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Abstract
The effects of the cytosolic expression of Escherichia coli pyrophosphatase (ppa) were investigated in the rosette leaves of transgenic Arabidopsis plants. During the daytime, glucose and fructose were found to accumulate at levels that were approximately two- to threefold higher in these plants than in the wild type. Interestingly, however, neither sucrose nor starch levels showed any distinctive build up in transgenic plants except under continuous white light growth conditions, during which they accumulated at high levels. Additionally, the leaves of transgenic Arabidopsis plants contain two- to threefold higher levels of inorganic phosphate (Pi) and two- to sixfold higher levels of uridine diphosphate-glucose than wild type plants during the diurnal cycle. In contrast, triose phosphate contents in the leaves of E. coli ppa transformants were either similar or slightly decreased when compared with wild type leaves. Furthermore, the photosynthetic activity of these transgenic plants was found to be reduced by 20-40% compared to normal levels. These results indicate that induction of ppa activity in the cytosol affects carbon partitioning between source and sink organs and also that the concomitant increase in Pi caused the accumulation of carbon metabolites and reduced photosynthetic activity.
... In a recent study, the ectopic expression of E. coli soluble pyrophosphatase in Arabidopsis thaliana was revealed to cause similar effects (Lee et al., 2005). The transgenic A. thaliana plants were shown to accumulate higher levels of glucose and fructose but have no change in sucrose or starch accumulation (Lee et al., 2005). ...
... In a recent study, the ectopic expression of E. coli soluble pyrophosphatase in Arabidopsis thaliana was revealed to cause similar effects (Lee et al., 2005). The transgenic A. thaliana plants were shown to accumulate higher levels of glucose and fructose but have no change in sucrose or starch accumulation (Lee et al., 2005). The overexpression of bacterial pyrophosphatase in A. thaliana caused no distinctive growth phenotype as in the case of overexpression in potato (Jelitto et al., 1992;Sonnewald, 1992;Lee et al., 2005). ...
... The transgenic A. thaliana plants were shown to accumulate higher levels of glucose and fructose but have no change in sucrose or starch accumulation (Lee et al., 2005). The overexpression of bacterial pyrophosphatase in A. thaliana caused no distinctive growth phenotype as in the case of overexpression in potato (Jelitto et al., 1992;Sonnewald, 1992;Lee et al., 2005). They were further characterized with lower levels of photosynthetic activity as compared to wild type plants (Lee et al., 2005), however, the transgenic Arabidopsis plants did not show an early germination phenotype as expected from the early sprouting behavior of transgenic potato plants (Jelitto et al., 1992;Sonnewald, 1992;Lee et al., 2005). ...
Although the activities of soluble pyrophosphatases were shown to be essential for a number of prokaryotes and eukaryotes, plants were assumed to lack cytoplasmic soluble pyrophosphatase activity and vacuolar membrane bound proton-pumping pyrophosphatase was accepted as the only enzyme responsible for the removal of pyrophosphate accumulated in the cytosol as a by-product of several biosynthetic pathways. On the contrary, Arabidopsis thaliana genome encodes six soluble pyrophosphatase isoforms (ASP1, ASP2A, ASP2B, ASP3, ASP4 and ASP5), which were shown to be highly conserved both in nucleic and in amino acid sequences. Microscopic analysis of leaves transiently transformed with C-terminal GFP tagged A. thaliana sPPase proteins revealed localization in the cytoplasm and/or nucleus for five isoforms (ASP1 to ASP4) and the in vivo plastidial localization of ASP5 could be shown. The analysis of stably transformed plants with promoter driven GUS expression revealed both tissue-specificity and developmental stage dependency of transcription for each A. thaliana soluble pyrophosphatase isoform. The promoter data were further confirmed by real time PCR analysis through which the active transcription of several A. thaliana soluble pyrophosphatase isoforms could be shown in all plant tissues, including later stages of development. The regulation of the expression of A. thaliana soluble pyrophosphatases by soluble sugars was analyzed using A. thaliana cell cultures and an in planta approach. A specific induction of the expression of ASP2B in response to sugar starvation was observed. Furthermore, the results indicate the possibility of sucrose regulation of the expression of ASP3, while the transcription of plastidial isoform (ASP5) was induced in response to 100 mM glucose in planta. The changes in the expression of A. thaliana soluble pyrophosphatases in response to ABA and different environmental stresses were analyzed using real time PCR. The data revealed an isoform- and/or tissue-specific and time dependent stress response suggesting specific roles for each isoform in vivo. Based on the data, the possible role of ASP3 in regulation