophytic relative species Arabidopsis thaliana under salt stress and performed transcriptomic analysis of S. parvula roots to identify possible gene regulatory networks underlying their physiological responses. Schrenkiella parvula roots do not avoid salt and experience less growth inhibition under salt stress. Salt-induced abscisic acid levels were higher in S. parvula roots compared with Arabidopsis. Root transcriptomic analysis of S. parvula revealed the induction of sugar transporters and genes regulating cell expansion and suberization under salt stress. 14 C-labeled carbon partitioning analyses showed that S. parvula continued allocating carbon to roots from shoots under salt stress while carbon barely allocated to Arabidopsis roots. Further physiological investigation revealed that S. parvula roots maintained root cell expansion and enhanced suberization under severe salt stress. In summary, roots of S. parvula deploy multiple physiological and developmental adjustments under salt stress to maintain growth, providing new avenues to improve salt tolerance of plants using root-specific strategies.
Assuntos
Arabidopsis , Brassicaceae , Arabidopsis/genética , Carbono , Brassicaceae/genética , Plantas Tolerantes a Sal , Tolerância ao Sal , Salinidade , Estresse Fisiológico/genética , Raízes de Plantas/genética , Regulação da Expressão Gênica de PlantasRESUMO
The pathway of starch synthesis in guard cells (GCs), despite the crucial role starch plays in stomatal movements, is not well understood. Here, we characterized starch dynamics in GCs of Arabidopsis (Arabidopsis thaliana) mutants lacking enzymes of the phosphoglucose isomerase-phosphoglucose mutase-ADP-glucose pyrophosphorylase starch synthesis pathway in leaf mesophyll chloroplasts or sugar transporters at the plastid membrane, such as glucose-6-phosphate/phosphate translocators, which are active in heterotrophic tissues. We demonstrate that GCs have metabolic features of both photoautotrophic and heterotrophic cells. GCs make starch using different carbon precursors depending on the time of day, which can originate both from GC photosynthesis and/or sugars imported from the leaf mesophyll. Furthermore, we unravel the major enzymes involved in GC starch synthesis and demonstrate that they act in a temporal manner according to the fluctuations of stomatal aperture, which is unique for GCs. Our work substantially enhances our knowledge on GC starch metabolism and uncovers targets for manipulating GC starch dynamics to improve stomatal behavior, directly affecting plant productivity.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Amido/metabolismoRESUMO
Starch in Arabidopsis (Arabidopsis thaliana) guard cells is rapidly degraded at the start of the day by the glucan hydrolases α-AMYLASE3 (AMY3) and ß-AMYLASE1 (BAM1) to promote stomatal opening. This process is activated via phototropin-mediated blue light signaling downstream of the plasma membrane H+-ATPase. It remains unknown how guard cell starch degradation integrates with light-regulated membrane transport processes in the fine control of stomatal opening kinetics. We report that H+, K+, and Cl- transport across the guard cell plasma membrane is unaltered in the amy3 bam1 mutant, suggesting that starch degradation products do not directly affect the capacity to transport ions. Enzymatic quantification revealed that after 30 min of blue light illumination, amy3 bam1 guard cells had similar malate levels as the wild type, but had dramatically altered sugar homeostasis, with almost undetectable amounts of Glc. Thus, Glc, not malate, is the major starch-derived metabolite in Arabidopsis guard cells. We further show that impaired starch degradation in the amy3 bam1 mutant resulted in an increase in the time constant for opening of 40 min. We conclude that rapid starch degradation at dawn is required to maintain the cytoplasmic sugar pool, clearly needed for fast stomatal opening. The conversion and exchange of metabolites between subcellular compartments therefore coordinates the energetic and metabolic status of the cell with membrane ion transport.
