RESUMEN
Genetic incompatibility between the cytoplasm and the nucleus is thought to be a major factor in species formation, but mechanistic understanding of this process is poor. In evening primroses (Oenothera spp.), a model plant for organelle genetics and population biology, hybrid offspring regularly display chloroplast-nuclear incompatibility. This usually manifests in bleached plants, more rarely in hybrid sterility or embryonic lethality. Hence, most of these incompatibilities affect photosynthetic capability, a trait that is under selection in changing environments. Here we show that light-dependent misregulation of the plastid psbB operon, which encodes core subunits of photosystem II and the cytochrome b6f complex, can lead to hybrid incompatibility, and this ultimately drives speciation. This misregulation causes an impaired light acclimation response in incompatible plants. Moreover, as a result of their different chloroplast genotypes, the parental lines differ in photosynthesis performance upon exposure to different light conditions. Significantly, the incompatible chloroplast genome is naturally found in xeric habitats with high light intensities, whereas the compatible one is limited to mesic habitats. Consequently, our data raise the possibility that the hybridization barrier evolved as a result of adaptation to specific climatic conditions.
Asunto(s)
Especiación Genética , Genoma del Cloroplasto , Oenothera biennis/genética , Operón , Fotosíntesis/genética , Aclimatación/genética , Complejo de Citocromo b6f/genética , Luz , Oenothera biennis/fisiología , Complejo de Proteína del Fotosistema II/genética , Proteínas de Plantas/genética , Plastidios/genética , Regiones Promotoras Genéticas , Edición de ARNRESUMEN
Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch [11A] of the atpB coding region (encoding the ß-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, nonfunctional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, we generated transplastomic tobacco lines in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We observed that insertion of two adenines was more efficiently corrected than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding, as an additional replacement of AAA lysine codon by AAG resulted in an albino phenotype. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in the presence of a mutation that otherwise would be lethal.
Asunto(s)
Cloroplastos/metabolismo , Mutación del Sistema de Lectura/genética , Genes de Plantas , Nicotiana/genética , Oenothera/genética , Proteínas de Plantas/genética , Biosíntesis de Proteínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cloroplastos/ultraestructura , ADN Complementario/genética , Escherichia coli/metabolismo , Genotipo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Péptidos/química , Péptidos/metabolismo , Fenotipo , Fotosíntesis , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , ReproducciónRESUMEN
De novo fatty acid biosynthesis in plants relies on a prokaryotic-type acetyl-CoA carboxylase (ACCase) that resides in the plastid compartment. The enzyme is composed of four subunits, one of which is encoded in the plastid genome, whereas the other three subunits are encoded by nuclear genes. The plastid gene (accD) encodes the ß-carboxyltransferase subunit of ACCase and is essential for cell viability. To facilitate the functional analysis of accD, we pursued a transplastomic knockdown strategy in tobacco (Nicotiana tabacum). By introducing point mutations into the translational start codon of accD, we obtained stable transplastomic lines with altered ACCase activity. Replacement of the standard initiator codon AUG with UUG strongly reduced AccD expression, whereas replacement with GUG had no detectable effects. AccD knockdown mutants displayed reduced ACCase activity, which resulted in changes in the levels of many but not all species of cellular lipids. Limiting fatty acid availability caused a wide range of macroscopic, microscopic, and biochemical phenotypes, including impaired chloroplast division, reduced seed set, and altered storage metabolism. Finally, while the mutants displayed reduced growth under photoautotrophic conditions, they showed exaggerated growth under heterotrophic conditions, thus uncovering an unexpected antagonistic role of AccD activity in autotrophic and heterotrophic growth.