of UGPase activity could be proposed. The wounding experiments using sugar beet (Beta vulgaris) plants overexpressing either Bsp1 (Beta vulgaris soluble pyrophosphatase isoform 1) or Bvp1 (Beta vulgaris vacuolar pyrophosphatase isoform 1) revealed the possibility of post-transcriptional regulation of mRNA levels of both soluble and vacuolar pyrophosphatases. Using recombinant Bsp1 protein expressed in Escherichia coli, the in vitro phosphorylation by protein kinase C could be shown and the possibility of post-translational regulation of plant soluble pyrophosphatase activity by phosphorylation was discussed. The overexpression of neither Bsp1 nor Bvp1 caused a significant effect on the sucrose accumulation of sugar beet taproots. On the other hand, the heterologous overexpression of not only Bvp1 but also Bsp1 revealed an impaired root growth and a possible contribution to the salt tolerance of A. thaliana. In summary, the results suggest that the plant soluble pyrophosphatases can perform multiple and vital functions during plant development and stress responses. In addition, the expression patterns of plant soluble pyrophosphatases indicate a strong link to the plant carbohydrate metabolism. Obwohl die Aktivität löslicher Pyrophosphatasen (sPPasen) essentiell für eine Reihe von Pro- und Eukaryonten ist, wurde für Pflanzen lange Zeit angenommen, dass sie keine im Zytosol löslichen Pyrophosphatasen besitzen und die membran-gebundene Protonen-transportierende vakuoläre Pyrophosphatase das einzige Enzym ist, welches das als Nebenprodukt verschiedener Biosynthesewege anfallende Pyrophosphat im Zytosol beseitigen könnte. Allerdings finden sich im Genom von Arabidopsis thaliana sechs lösliche Pyrophosphatase-Isoformen (ASP1, ASP2A, ASP2B, ASP3, ASP4 und ASP5), welche sowohl auf Nukleinsäure- als auch auf Aminosäure-Ebene hoch konserviert sind. Die mikroskopische Analyse von Blätten, welche transient mit C-terminalem GFP-Fusionsprotein der Arabidopsis sPPasen transformiert wurden, zeigt dass fünf Isoformen (ASP1-ASP4) im Zytoplasma und/oder dem Kern und eine Isoform (ASP5) in den Plastiden vorkommen. Durch Analyse von stabil mit Promotor:GUS-Konstrukten transformierten Pflanzen lässt sich die Gewebs-spezifische und entwicklungsabhängige Transkription für jede sPPase aus A. thaliana zeigen. Diese Daten werden unterstützt durch eine quantitative "real time PCR"-Analyse, über die Transkripte der verschiedenen sPPase-Isoformen in allen untersuchten Pflanzenorganen (auch in späten Entwicklungsstadien) nachgewiesen werden konnten. Die Regulation der Expression von sPPasen aus A. thaliana durch lösliche Zucker wurde mit Hilfe von Arabidopsis-Zellkulturen und einem in planta-Ansatz untersucht. Dabei zeigte sich, dass ASP2B spezifisch durch Zucker-Mangel induziert wird. Außerdem deuten die Ergebnisse auf eine mögliche Saccharose-Regulation der Expression von ASP3, während die Transkription der plastidären Isoform (ASP5) in planta durch 100mM Glukose induziert wird. Mittels "real time PCR" wurden auch Änderungen in der Expression der A. thaliana sPPasen in Antwort auf ABA und unterschiedliche Stresssituationen analysiert. Die Ergebnisse zeigen sowohl eine Isoform- und Gewebe-spezifische als auch eine zeitabhängige Stress-Antwort, was eine spezifische Rolle jeder einzelnen Isoform in vivo nahe legt. Anhand der Daten lässt sich eine mögliche Rolle der ASP3-Isoform, reguliert durch die Aktivität der UGPase, vorschlagen. Verwundungsexperimente mit Zuckerrüben-Pflanzen (Beta vulgaris) die entweder Bsp1 (Beta vulgaris soluble pyrophosphatase isoform 1) oder Bvp1 (Beta vulgaris vacuolar pyrophosphatase isoform 1) überexprimieren zeigen die Möglichkeit der post-transkriptionalen Regulation der mRNA-Mengen von beiden Genen. Rekombinant in Escherichia coli hergestelltes BSP1-Protein wird in vitro durch Protein Kinase C phosphoryliert und die mögliche post-translationale Regulation löslicher Pyrophosphatase-Aktivität durch Phosphosylierung wird diskutiert. Weder die Überexpression von Bsp1 noch von Bvp1 führte zu einem signifikanten Effekt auf die Saccharose-Akkumulation in Zuckerrüben-Speicherwurzeln. Auf der anderen Seite führte die heterologe Expression von Bvp1 und auch von Bsp1 zu einem veränderten Wurzelwachstum und möglicherweise zu veränderter Salztoleranz in A. thaliana. Zusammenfassend lässt sich sagen, dass die Ergebnisse vielfältige und lebenswichtige Funktionen pflanzlicher, löslicher Pyrophosphatasen während der Entwicklung und während Stressantworten nahe legen. Außerdem deuten die Expressionsmuster der löslichen Pyrophosphatasen auf eine starke Vernetzung mit dem pflanzlichen Kohlenhydrat-Metabolismus.
... Approximately 100 mg of leaves were sampled from 6-week-old rice plants grown under a 14/10 light cycle in a greenhouse. Each sample was harvested at zeitgeber (ZT) 0, 8, 16, 18, 20, and 22. Glucose, fructose, sucrose, and starch were measured in the soluble and insoluble fractions of ethanol-water extracts using enzymatic methods (Lee et al., 2005). The measured metabolite contents were normalized to fresh weights (n = 3, biological replicates). ...