Assuntos
Arabidopsis/citologia , Arabidopsis/fisiologia , Glucose/metabolismo , Estômatos de Plantas/fisiologia , Amido/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Cloretos/metabolismo , Escuridão , Luz , Malatos/metabolismo , Mutação , Fotossíntese , Células Vegetais/metabolismo , Potássio/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , PrótonsRESUMO
Guard cells on the leaf epidermis regulate stomatal opening for gas exchange between plants and the atmosphere, allowing a balance between photosynthesis and transpiration. Given that guard cells possess several characteristics of sink tissues, their metabolic activities should largely depend on mesophyll-derived sugars. Early biochemical studies revealed sugar uptake into guard cells. However, the transporters that are involved and their relative contribution to guard cell function are not yet known. Here, we identified the monosaccharide/proton symporters Sugar Transport Protein 1 and 4 (STP1 and STP4) as the major plasma membrane hexose sugar transporters in the guard cells of Arabidopsis thaliana. We show that their combined action is required for glucose import to guard cells, providing carbon sources for starch accumulation and light-induced stomatal opening that are essential for plant growth. These findings highlight mesophyll-derived glucose as an important metabolite connecting stomatal movements with photosynthesis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Carbono , Glucose , Luz , Estômatos de PlantasRESUMO
Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that offers the potential to engineer improved water-use efficiency (WUE) and drought resilience in C3 plants while sustaining productivity in the hotter and drier climates that are predicted for much of the world. CAM species show an inverted pattern of stomatal opening and closing across the diel cycle, which conserves water and provides a means of maintaining growth in hot, water-limited environments. Recent genome sequencing of the constitutive model CAM species Kalanchoë fedtschenkoi provides a platform for elucidating the ensemble of proteins that link photosynthetic metabolism with stomatal movement, and that protect CAM plants from harsh environmental conditions. We describe a large-scale proteomics analysis to characterize and compare proteins, as well as diel changes in their abundance in guard cell-enriched epidermis and mesophyll cells from leaves of K. fedtschenkoi. Proteins implicated in processes that encompass respiration, the transport of water and CO2 , stomatal regulation, and CAM biochemistry are highlighted and discussed. Diel rescheduling of guard cell starch turnover in K. fedtschenkoi compared with that observed in Arabidopsis is reported and tissue-specific localization in the epidermis and mesophyll of isozymes implicated in starch and malate turnover are discussed in line with the contrasting roles for these metabolites within the CAM mesophyll and stomatal complex. These data reveal the proteins and the biological processes enriched in each layer and provide key information for studies aiming to adapt plants to hot and dry environments by modifying leaf physiology for improved plant sustainability.
Assuntos
Metabolismo Ácido das Crassuláceas , Kalanchoe/metabolismo , Células do Mesofilo/metabolismo , Epiderme Vegetal/metabolismo , Proteínas de Plantas/metabolismo , Especificidade de Órgãos , Fotossíntese , Proteoma/metabolismoRESUMO
Guard cell membrane ion transport and metabolism are deeply interconnected, and their coordinated regulation is integral to stomatal opening. Whereas ion transport is exceptionally well understood, how guard cell metabolism influences stomatal movements is less well known. Organic metabolites, such as malate and sugars, fulfill several functions in guard cells during stomatal opening as allosteric activators, counter-ions, energy source and osmolytes. However, their origin and exact fate remain debated. Recent work revealed that the guard cell carbon pool regulating stomatal function and plant growth is mostly of mesophyll origin, highlighting a tight correlation between mesophyll and guard cell metabolism. This review discusses latest research into guard cell carbon metabolism and its impact on stomatal function and whole plant physiology.