Asunto(s)
Acetil-CoA Carboxilasa/metabolismo , Cloroplastos/metabolismo , Nicotiana/metabolismo , Hojas de la Planta/metabolismo , Plastidios/metabolismo , Acetil-CoA Carboxilasa/genética , Núcleo Celular/metabolismo , Plastidios/genética , Semillas/metabolismoRESUMEN
Chloroplast nucleoids are large, compact nucleoprotein structures containing multiple copies of the plastid genome. Studies on structural and quantitative changes of plastid DNA (ptDNA) during leaf development are scarce and have produced controversial data. We have systematically investigated nucleoid dynamics and ptDNA quantities in the mesophyll of Arabidopsis, tobacco, sugar beet, and maize from the early post-meristematic stage until necrosis. DNA of individual nucleoids was quantified by DAPI-based supersensitive epifluorescence microscopy. Nucleoids occurred in scattered, stacked, or ring-shaped arrangements and in recurring patterns during leaf development that was remarkably similar between the species studied. Nucleoids per organelle varied from a few in meristematic plastids to >30 in mature chloroplasts (corresponding to about 20-750 nucleoids per cell). Nucleoid ploidies ranged from haploid to >20-fold even within individual organelles, with average values between 2.6-fold and 6.7-fold and little changes during leaf development. DNA quantities per organelle increased gradually from about a dozen plastome copies in tiny plastids of apex cells to 70-130 copies in chloroplasts of about 7 µm diameter in mature mesophyll tissue, and from about 80 plastome copies in meristematic cells to 2600-3300 copies in mature diploid mesophyll cells without conspicuous decline during leaf development. Pulsed-field electrophoresis, restriction of high-molecular-weight DNA from chloroplasts and gerontoplasts, and CsCl equilibrium centrifugation of single-stranded and double-stranded ptDNA revealed no noticeable fragmentation of the organelle DNA during leaf development, implying that plastid genomes in mesophyll tissues are remarkably stable until senescence.
Asunto(s)
Genoma de Plastidios/genética , Magnoliopsida/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Beta vulgaris/genética , Beta vulgaris/crecimiento & desarrollo , Cloroplastos/genética , Magnoliopsida/crecimiento & desarrollo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Plastidios/genética , Nicotiana/genética , Nicotiana/crecimiento & desarrollo , Zea mays/genética , Zea mays/crecimiento & desarrolloRESUMEN
Primary carbohydrate metabolism in plants includes several sugar and sugar-derivative transport processes. Over recent years, evidences have shown that in starch-related transport processes, in addition to glucose 6-phosphate, maltose, glucose and triose-phosphates, glucose 1-phosphate also plays a role and thereby increases the possible fluxes of sugar metabolites in planta. In this study, we report the characterization of two highly similar transporters, At1g34020 and At4g09810, in Arabidopsis thaliana, which allow the import of glucose 1-phosphate through the plasma membrane. Both transporters were expressed in yeast and were biochemically analyzed to reveal an antiport of glucose 1-phosphate/phosphate. Furthermore, we showed that the apoplast of Arabidopsis leaves contained glucose 1-phosphate and that the corresponding mutant of these transporters had higher glucose 1-phosphate amounts in the apoplast and alterations in starch and starch-related metabolism.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Glucofosfatos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico/fisiología , Metabolismo de los Hidratos de Carbono , Escherichia coli/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Transporte de Membrana/genética , Mutación , Hojas de la Planta/metabolismo , Protoplastos , Almidón/metabolismo , TranscriptomaRESUMEN
Starch phosphorylation by starch-related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50-kDa starch-binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various in vitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, α-glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfotransferasas (Aceptores Pareados)/metabolismo , Almidón/metabolismo , Arabidopsis/enzimología , Clonación Molecular , FosforilaciónRESUMEN
The process of starch granule formation in leaves of Arabidopsis (Arabidopsis thaliana) is obscure. Besides STARCH SYNTHASE4 (SS4), the PLASTIDIAL PHOSPHORYLASE (PHS1) also seems to be involved, since dpe2-1/phs1a double mutants lacking both PHS1 and the cytosolic DISPROPORTIONATING ENZYME2 (DPE2) displayed only one starch granule per chloroplast under normal growth conditions. For further studies, a dpe2-1/phs1a/ss4 triple mutant and various combinations of double mutants were generated and metabolically analyzed with a focus on starch metabolism. The dpe2-1/phs1a/ss4 mutant revealed a massive starch excess phenotype. Furthermore, these plants grown under 12 h of light/12 h of dark harbored a single large and spherical starch granule per plastid. The number of starch granules was constant when the light/dark regime was altered, but this was not observed in the parental lines. With regard to growth, photosynthetic parameters, and metabolic analyses, the triple mutant additionally displayed alterations in comparison with ss4 and dpe2-1/phs1a The results clearly illustrate that PHS1 and SS4 are differently involved in starch granule formation and do not act in series. However, SS4 appears to exert a stronger influence. In connection with the characterized double mutants, we discuss the generation of starch granules and the observed formation of spherical starch granules.