Starch granules in the endodermis of plant hypocotyls act as statoliths that promote hypocotyl negative gravitropism—the directional growth of hypocotyls against gravity—in the dark. To identify the molecular components that regulate hypocotyl negative gravitropism, we performed a mutagenesis screen and isolated reduced gravitropic 1 (rgv1) mutants that lack starch granules in their hypocotyl endodermis and show reduced hypocotyl negative gravitropism in the dark. Using whole genome sequencing, we identified three different rgv1 mutants that are allelic to the previously reported early starvation 1 mutant, which is rapidly depleted of starch just before the dawn. ESV1 orthologs are present in starch-producing green organisms, suggesting ESV1 is a functionally conserved protein necessary for the formation of starch granules. Consistent with this, we found that liverwort and rice ESV1 can complement the Arabidopsis ESV1 mutant phenotype for both starch granules and hypocotyl negative gravitropism. To further investigate the function of ESV1 in other plants, we isolated rice ESV1 mutants and found that they show reduced levels of starch in their leaves and loosely packed starch granules in their grains. Both Arabidopsis and rice ESV1 mutants also lack starch granules in root columella and show reduced root gravitropism. Together, these results indicate ESV1 is a functionally conserved protein that promotes gravitropic responses in plants via its role in starch granule formation.
... Approximately 50 mg of palea/lemma at an early developmental stage and rice seeds at the pre-storage stage (three to six days after fertilization [DAF]), respectively, were harvested from plants grown in the LMO paddy field. Glucose, fructose, sucrose, and starch were measured in the soluble and insoluble fractions using enzymatic methods after preparing ethanol-water extracts ( Lee et al., 2005). The measured metabolite contents were normalized to fresh weights. ...
Sink strength optimizes sucrose import, which is fundamental to supporting developing seed grains and increasing crop yields, including those of rice (Oryza sativa). In this regard, little is known about the function of vacuolar invertase (VIN) in controlling sink strength and thereby seed size. Here, in rice we analyzed mutants of two VINs, OsVIN1 and OsVIN2, to examine their role during seed development. In a phenotypic analysis of the T-DNA insertion mutants, only the OsVIN2 mutant osvin2-1 exhibited reduced seed size and grain weight. Scanning electron microscopy analysis revealed that the small seed grains of osvin2-1 can be attributed to a reduction in spikelet size. A significant decrease in VIN activity and hexose level in the osvin2-1 spikelets interfered with spikelet growth. In addition, a significant reduction in starch and increase in sucrose, characteristic features of reduced turnover and flux of sucrose due to impaired sink strength, was evident in the pre-storage stage of osvin2-1 developing grains. In situ hybridization analysis found that expression of OsVIN2 was predominant in the endocarp of developing grains. A genetically complemented line with a native genomic clone of OsVIN2 rescued reduced VIN activity and seed size. Two additional mutants, osvin2-2 and osvin2-3, generated by the CRISPR/Cas9 method exhibited phenotypes similar to those of osvin2-1 in spikelet and seed size, VIN activity, and sugar metabolites. These results clearly demonstrate an important role of OsVIN2 as sink strength modulator that is critical for the maintenance of sucrose flux into developing seed grains.
... Our proteomic analysis showed that soluble inorganic pyrophosphatase (spot 42), which hydrolyzes pyrophosphate (PPi) to orthophosphate (Pi), is down-regulated in peach fruit at 5 • C. In transgenic Arabidopsis plants expressing Escherichia coli pyrophosphatase, sucrose content increased under continuous Table 1 Identification of 60 protein spots in peach fruit stored at 5 • C and 10 • C for 0, 14 and 28 days. white light growth conditions (Lee et al., 2005). Our observation that soluble inorganic pyrophosphatase is less accumulation in fruit at 5 • C is consistent with lowered sucrose content. ...
... Our proteomic analysis showed that soluble inorganic pyrophosphatase (spot 42), which hydrolyzes pyrophosphate (PPi) to orthophosphate (Pi), is down-regulated in peach fruit at 5 @BULLET C. In transgenic Arabidopsis plants expressing Escherichia coli pyrophosphatase, sucrose content increased under continuousTable 1 Identification of 60 protein spots in peach fruit stored at 5 @BULLET C and 10 @BULLET C for 0, 14 and 28 days. white light growth conditions (Lee et al., 2005). Our observation that soluble inorganic pyrophosphatase is less accumulation in fruit at 5 @BULLET C is consistent with lowered sucrose content. ...
Proteome patterns from peach fruit subjected to chilling stress (5◦C) and non-chilling stress (10◦C)temperatures were compared by 2-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF massspectrometry. Among the 60 differentially expressed proteins detected by 2-DE, those involved in stressresponse and defense were the most abundant. Proteins associated with membrane stability, as wellas sucrose content were reduced in fruit stored at 5◦C, possibly contributing to the development ofchilling injury (CI) seen at 5◦C. In addition, small heat shock proteins and components of the antioxidantdefense system were expressed at higher levels under chilling stress. In contrast, storage at non-chillingtemperature (10◦C) promoted the softening of peach fruit accompanied by increased levels of expansinand pectate lyase. The expression of pathogenesis-related proteins also increased at 10◦C, in parallel withthe relatively higher incidence of decay observed at this temperature.
... Suc, Glc, and Fru were analyzed by an HPLC system (Agilent Technologies 1200 series) as previously described (Eom et al., 2011). Starch was measured enzymatically (Lee et al., 2005). Total carbon and nitrogen in dried rice flour and leaves were determined using a TOC/TNb analyzer (Elementar Analysensysteme GmbH), and protein content was calculated by multiplying the percentage of total nitrogen by a conversion factor of 5.26 for rice grains and leaves (Fujihara et al., 2008). ...