Assuntos
Fotossíntese , Estômatos de Plantas , Carbono , AçúcaresRESUMO
BACKGROUND: ß-Amylases (BAMs) are a multigene family of glucan hydrolytic enzymes playing a key role not only for plant biology but also for many industrial applications, such as the malting process in the brewing and distilling industries. BAMs have been extensively studied in Arabidopsis thaliana where they show a surprising level of complexity in terms of specialization within the different isoforms as well as regulatory functions played by at least three catalytically inactive members. Despite the importance of BAMs and the fact that multiple BAM proteins are also present in other angiosperms, little is known about their phylogenetic history or functional relationship. RESULTS: Here, we examined 961 ß-amylase sequences from 136 different algae and land plant species, including 66 sequenced genomes and many transcriptomes. The extraordinary number and the diversity of organisms examined allowed us to reconstruct the main patterns of ß-amylase evolution in land plants. We identified eight distinct clades in angiosperms, which results from extensive gene duplications and sub- or neo-functionalization. We discovered a novel clade of BAM, absent in Arabidopsis, which we called BAM10. BAM10 emerged before the radiation of seed plants and has the feature of an inactive enzyme. Furthermore, we report that BAM4 - an important protein regulating Arabidopsis starch metabolism - is absent in many relevant starch-accumulating crop species, suggesting that starch degradation may be differently regulated between species. CONCLUSIONS: BAM proteins originated sometime more than 400 million years ago and expanded together with the differentiation of plants into organisms of increasing complexity. Our phylogenetic analyses provide essential insights for future functional studies of this important class of storage glucan hydrolases and regulatory proteins.
Assuntos
Embriófitas/enzimologia , Evolução Molecular , Proteínas de Plantas/genética , beta-Amilase/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Embriófitas/genética , Duplicação Gênica , Perfilação da Expressão Gênica , Genoma de Planta , Família Multigênica , Filogenia , Proteínas de Plantas/fisiologia , beta-Amilase/fisiologiaRESUMO
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/fisiologiaRESUMO
Contents 943 I. 943 II. 944 III. 945 IV. 945 V. 948 VI. 949 950 References 950 SUMMARY: Abiotic stresses, such as drought, high salinity and extreme temperatures, pose one of the most important constraints to plant growth and productivity in many regions of the world. A number of investigations have shown that plants, including several important crops, remobilize their starch reserve to release energy, sugars and derived metabolites to help mitigate the stress. This is an essential process for plant fitness with important implications for plant productivity under challenging environmental conditions. In this Tansley insight, we evaluate the current literature on starch metabolism in response to abiotic stresses, and discuss the key enzymes involved and how they are regulated.
Assuntos
Aptidão Genética , Plantas/metabolismo , Amido/metabolismo , Estresse Fisiológico , Modelos Biológicos , Plantas/enzimologiaRESUMO
Stomata control gaseous fluxes between the internal leaf air spaces and the external atmosphere and, therefore, play a pivotal role in regulating CO2 uptake for photosynthesis as well as water loss through transpiration. Guard cells, which flank the stomata, undergo adjustments in volume, resulting in changes in pore aperture. Stomatal opening is mediated by the complex regulation of ion transport and solute biosynthesis. Ion transport is exceptionally well understood, whereas our knowledge of guard cell metabolism remains limited, despite several decades of research. In this review, we evaluate the current literature on metabolism in guard cells, particularly the roles of starch, sucrose, and malate. We explore the possible origins of sucrose, including guard cell photosynthesis, and discuss new evidence that points to multiple processes and plasticity in guard cell metabolism that enable these cells to function effectively to maintain optimal stomatal aperture. We also discuss the new tools, techniques, and approaches available for further exploring and potentially manipulating guard cell metabolism to improve plant water use and productivity.