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Plastidios/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Almidón Sintasa/genética , Almidón Sintasa/metabolismo , Almidón/metabolismo , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Sistema de la Enzima Desramificadora del Glucógeno/genética , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Luz , Microscopía Electrónica , Mutación , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de la radiación , Proteínas Tirosina Fosfatasas/genética , Almidón/ultraestructuraRESUMEN
In leaves of two starch-related single-knockout lines lacking either the cytosolic transglucosidase (also designated as disproportionating enzyme 2, DPE2) or the maltose transporter (MEX1), the activity of the plastidial phosphorylase isozyme (PHS1) is increased. In both mutants, metabolism of starch-derived maltose is impaired but inhibition is effective at different subcellular sites. Two constitutive double knockout mutants were generated (designated as dpe2-1×phs1a and mex1×phs1b) both lacking functional PHS1. They reveal that in normally grown plants, the plastidial phosphorylase isozyme participates in transitory starch degradation and that the central carbon metabolism is closely integrated into the entire cell biology. All plants were grown either under continuous illumination or in a light-dark regime. Both double mutants were compromised in growth and, compared with the single knockout plants, possess less average leaf starch when grown in a light-dark regime. Starch and chlorophyll contents decline with leaf age. As revealed by transmission electron microscopy, mesophyll cells degrade chloroplasts, but degradation is not observed in plants grown under continuous illumination. The two double mutants possess similar but not identical phenotypes. When grown in a light-dark regime, mesophyll chloroplasts of dpe2-1×phs1a contain a single starch granule but under continuous illumination more granules per chloroplast are formed. The other double mutant synthesizes more granules under either growth condition. In continuous light, growth of both double mutants is similar to that of the parental single knockout lines. Metabolite profiles and oligoglucan patterns differ largely in the two double mutants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Técnicas de Inactivación de Genes , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Mutación/genética , Plastidios/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Almidón/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/ultraestructura , Biomasa , Metabolismo de los Hidratos de Carbono , Carbono/metabolismo , Clorofila/metabolismo , Cromatografía de Afinidad , Cruzamientos Genéticos , Isoenzimas/metabolismo , Maltosa/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Células del Mesófilo/metabolismo , Células del Mesófilo/ultraestructura , Metabolómica , Fenotipo , Fotoperiodo , Plastidios/ultraestructura , Sacarosa/metabolismoRESUMEN
Carbohydrate metabolism in plants is tightly linked to photosynthesis and is essential for energy and carbon skeleton supply of the entire organism. Thus, the hexose phosphate pools of the cytosol and the chloroplast represent important metabolic resources that are maintained through action of phosphoglucose isomerase (PGI) and phosphoglucose mutase interconverting glucose 6-phosphate, fructose 6-phosphate, and glucose 1-phosphate. Here, we investigated the impact of disrupted cytosolic PGI (cPGI) function on plant viability and metabolism. Overexpressing an artificial microRNA targeted against cPGI (amiR-cpgi) resulted in adult plants with vegetative tissue essentially free of cPGI activity. These plants displayed diminished growth compared with the wild type and accumulated excess starch in chloroplasts but maintained low sucrose content in leaves at the end of the night. Moreover, amiR-cpgi plants exhibited increased nonphotochemical chlorophyll a quenching during photosynthesis. In contrast to amiR-cpgi plants, viable transfer DNA insertion mutants disrupted in cPGI function could only be identified as heterozygous individuals. However, homozygous transfer DNA insertion mutants could be isolated among plants ectopically expressing cPGI. Intriguingly, these plants were only fertile when expression was driven by the ubiquitin10 promoter but sterile when the seed-specific unknown seed protein promoter or the Cauliflower mosaic virus 35S promoter were employed. These data show that metabolism is apparently able to compensate for missing cPGI activity in adult amiR-cpgi plants and indicate an essential function for cPGI in plant reproduction. Moreover, our data suggest a feedback regulation in amiR-cpgi plants that fine-tunes cytosolic sucrose metabolism with plastidic starch turnover.