Yield in cereals is a function of grain number and size. Suc, the main carbohydrate product of photosynthesis in higher plants, is transported long distances from source leaves to sink organs such as seeds and roots. Here, we report that transgenic rice plants expressing the Arabidopsis phloem-specific sucrose transporter AtSUC2, which loads Suc into the phloem, showed an increase in grain yield of up to 16% relative to wild-type plants in field trials. Compared to wild-type plants, pPP2::AtSUC2 plants had larger spikelet hulls and larger and heavier grains. Grain filling was accelerated in the transgenic plants, and more photoassimilate was transported from the leaves to the grain. In addition, microarray analyses revealed that carbohydrate, amino acid, and lipid metabolism was enhanced in the leaves and grain of pPP2::AtSUC2 plants. Thus, enhancing sucrose loading represents a promising strategy to improve rice yield to feed the global population.
... Leaf blades were harvested both at the end of day and night. Sucrose, glucose, fructose and starch were then mea- sured in the soluble and residual fractions of ethanol-water extracts ( Lee et al. 2005). Soluble sugars and starch were determined spectrophotometrically (Jelitto et al. 1992). ...
We characterized a rice monoculm mutant moc2, which showed significantly reduced tiller numbers, pale-green leaves, a reduced growth rate, and a consequent dwarf phenotype. The monoculm feature was attributed to a deficiency in the efficient outgrowth of tiller buds, although the moc2 mutant produced tiller buds. Inconsistent change was observed in the expression of genes involved in tiller bud outgrowth, suggesting that the moc2 mutant has a defective function necessary for the tiller bud outgrowth. The gene responsible for the moc2 mutant was mapped to a locus encoding cytosolic fructose- 1,6-bisphosphatase 1 (FBP1), in which a Tos17 retrotransposon was inserted in exon 4. Reverse-transcription PCR for the FBP1 gene amplified a shorter transcript from the moc2 mutant than from the wild-type plant. The sequence of the shorter transcript revealed a deletion of exon 4 by abnormal splicing, and the resulting frameshift generated a new translation termination signal. The moc2 mutant showed a very low level of FBPase activity, suggesting that it involves a loss-of-function mutation of FBP1. Cytosolic FBPase is considered a key enzyme in the sucrose biosynthesis pathway. Defective FBPase activity is anticipated to lead a shortage of sucrose supply, which probably causes the inhibition of tiller bud outgrowth in the moc2 mutant. The monoculm phenotype of the moc2 mutant supports the idea that sucrose supply may be an important cue to outgrow tiller buds. © 2013 The Japanese Society for Plant Cell and Molecular Biology.
... PPi is generally removed by inorganic pyrophosphatases, which hydrolyze PPi to orthophosphate (Pi). Heterologous expression of the Escherichia coli pyrophosphatase in an untargeted manner, conferring cytosolic localization of the encoded protein, showed an important role in the partitioning between sucrose (Suc) and starch (Sonnewald, 1992; Farre et al., 2001; Lee et al., 2005). In contrast, expression of the E. coli pyrophosphatase targeted to the plastid displayed only minor changes in metabolites levels (Farre et al., 2006). ...
Ascorbate (vitamin C) deficiency leads to low immunity, scurvy, and other human diseases and is therefore a global health problem. Given that plants are major ascorbate sources for humans, biofortification of this vitamin in our foodstuffs is of considerable importance. Ascorbate is synthetized by a number of alternative pathways: (i) from the glycolytic intermediates D-glucose-6P (the key intermediates are GDP-D-mannose and L-galactose), (ii) from the breakdown of the cell wall polymer pectin which uses the methyl ester of D-galacturonic acid as precursor, and (iii) from myo-inositol as precursor via myo-inositol oxygenase. We report here the engineering of fruit-specific overexpression of a bacterial pyrophosphatase, which hydrolyzes the inorganic pyrophosphate (PPi) to orthophosphate (Pi). This strategy resulted in increased vitamin C levels up to 2.5-fold in ripe fruit as well as increasing in the major sugars, sucrose, and glucose, yet decreasing the level of starch. When considered together, these finding indicate an intimate linkage between ascorbate and sugar biosynthesis in plants. Moreover, the combined data reveal the importance of PPi metabolism in tomato fruit metabolism and development.
... La síntesis de sacarosa a partir de glucosa y fructosa fosforiladas se realiza principalmente gracias a la acción enzimática concertada de SPS, sacarosa fosfato fosfatasa y sacarosa sintetasa. Sin embargo la sobreexpresión de otras enzimas de la ruta de la sacarosa, fosfoglucomutasa (PGM) y pirofosfatasa, tiene efectos pleiotrópicos sobre la fotosíntesis, crecimiento y metabolismo heterotrófico de Arabidopsis, papa y otros cultivos (Rung et al., 2004; Lee et al., 2005). El reparto del carbono puede alterarse por la acción individual o conjunta del suministro inadecuado de nutrientes, cambios en la actividad de enzimas claves en el control metabólico y por cambios en la fenología de la fuente (Wissuwa et al., 2005). ...