Assuntos
Estômatos de Plantas/citologia , Estômatos de Plantas/metabolismo , Carbono/metabolismo , Ácidos Carboxílicos/metabolismo , Fotossíntese , Amido/metabolismo , Sacarose/metabolismoRESUMO
The accumulation of starch within photosynthetic tissues and within dedicated storage organs has been characterized extensively in many species, and a function in buffering carbon availability or in fueling later growth phases, respectively, has been proposed. However, developmentally regulated starch turnover within heterotrophic tissues other than dedicated storage organs is poorly characterized, and its function is not well understood. Here, we report on the characterization of starch turnover during flower, early embryo, and silique development in Arabidopsis (Arabidopsis thaliana) using a combined clearing-staining technique on whole-mount tissue. Besides the two previously documented waves of transient starch accumulation in the stamen envelope, occurring during meiosis and pollen mitosis I, we identified a novel, third wave of starch amylogenesis/amylolysis during the last stages of stamen development. To gain insights into the underlying molecular mechanisms, we analyzed publicly available microarray data, which revealed a developmentally coordinated expression of carbohydrate transport and metabolism genes during these waves of transient starch accumulation. Based on this analysis, we characterized starch dynamics in mutants affecting hexose phosphate metabolism and translocation, and identified the Glc-6-phosphate/phosphate antiporter GPT1 as the putative translocator of Glc-6-phosphate for starch biosynthesis in reproductive tissues. Based on these results, we propose a model of starch synthesis within the pollen grain and discuss the nutrient transport route feeding the embryo within the developing seed.
Assuntos
Arabidopsis/embriologia , Arabidopsis/metabolismo , Flores/embriologia , Flores/metabolismo , Sementes/embriologia , Amido/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Vias Biossintéticas/genética , Metabolismo dos Carboidratos/genética , Proliferação de Células , Simulação por Computador , Regulação para Baixo/genética , Flores/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Modelos Biológicos , Mutação/genética , Especificidade de Órgãos/genética , Pólen/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sementes/genética , Zigoto/citologia , Zigoto/metabolismoRESUMO
Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Fosfatases de Especificidade Dupla/química , Glicogênio/química , Amido/química , Motivos de Aminoácidos , Humanos , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases não Receptoras/químicaRESUMO
During photosynthesis of higher plants, absorbed light energy is converted into chemical energy that, in part, is accumulated in the form of transitory starch within chloroplasts. In the following night, transitory starch is mobilized to sustain the heterotrophic metabolism of the plant. ß-amylases are glucan hydrolases that cleave α-1,4-glycosidic bonds of starch and release maltose units from the non-reducing end of the polysaccharide chain. In Arabidopsis, nocturnal degradation of transitory starch involves mainly ß-amylase-3 (BAM3). A second ß-amylase isoform, ß-amylase-1 (BAM1), is involved in diurnal starch degradation in guard cells, a process that sustains stomata opening. However, BAM1 also contributes to diurnal starch turnover in mesophyll cells under osmotic stress. With the aim of dissecting the role of ß-amylases in osmotic stress responses in Arabidopsis, mutant plants lacking either BAM1 or BAM3 were subject to a mild (150mM mannitol) and prolonged (up to one week) osmotic stress. We show here that leaves of osmotically-stressed bam1 plants accumulated more starch and fewer soluble sugars than both wild-type and bam3 plants during the day. Moreover, bam1 mutants were impaired in proline accumulation and suffered from stronger lipid peroxidation, compared with both wild-type and bam3 plants. Taken together, these data strongly suggest that carbon skeletons deriving from BAM1 diurnal degradation of transitory starch support the biosynthesis of proline required to face the osmotic stress. We propose the transitory-starch/proline interplay as an interesting trait to be tackled by breeding technologies aimingto improve drought tolerance in relevant crops.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Secas , Prolina/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Amido/metabolismo , Estresse Fisiológico , Arabidopsis/genética , Luz , Peroxidação de Lipídeos/efeitos da radiação , Pressão Osmótica/efeitos da radiação , Transpiração Vegetal/fisiologia , Transpiração Vegetal/efeitos da radiação , Plantas Geneticamente Modificadas , Polissacarídeos/metabolismo , Regiões Promotoras Genéticas/genética , Solubilidade , Estresse Fisiológico/genética , Estresse Fisiológico/efeitos da radiaçãoRESUMO