Asunto(s)
Arabidopsis/metabolismo , Metabolismo de los Hidratos de Carbono , Citosol/enzimología , Glucosa-6-Fosfato Isomerasa/metabolismo , Hojas de la Planta/metabolismo , Arabidopsis/enzimología , Arabidopsis/fisiología , Clorofila/metabolismo , Clorofila A , ADN Bacteriano/genética , Isoenzimas/metabolismo , Mutación , Fracciones Subcelulares/enzimología , Fracciones Subcelulares/metabolismoRESUMEN
Plants metabolize transitory starch by precisely coordinated plastidial and cytosolic processes. The latter appear to include the action of water-soluble heteroglycans (SHGin ) whose monosaccharide pattern is similar to that of apoplastic glycans (SHGex ) but, unlike SHGex , SHGin strongly interacts with glucosyl transferases. In this study, we analyzed starch metabolism using mesophyll protoplasts from wild-type plants and two knock-out mutants [deficient in the cytosolic transglucosidase, disproportionating isoenzyme 2 (DPE2) or the plastidial phosphoglucomutase (PGM1)] from Arabidopsis thaliana. Protoplasts prelabeled by photosynthetic (14) CO2 fixation were transferred to an unlabeled medium and were darkened or illuminated. Carbon transitions from the Calvin cycle or from starch to both SHGin and SHGex were analyzed. In illuminated protoplasts, starch turn-over was undetectable but darkened protoplasts continuously degraded starch. During illumination, neither the total (14) C content nor the labeling patterns of the sugar residues of SHGin were significantly altered but both the total amount and the labeling of the constituents of SHGex increased with time. In darkened protoplasts, the (14) C-content of most of the sugar residues of SHGin transiently and strongly increased and then declined. This effect was not observed in any SHGex constituent. In darkened DPE2-deficient protoplasts, none of the SHGin constituents exhibited an essential transient increase in labeling. In contrast, some residues of SHGin from the PGM1 mutant exhibited a transient increase in label but this effect significantly differed from that of the wild type. Two conclusions are reached: first, SHGin and SHGex exert different metabolic functions and second, SHGin is directly involved in starch degradation.
Asunto(s)
Arabidopsis/metabolismo , Carbono/metabolismo , Fotosíntesis/fisiología , Polisacáridos/metabolismo , Almidón/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Metabolismo de los Hidratos de Carbono , Radioisótopos de Carbono , Oscuridad , Técnicas de Inactivación de Genes , Marcaje Isotópico , Luz , Células del Mesófilo/metabolismo , Mutación , Fosfoglucomutasa/genética , Fosfoglucomutasa/metabolismo , Plantas Modificadas Genéticamente , Polisacáridos/química , SolubilidadRESUMEN
Almost all glucosyl transfer reactions rely on glucose-1-phosphate (Glc-1-P) that either immediately acts as glucosyl donor or as substrate for the synthesis of the more widely used Glc dinucleotides, ADPglucose or UDPglucose. In this communication, we have analyzed two Glc-1-P-related processes: the carbon flux from externally supplied Glc-1-P to starch by either mesophyll protoplasts or intact chloroplasts from Arabidopsis (Arabidopsis thaliana). When intact protoplasts or chloroplasts are incubated with [U-(14)C]Glc-1-P, starch is rapidly labeled. Incorporation into starch is unaffected by the addition of unlabeled Glc-6-P or Glc, indicating a selective flux from Glc-1-P to starch. However, illuminated protoplasts incorporate less (14)C into starch when unlabeled bicarbonate is supplied in addition to the (14)C-labeled Glc-1-P. Mesophyll protoplasts incubated with [U-(14)C]Glc-1-P incorporate (14)C into the plastidial pool of adenosine diphosphoglucose. Protoplasts prepared from leaves of mutants of Arabidopsis that lack either the plastidial phosphorylase or the phosphoglucomutase isozyme incorporate (14)C derived from external Glc-1-P into starch, but incorporation into starch is insignificant when protoplasts from a mutant possessing a highly reduced ADPglucose pyrophosphorylase activity are studied. Thus, the path of assimilatory starch biosynthesis initiated by extraplastidial Glc-1-P leads to the plastidial pool of adenosine diphosphoglucose, and at this intermediate it is fused with the Calvin cycle-driven route. Mutants lacking the plastidial phosphoglucomutase contain a small yet significant amount of transitory starch.
Asunto(s)
Arabidopsis/metabolismo , Cloroplastos/metabolismo , Glucofosfatos/metabolismo , Hojas de la Planta/metabolismo , Protoplastos/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Isótopos de Carbono/análisis , Cloroplastos/enzimología , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Mutación , Almidón/biosíntesisRESUMEN
Starch is the primary storage carbohydrate in most photosynthetic organisms and allows the accumulation of carbon and energy in form of an insoluble and semi-crystalline particle. In the last decades large progress, especially in the model plant Arabidopsis thaliana, was made in understanding the structure and metabolism of starch and its conjunction. The process underlying the initiation of starch granules remains obscure, although this is a fundamental process and seems to be strongly regulated, as in Arabidopsis leaves the starch granule number per chloroplast is fixed with 5-7. Several single, double, and triple mutants were reported in the last years that showed massively alterations in the starch granule number per chloroplast and allowed further insights in this important process. This mini review provides an overview of the current knowledge of processes involved in the initiation and formation of starch granules. We discuss the central role of starch synthase 4 and further proteins for starch genesis and affecting metabolic factors.