In higher plants, starch biosynthesis is highly sensitive to stress acting on the supply of carbohydrates from source organs, sink activity, and the activities of associated enzymes. Whole plant measurements in vivo have demonstrated that carbon flow rates are additionally dependent on the properties of sink, as well as those of whole transport system. In this article, some issues of the adjustment of this interaction through the complex of cellular responses to abiotic stress are reviewed. The aspects of pathway of stress signaling and its influence on the genetic program of starch expression are also elucidated. Additionally, the pathway, by which plant senses and responds to environmental cues and endogenous signals through changes in carbon partitioning and increases in soluble sugar cell concentration, and other adaptive physiological strategies are considered.
... La síntesis de sacarosa a partir de glucosa y fructosa fosforiladas se realiza principalmente gracias a la acción enzimática concertada de SPS, sacarosa fosfato fosfatasa y sacarosa sintetasa. Sin embargo, la sobreexpresión de otras enzimas de la ruta de la sacarosa, fosfoglucomutasa (PGM) y pirofosfatasa, tiene efectos pleiotrópicos sobre la fotosíntesis, crecimiento y metabolismo heterotrófico de Arabidopsis, papa y otros cultivos (Rung et al., 2004; Lee et al., 2005). El reparto del carbono puede alterarse por la acción individual o conjunta del suministro inadecuado de nutrientes, cambios en la actividad de enzimas claves en el control metabólico y por cambios en la fenología de la fuente (Wissuwa et al., 2005). ...
En las plantas superiores la biosíntesis del almidón es sensible al efecto de condiciones ambientales adversas, las cuales actúan sobre el suministro de carbohidratos desde los órganos fuente, la actividad de los vertederos y de las enzimas biosintéticas asociadas. Mediciones de la planta entera in vivo han demos- trado que la tasa de flujo de carbono depende además de las propiedades del vertedero y de las características del sistema de transporte. En este documento también se revisan los aspectos del ajuste de esta interacción por el complejo de respuestas celulares inducidas por los estreses abióticos. Además de la problemática de la ruta de señalización del estrés, se elucida su influencia sobre la expresión genética del metabolismo del almidón. Por otro lado, se considera la manera como la planta percibe y responde a las señales ambientales a través de modifi - caciones en la partición del carbono, mediante el incremento de los niveles de los azúcares solubles y otras estrategias fisiológicas de aclimatación al estrés abiótico. In higher plants, starch biosynthesis is highly sensitive to stress acting on the supply of carbohydrates from source organs, sink activity, and the activities of associated enzymes. Whole plant measurements in vivo have demonstrated that carbon flow rates are additionally dependent on the properties of sink, as well as those of whole transport system. In this article, some issues of the adjustment of this interaction through the complex of cellular responses to abiotic stress are reviewed. The aspects of pathway of stress signaling and its influence on the genetic program of starch expression are also elucidated. Additionally, the pathway, by which a plant senses and responds to environmental cues and endogenous signals through changes in carbon partitioning and increases in soluble sugar cell concentration, and other adaptive physiological strategies are considered.
... La síntesis de sacarosa a partir de glucosa y fructosa fosforiladas se realiza principalmente gracias a la acción enzimática concertada de SPS, sacarosa fosfato fosfatasa y sacarosa sintetasa. Sin embargo, la sobreexpresión de otras enzimas de la ruta de la sacarosa, fosfoglucomutasa (PGM) y pirofosfatasa, tiene efectos pleiotrópicos sobre la fotosíntesis, crecimiento y metabolismo heterotrófico de Arabidopsis, papa y otros cultivos (Rung et al., 2004; Lee et al., 2005). El reparto del carbono puede alterarse por la acción individual o conjunta del suministro inadecuado de nutrientes, cambios en la actividad de enzimas claves en el control metabólico y por cambios en la fenología de la fuente (Wissuwa et al., 2005). ...
... In cytosolic energy metabolism , PPi levels are modulated by the combined activity of UGPase, PFP, and membrane-bound, vacuolar H + -translocating PPiases (Heinonen, 2001). The role of cytosolic PPi in plant carbohydrate metabolism has been investigated using transgenic plants expressing an E. coli PPiase gene product in the cytosol (Jelitto et al., 1992; Sonnewald, 1992; Lee et al., 2005). Constitutive expression leads to pleiotropic growth defects and increased accumulation of Suc, hexoses, nucleotide sugars, and transient starch in photosynthetic tissues. ...
This study describes a dominant low-seed-oil mutant (lo15571) of Arabidopsis (Arabidopsis thaliana) generated by enhancer tagging. Compositional analysis of developing siliques and mature seeds indicated reduced conversion of photoassimilates to oil. Immunoblot analysis revealed increased levels of At1g01050 protein in developing siliques of lo15571. At1g01050 encodes a soluble, cytosolic pyrophosphatase and is one of five closely related genes that share predicted cytosolic localization and at least 70% amino acid sequence identity. Expression of At1g01050 using a seed-preferred promoter recreated most features of the lo15571 seed phenotype, including low seed oil content and increased levels of transient starch and soluble sugars in developing siliques. Seed-preferred RNA interference-mediated silencing of At1g01050 and At3g53620, a second cytosolic pyrophosphatase gene that shows expression during seed filling, led to a heritable oil increase of 1% to 4%, mostly at the expense of seed storage protein. These results are consistent with a scenario in which the rate of mobilization of sucrose, for precursor supply of seed storage lipid biosynthesis by cytosolic glycolysis, is strongly influenced by the expression of endogenous pyrophosphatase enzymes. This emphasizes the central role of pyrophosphate-dependent reactions supporting cytosolic glycolysis during seed maturation when ATP supply is low, presumably due to hypoxic conditions. This route is the major route providing precursors for seed oil biosynthesis. ATP-dependent reactions at the entry point of glycolysis in the cytosol or plastid cannot fully compensate for the loss of oil content observed in transgenic events with increased expression of cytosolic pyrophosphatase enzyme in the cytosol. These findings shed new light on the dynamic properties of cytosolic pyrophosphate pools in developing seed and their influence on carbon partitioning during seed filling. Finally, our work uniquely demonstrates that genes encoding cytosolic pyrophosphatase enzymes provide novel targets to improve seed composition for plant biotechnology applications.