Starch is a water-insoluble, Glc-based biopolymer that is used for energy storage and is synthesized and degraded in a diurnal manner in plant leaves. Reversible phosphorylation is the only known natural starch modification and is required for starch degradation in planta. Critical to starch energy release is the activity of glucan phosphatases; however, the structural basis of dephosphorylation by glucan phosphatases is unknown. Here, we describe the structure of the Arabidopsis thaliana starch glucan phosphatase like sex four2 (LSF2) both with and without phospho-glucan product bound at 2.3Å and 1.65Å, respectively. LSF2 binds maltohexaose-phosphate using an aromatic channel within an extended phosphatase active site and positions maltohexaose in a C3-specific orientation, which we show is critical for the specific glucan phosphatase activity of LSF2 toward native Arabidopsis starch. However, unlike other starch binding enzymes, LSF2 does not possess a carbohydrate binding module domain. Instead we identify two additional glucan binding sites located within the core LSF2 phosphatase domain. This structure is the first of a glucan-bound glucan phosphatase and provides new insights into the molecular basis of this agriculturally and industrially relevant enzyme family as well as the unique mechanism of LSF2 catalysis, substrate specificity, and interaction with starch granules.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Fosfatases de Especificidade Dupla/metabolismo , Glucanos/metabolismo , Amido/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação/genética , Cristalografia por Raios X , Fosfatases de Especificidade Dupla/química , Fosfatases de Especificidade Dupla/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Fosfatos/química , Fosfatos/metabolismo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Fructose (Fru) is a major storage form of sugars found in vacuoles, yet the molecular regulation of vacuolar Fru transport is poorly studied. Although SWEET17 (for SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS17) has been characterized as a vacuolar Fru exporter in leaves, its expression in leaves is low. Here, RNA analysis and SWEET17-ß-glucuronidase/-GREEN FLUORESCENT PROTEIN fusions expressed in Arabidopsis (Arabidopsis thaliana) reveal that SWEET17 is highly expressed in the cortex of roots and localizes to the tonoplast of root cells. Expression of SWEET17 in roots was inducible by Fru and darkness, treatments that activate accumulation and release of vacuolar Fru, respectively. Mutation and ectopic expression of SWEET17 led to increased and decreased root growth in the presence of Fru, respectively. Overexpression of SWEET17 specifically reduced the Fru content in leaves by 80% during cold stress. These results intimate that SWEET17 functions as a Fru-specific uniporter on the root tonoplast. Vacuoles overexpressing SWEET17 showed increased [14C]Fru uptake compared with the wild type. SWEET17-mediated Fru uptake was insensitive to ATP or treatment with NH4Cl or carbonyl cyanide m-chlorophenyl hydrazone, indicating that SWEET17 functions as an energy-independent facilitative carrier. The Arabidopsis genome contains a close paralog of SWEET17 in clade IV, SWEET16. The predominant expression of SWEET16 in root vacuoles and reduced root growth of mutants under Fru excess indicate that SWEET16 also functions as a vacuolar transporter in roots. We propose that in addition to a role in leaves, SWEET17 plays a key role in facilitating bidirectional Fru transport across the tonoplast of roots in response to metabolic demand to maintain cytosolic Fru homeostasis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Frutose/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Vacúolos/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico/genética , Temperatura Baixa , Frutose/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana Transportadoras/genética , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Vacúolos/efeitos dos fármacosRESUMO