RESUMEN
An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5-7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology.
Asunto(s)
Proteínas de Arabidopsis/genética , Cloroplastos/genética , Sistema de la Enzima Desramificadora del Glucógeno/genética , Fosfotransferasas (Aceptores Pareados)/genética , Proteínas Tirosina Fosfatasas/genética , Almidón/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Cloroplastos/metabolismo , Cloroplastos/efectos de la radiación , Cloroplastos/ultraestructura , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/efectos de la radiación , Gránulos Citoplasmáticos/ultraestructura , Expresión Génica , Genotipo , Sistema de la Enzima Desramificadora del Glucógeno/deficiencia , Hidrólisis , Luz , Mutación , Fenotipo , Fosfotransferasas (Aceptores Pareados)/deficiencia , Fotoperiodo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de la radiación , Hojas de la Planta/ultraestructura , Proteínas Tirosina Fosfatasas/deficiencia , Almidón/biosíntesisRESUMEN
Tubers of potato (Solanum tuberosum L.), one of the most important crops, are a prominent example for an efficient production of storage starch. Nevertheless, the synthesis of this storage starch is not completely understood. The plastidial phosphorylase (Pho1; EC 2.4.1.1) catalyzes the reversible transfer of glucosyl residues from glucose-1-phosphate to the non-reducing end of α-glucans with the release of orthophosphate. Thus, the enzyme is in principle able to act during starch synthesis. However, so far under normal growth conditions no alterations in tuber starch metabolism were observed. Based on analyses of other species and also from in vitro experiments with potato tuber slices it was supposed, that Pho1 has a stronger impact on starch metabolism, when plants grow under low temperature conditions. Therefore, we analyzed the starch content, granule size, as well as the internal structure of starch granules isolated from potato plants grown under low temperatures. Besides wild type, transgenic potato plants with a strong reduction in the Pho1 activity were analyzed. No significant alterations in starch content and granule size were detected. In contrast, when plants were cultivated at low temperatures the chain length distributions of the starch granules were altered. Thus, the granules contained more short glucan chains. That was not observed in the transgenic plants, revealing that Pho1 in wild type is involved in the formation of the short glucan chains, at least at low temperatures.
Asunto(s)
Frío , Fosforilasas/biosíntesis , Proteínas de Plantas/biosíntesis , Tubérculos de la Planta/crecimiento & desarrollo , Plastidios/metabolismo , Solanum tuberosum/crecimiento & desarrollo , Almidón/biosíntesisRESUMEN
Phosphoglucomutase (PGM) catalyses the interconversion of glucose 1-phosphate (G1P) and glucose 6-phosphate (G6P) and exists as plastidial (pPGM) and cytosolic (cPGM) isoforms. The plastidial isoform is essential for transitory starch synthesis in chloroplasts of leaves, whereas the cytosolic counterpart is essential for glucose phosphate partitioning and, therefore, for syntheses of sucrose and cell wall components. In Arabidopsis two cytosolic isoforms (PGM2 and PGM3) exist. Both PGM2 and PGM3 are redundant in function as single mutants reveal only small or no alterations compared to wild type with respect to plant primary metabolism. So far, there are no reports of Arabidopsis plants lacking the entire cPGM or total PGM activity, respectively. Therefore, amiRNA transgenic plants were generated and used for analyses of various parameters such as growth, development, and starch metabolism. The lack of the entire cPGM activity resulted in a strongly reduced growth revealed by decreased rosette fresh weight, shorter roots, and reduced seed production compared to wild type. By contrast content of starch, sucrose, maltose and cell wall components were significantly increased. The lack of both cPGM and pPGM activities in Arabidopsis resulted in dwarf growth, prematurely die off, and inability to develop a functional inflorescence. The combined results are discussed in comparison to potato, the only described mutant with lack of total PGM activity.