... Approximately 100 mg of leaf blades was harvested at the end of day and night from 4-week-old plants grown in the greenhouse. Suc, Glc, Fru, and starch were measured using NAD(P)H-coupled enzymatic methods in the soluble and residual fractions of ethanol-water extracts (Lee et al., 2005). The measured metabolite contents were normalized to the leaf fresh weights. ...
Physiological functions of sucrose (Suc) transporters (SUTs) localized to the tonoplast in higher plants are poorly understood. We here report the isolation and characterization of a mutation in the rice (Oryza sativa) OsSUT2 gene. Expression of OsSUT2-green fluorescent protein in rice revealed that OsSUT2 localizes to the tonoplast. Analysis of the OsSUT2 promoter::β-glucuronidase transgenic rice indicated that this gene is highly expressed in leaf mesophyll cells, emerging lateral roots, pedicels of fertilized spikelets, and cross cell layers of seed coats. Results of Suc transport assays in yeast were consistent with a H(+)-Suc symport mechanism, suggesting that OsSUT2 functions in Suc uptake from the vacuole. The ossut2 mutant exhibited a growth retardation phenotype with a significant reduction in tiller number, plant height, 1,000-grain weight, and root dry weight compared with the controls, the wild type, and complemented transgenic lines. Analysis of primary carbon metabolites revealed that ossut2 accumulated more Suc, glucose, and fructose in the leaves than the controls. Further sugar export analysis of detached leaves indicated that ossut2 had a significantly decreased sugar export ability compared with the controls. These results suggest that OsSUT2 is involved in Suc transport across the tonoplast from the vacuole lumen to the cytosol in rice, playing an essential role in sugar export from the source leaves to sink organs.
... Due to a lack of adverse phenotypic alterations found when altering the expression of these enzymes in autotrophic sink metabolism, a considerable degree of redundancy in cytosolic PP i catabolism has been proposed (Hajirezaei et al., 1994; Paul et al., 1995). Heterologous studies aimed at elucidating the biological role of PP i in plants demonstrated to date that cytosolically expressed Escherichia coli sPPase leads to major effects on carbohydrate partitioning between Suc and starch (Sonnewald, 1992; Geigenberger et al., 1998; Farré et al., 2000; Lee et al., 2005). In contrast, tuber-specific plastid-targeted E. coli sPPase led to only minor changes in metabolite levels (Farré et al., 2006). ...
The role of pyrophosphate in primary metabolism is poorly understood. Here, we report on the transient down-regulation of plastid-targeted soluble inorganic pyrophosphatase in Nicotiana benthamiana source leaves. Physiological and metabolic perturbations were particularly evident in chloroplastic central metabolism, which is reliant on fast and efficient pyrophosphate dissipation. Plants lacking plastidial soluble inorganic pyrophosphatase (psPPase) were characterized by increased pyrophosphate levels, decreased starch content, and alterations in chlorophyll and carotenoid biosynthesis, while constituents like amino acids (except for histidine, serine, and tryptophan) and soluble sugars and organic acids (except for malate and citrate) remained invariable from the control. Furthermore, translation of Rubisco was significantly affected, as observed for the amounts of the respective subunits as well as total soluble protein content. These changes were concurrent with the fact that plants with reduced psPPase were unable to assimilate carbon to the same extent as the controls. Furthermore, plants with lowered psPPase exposed to mild drought stress showed a moderate wilting phenotype and reduced vitality, which could be correlated to reduced abscisic acid levels limiting stomatal closure. Taken together, the results suggest that plastidial pyrophosphate dissipation through psPPase is indispensable for vital plant processes.
... The present findings suggest that either starch degradation or transport pathways might be impaired in the source tissues of phyA phyB phyC triple mutants. In addition, it has been proposed that an increase in Pi levels disrupted plastidic membrane phosphate transport and sugar transport systems in a transgenic Arabidopsis plants expressing E. coli pyrophosphatase (22). As a result, intercellular metabolism had changed, including increased hexose phosphates, carbohydrate accumulation and decreased photosynthetic activity. ...