α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. In vascular plants, α-amylases can be classified into three subfamilies. Arabidopsis has one member of each subfamily. Among them, only AtAMY3 is localized in the chloroplast. We expressed and purified AtAMY3 from Escherichia coli and carried out a biochemical characterization of the protein to find factors that regulate its activity. Recombinant AtAMY3 was active toward both insoluble starch granules and soluble substrates, with a strong preference for ß-limit dextrin over amylopectin. Activity was shown to be dependent on a conserved aspartic acid residue (Asp(666)), identified as the catalytic nucleophile in other plant α-amylases such as the barley AMY1. AtAMY3 released small linear and branched glucans from Arabidopsis starch granules, and the proportion of branched glucans increased after the predigestion of starch with a ß-amylase. Optimal rates of starch digestion in vitro was achieved when both AtAMY3 and ß-amylase activities were present, suggesting that the two enzymes work synergistically at the granule surface. We also found that AtAMY3 has unique properties among other characterized plant α-amylases, with a pH optimum of 7.5-8, appropriate for activity in the chloroplast stroma. AtAMY3 is also redox-regulated, and the inactive oxidized form of AtAMY3 could be reactivated by reduced thioredoxins. Site-directed mutagenesis combined with mass spectrometry analysis showed that a disulfide bridge between Cys(499) and Cys(587) is central to this regulation. This work provides new insights into how α-amylase activity may be regulated in the chloroplast.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Cloroplastos/metabolismo , Cloroplastos/enzimologia , alfa-Amilases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Cloroplastos/química , Proteínas de Cloroplastos/genética , Cloroplastos/genética , Hordeum/enzimologia , Hordeum/genética , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Amido/química , Amido/genética , Amido/metabolismo , alfa-Amilases/química , alfa-Amilases/genéticaRESUMO
Starch contains phosphate covalently bound to the C6-position (70 to 80% of total bound phosphate) and the C3-position (20 to 30%) of the glucosyl residues of the amylopectin fraction. In plants, the transient phosphorylation of starch renders the granule surface more accessible to glucan hydrolyzing enzymes and is required for proper starch degradation. Phosphate also confers desired properties to starch-derived pastes for industrial applications. In Arabidopsis thaliana, the removal of phosphate by the glucan phosphatase Starch Excess4 (SEX4) is essential for starch breakdown. We identified a homolog of SEX4, LSF2 (Like Sex Four2), as a novel enzyme involved in starch metabolism in Arabidopsis chloroplasts. Unlike SEX4, LSF2 does not have a carbohydrate binding module. Nevertheless, it binds to starch and specifically hydrolyzes phosphate from the C3-position. As a consequence, lsf2 mutant starch has elevated levels of C3-bound phosphate. SEX4 can release phosphate from both the C6- and the C3-positions, resulting in partial functional overlap with LSF2. However, compared with sex4 single mutants, the lsf2 sex4 double mutants have a more severe starch-excess phenotype, impaired growth, and a further change in the proportion of C3- and C6-bound phosphate. These findings significantly advance our understanding of the metabolism of phosphate in starch and provide innovative options for tailoring novel starches with improved functionality for industry.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , Amido/metabolismo , Amilopectina/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , Fosfatases de Especificidade Dupla/genética , Glucanos/metabolismo , Modelos Moleculares , Mutação , Fenótipo , Fosfatos/metabolismo , Conformação Proteica , Homologia de Sequência de Aminoácidos , Amido/química , Especificidade por SubstratoRESUMO
Starch in the stomatal guard cells is largely synthesized using carbon precursors originating from sugars imported from the leaf mesophyll. Such heterotrophic nature of guard cell starch synthesis prompted us to investigate the role of cytosolic sucrose synthases (SUS) in this pathway. Out of the six members of the Arabidopsis SUS gene family, SUS3 was the most highly expressed isoform in guard cells. The Arabidopsis sus3 mutant displayed changes in guard cell starch contents comparable to the Wild Type (WT) up until 6 h into the day. After this time point, sus3 guard cells surprisingly started to accumulate starch at very high rates, reaching the end of the day with significantly more starch than WT. Based on the phenotype of the sus3 mutant, we suggest that in guard cells, SUS3 is involved in the regulation of carbon fluxes towards starch synthesis during the second half of the day. SUS3 may be part of a previously predicted guard cell futile cycle of metabolic reactions, in which sucrose is re-synthesized from UDP-glucose to avoid excessive starch synthesis toward the end of the day. This is in contrast to typical storage organs, in which cytosolic SUS is required to produce ADP-glucose for starch synthesis.