Asunto(s)
Arabidopsis/fisiología , Metabolismo de los Hidratos de Carbono , Citosol/metabolismo , Fosfoglucomutasa/metabolismo , Raíces de Plantas/metabolismo , Semillas/metabolismo , Activación Enzimática , Isoenzimas , Metaboloma , Metabolómica , Fenotipo , Fosfoglucomutasa/genética , Plantas Modificadas Genéticamente , Almidón/metabolismoRESUMEN
Both photoautotrophic and heterotrophic plant cells are capable of accumulating starch inside the plastid. However, depending on the metabolic state of the respective cell the starch-related carbon fluxes are different. The vast majority of the transitory starch biosynthesis relies on the hexose phosphate pools derived from the reductive pentose phosphate cycle and, therefore, is restricted to ongoing photosynthesis. Transitory starch is usually degraded in the subsequent dark period and mainly results in the formation of neutral sugars, such as glucose and maltose, that both are exported into the cytosol. The cytosolic metabolism of the two carbohydrates includes reversible glucosyl transfer reactions to a heteroglycan that are mediated by two glucosyl transferases, DPE2 and PHS2 (or, in all other species, Pho2). In heterotrophic cells, accumulation of starch mostly depends on the long distance transport of reduced carbon compounds from source to sink organs and, therefore, includes as an essential step the import of carbohydrates from the cytosol into the starch forming plastids. In this communication, we focus on starch metabolism in heterotrophic tissues from Arabidopsis thaliana wild type plants (and in various starch-related mutants as well). By using hydroponically grown A. thaliana plants, we were able to analyse starch-related biochemical processes in leaves and roots from the same plants. Within the roots we determined starch levels and the morphology of native starch granules. Cytosolic and apoplastic heteroglycans were analysed in roots and compared with those from leaves of the same plants. A. thaliana mutants lacking functional enzymes either inside the plastid (such as phosphoglucomutase) or in the cytosol (disproportionating isoenzyme 2 or the phosphorylase isozyme, PHS2) were included in this study. In roots and leaves from the three mutants (and from the respective wild type organ as well), starch and heteroglycans as well as enzyme patterns were analysed.
Asunto(s)
Arabidopsis/metabolismo , Citosol/metabolismo , Raíces de Plantas/metabolismo , Almidón/metabolismo , Arabidopsis/citología , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Procesos Autotróficos , Carbono/metabolismo , Electroforesis en Gel de Poliacrilamida , Hidroponía , Monosacáridos/metabolismo , Hojas de la Planta/ultraestructura , Raíces de Plantas/ultraestructura , Solubilidad , Almidón/ultraestructura , AguaRESUMEN
Both photoautotrophic and heterotrophic tissues from plants are capable of synthesizing and degrading starch. To analyse starch metabolism in the two types of tissue from the same plant, several starch-related mutants from Arabidopsis thaliana were grown hydroponically together with the respective wild type control. Starch contents, patterns of starch-related enzymes, and the monomer patterns of the cytosolic starch-related heteroglycans were determined. Based on the phenotypical data obtained, three comparisons were made: First, data from leaves and roots of the mutants were compared with the respective wild type controls. Secondly, data from leaves and roots from the same plant were compared. Third, we included data obtained from soil-grown plants and compared them with those from hydroponically grown plants. Thus, phenotypical features reflecting altered gene expression can be distinguished from those that are due to the specific growth conditions. Implications on the carbon fluxes in photoautotrophic and heterotrophic cells are discussed.
Asunto(s)
Arabidopsis/metabolismo , Ciclo del Carbono/fisiología , Hojas de la Planta/metabolismo , Raíces de Plantas/metabolismo , Almidón/metabolismo , Ciclo del Carbono/genéticaRESUMEN
In plants several 'starch-related' enzymes exist as plastid- and cytosol-specific isoforms and in some cases the extraplastidial isoforms represent the majority of the enzyme activity. Due to the compartmentation of the plant cells, these extraplastidial isozymes have no access to the plastidial starch granules and, therefore, their in vivo function remained enigmatic. Recently, cytosolic heteroglycans have been identified that possess a complex pattern of the monomer composition and glycosidic bonds. The glycans act both as acceptors and donors for cytosolic glucosyl transferases. In autotrophic tissues the heteroglycans are essential for the nocturnal starch-sucrose conversion. In this review we summarize the current knowledge of these glycans, their interaction with glucosyl transferases and their possible cellular functions. We include data on the heteroglycans in heterotrophic plant tissues and discuss their role in intracellular carbon fluxes that originate from externally supplied carbohydrates.