The phytochrome photoreceptors regulate plant growth and development throughout their life cycle. Rice (Oryza sativa) possesses three phytochromes, phyA, phyB, and phyC. Physiological, genetic, and biochemical analyses of null mutants of each phytochrome have revealed the function of each in rice. However, few studies have investigated the relationship between phytochrome signaling and metabolism. In the present study, non-targeted metabolite analysis by gas chromatography time-of-flight mass spectrometry (GC/TOF-MS) and targeted metabolite analysis by capillary electrophoresis electrospray ionization mass spectrometry (CE/ESI-MS) were employed to investigate metabolic changes in rice phyA phyB phyC triple mutants. Distinct metabolic profiles between phyA phyB phyC triple mutants and the wild type (WT), as well as those between young and mature leaf blades, could be clearly observed by principal component analysis (PCA). The metabolite profiles indicated high accumulation of amino acids, organic acids, sugars, sugar phosphates, and nucleotides in the leaf blades of phyA phyB phyC triple mutants, especially in the young leaves, compared with those in the WT. Remarkable overaccumulation of monosaccharide, such as glucose (53.4-fold), fructose (42.5-fold), and galactose (24.5-fold), was observed in young leaves of phyA phyB phyC triple mutants. These metabolic phenotypes suggest that sugar metabolism, carbon partitioning, sugar transport, or some combination of these is impaired in the phyA phyB phyC triple mutants, and conversely, that phytochromes have crucial roles in sugar metabolism.
... Leaf blades were harvested both at the end of day and night. Sucrose, glucose, fructose and starch were then measured in the soluble and residual fractions of ethanol-water extracts ( Lee et al. 2005). Soluble sugars and starch were determined spectrophotometrically (Jelitto et al. 1992). ...
During photosynthesis, triose-phosphates (trioseP) exported from the chloroplast to the cytosol are converted to sucrose via cytosolic fructose-1,6-bisphosphatase (cFBPase). Expression analysis in rice suggests that OscFBP1 plays a major role in the cytosolic conversion of trioseP to sucrose in leaves during the day. The isolated OscFBP1 mutants exhibited markedly decreased photosynthetic rates and severe growth retardation with reduced chlorophyll content, which results in plant death. Analysis of primary carbon metabolites revealed both significantly reduced levels of sucrose, glucose, fructose and starch in leaves of these mutants, and a high accumulation of sucrose to starch in leaves of rice plants. In the oscfbp1 mutants, products of glycolysis and the TCA cycle were significantly increased. A partitioning experiment of (14)C-labelled photoassimilates revealed altered carbon distributions including a slight increase in the insoluble fraction representing transitory starch, a significant decrease in the neutral fraction corresponding to soluble sugars and a high accumulation of phosphorylated intermediates and carboxylic acid fractions in the oscfbp1 mutants. These results indicate that the impaired synthesis of sucrose in rice cannot be sufficiently compensated for by the transitory starch-mediated pathways that have been found to facilitate plant growth in the equivalent Arabidopsis mutants.
... Starch contents were measured in the insoluble fractions of ethanol–water extracts (Lee et al. 2005), and determined spectrophotometrically as described previously (Jelitto et al. 1992). Preparation of insoluble starch from rice endosperm ...
To elucidate the role of SSIIIa during starch synthesis in rice (Oryza sativa L.) endosperm, we characterized null mutants of this gene, generated by T-DNA insertions. Scanning electron microscope (SEM) analysis revealed that the starch granules in these mutants are smaller and rounder compared with the wild type controls, and that the mutant endosperm is characterized by a loosely packed central portion exhibiting a floury-like phenotype. Hence, the OsSSIIIa (Oryza sativa SSIIIa) mutations are referred to as white-core floury endosperm 5-1 (flo5-1) and flo5-2. Based upon their X-ray diffraction patterns, the crystallinity of the starch in the flo5 mutant endosperm is decreased compared with wild type. Through determination of the chain-length distribution of the mutant endosperm starch, we found that flo5-1 and flo5-2 mutants have reduced the content of long chains with degree of polymerization (DP) 30 or greater compared with the controls. This suggests that OsSSIIIa/Flo5 plays an important role in generating relatively long chains in rice endosperm. In addition, DP 6 to 8 and DP 16 to 20 appeared to be reduced in endosperm starch of flo5-1 and flo5-2, whereas DP 9 to 15 and DP 22 to 29 were increased in these mutants. By the use of differential scanning calorimetry (DSC), the gelatinization temperatures of endosperm starch were found to be 1-5 degrees C lower than those of the control. We propose a distinct role for OsSSIIIa/Flo5 and the coordinated action of other SS isoforms during starch synthesis in the seed endosperm of rice.
... Ten-week-old leaf blades harvested at the end of day and fully developed endosperms were analyzed. Starch contents were measured in the insoluble fractions of ethanolwater extracts ( Lee et al. 2005) and determined spectrophotometrically as described previously ( Jelitto et al. 1992). ...
ADP-glucose pyrophosphorylase (AGP) catalyzes the first committed step of starch biosynthesis in higher plants. To identify AGP isoforms essential for this biosynthetic process in sink and source tissues of rice plants, we analyzed the rice AGP gene family which consists of two genes, OsAGPS1 and OsAGPS2, encoding small subunits (SSU) and four genes, OsAGPL1, OsAGPL2, OsAGPL3 and OsAGPL4, encoding large subunits (LSU) of this enzyme heterotetrameric complex. Subcellular localization studies using green fluorescent protein (GFP) fusion constructs indicate that OsAGPS2a, the product of the leaf-preferential transcript of OsAGPS2, and OsAGPS1, OsAGPL1, OsAGPL3, and OsAGPL4 are plastid-targeted isoforms. In contrast, two isoforms, SSU OsAGPS2b which is a product of a seed-specific transcript of OsAGPS2, and LSU OsAGPL2, are localized in the cytosol. Analysis of osagps2 and osagpl2 mutants revealed that a lesion of one of the two cytosolic isoforms, OsAGPL2 and OsAGPS2b, causes a shrunken endosperm due to a remarkable reduction in starch synthesis. In leaves, however, only the osagps2 mutant appears to severely reduce the transitory starch content. Interestingly, the osagps2 mutant was indistinguishable from wild type during vegetative plant growth. Western blot analysis of the osagp mutants and wild type plants demonstrated that OsAGPS2a is an SSU isoform mainly present in leaves, and that OsAGPS2b and OsAGPL2 are the major SSU and LSU isoforms, respectively, in the endosperm. Finally, we propose a spatiotemporal complex model of OsAGP SSU and LSU isoforms in leaves and in developing endosperm of rice plants.
Pyrophosphatases (PPases) are enzymes that catalyze the hydrolysis of pyrophosphate (PPi), a byproduct of the synthesis and degradation of diverse biomolecules. The accumulation of PPi in the cell can result in cell death. Although the substrate is the same, there are variations in the catalysis and features of these enzymes. Two enzyme forms have been identified in bacteria: cytoplasmic or soluble pyrophosphatases and membrane-bound pyrophosphatases, which play major roles in cell bioenergetics. In eukaryotic cells, cytoplasmic enzymes are the predominant form of PPases (c-PPases), while membrane enzymes (m-PPases) are found only in protists and plants. The study of bacterial cytoplasmic and membrane-bound pyrophosphatases has slowed in recent years. These enzymes are central to cell metabolism and physiology since phospholipid and nucleic acid synthesis release important amounts of PPi that must be removed to allow biosynthesis to continue. In this review, two aims were pursued: first, to provide insight into the structural features of PPases known to date and that are well characterized, and to provide examples of enzymes with novel features. Second, the scientific community should continue studying these enzymes because they have many biotechnological applications. Additionally, in this review, we provide evidence that there are m-PPases present in fungi; to date, no examples have been characterized. Therefore, the diversity of PPase enzymes is still a fruitful field of research. Additionally, we focused on the roles of H ⁺ /Na ⁺ pumps and m-PPases in cell bioenergetics. Finally, we provide some examples of the applications of these enzymes in molecular biology and biotechnology, especially in plants. This review is valuable for professionals in the biochemistry field of protein structure–function relationships and experts in other fields, such as chemistry, nanotechnology, and plant sciences.
Sucrose has been known as pivotal carbon source for plant nutrient metabolism including ammonium (NH4+) assimilation. However, the correlation between rice root NH4+ assimilation and gene expression responsible for sucrose allocation has not been investigated. Here, we reported the transcriptional regulation of OsSUTs and OsSPSs by exogenously supplied sucrose and the response of NH4+ assimilation in rice roots. Spraying sucrose to mature leaves of rice (Oryza sativa L. cv. indica 9311) up-regulated transcript abundances of leaf OsSUT1, OsSUT2, OsSUT3, OsSUT4, OsSPS6, and OsSPS11, down-regulated mRNA expression of leaf OsSUT5, OsSPS1, OsSPS2, and OsSPS8, increased soluble sugar contents in leaves and roots, and promoted root NH4+ assimilation. The similar responses, except for leaf OsSPS1 mRNA expression, were observed when sucrose was fed to hydroponic media. Rice (indica 9311) treated by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) exhibited altered transcript abundances of leaf OsSUTs and OsSPSs, lower contents of soluble sugars in leaves and roots, and reduced capacity of root NH4+ assimilation, which was partially reversed by sucrose supply. The results indicated that the exogenously supplied sucrose coordinately regulated leaf OsSUTs and OsSPSs mRNA expression and root NH4+ assimilation in rice seedlings.
We prepared Arabidopsis thaliana lines expressing a functional green fluorescent protein (GFP)-linked vacuolar H(+)-pyrophosphatase (H(+)-PPase) under the control of its own promoter to investigate morphological dynamics of vacuoles and tissue-specific expression of H(+)-PPase. The lines obtained had spherical structures in vacuoles with strong fluorescence, which are referred to as bulbs. Quantitative analyses revealed that the occurrence of the bulbs correlated with the amount of GFP. Next, we prepared a construct of H(+)-PPase linked with a nondimerizing GFP (mGFP); we detected no bulbs. These results indicate that the membranes adhere face-to-face by antiparallel dimerization of GFP, resulting in the formation of bulbs. In plants expressing H(+)-PPase-mGFP, intravacuolar spherical structures with double membranes, which differed from bulbs in fluorescence intensity and intermembrane spacing, were still observed in peripheral endosperm, pistil epidermis and hypocotyls. Four-dimensional imaging revealed the dynamics of formation, transformation, and disappearance of intravacuolar spherical structures and transvacuolar strands in living cells. Visualization of H(+)-PPase-mGFP revealed intensive accumulation of the enzyme, not only in dividing and elongating cells but also in mesophyll, phloem, and nectary cells, which may have high sugar content. Dynamic morphological changes including transformation of vacuolar structures between transvacuolar strands, intravacuolar sheet-like structures, and intravacuolar spherical structures were also revealed.
In higher plants, starch biosynthesis is highly sensitive to stress acting on the supply of carbohydrates from source organs, sink activity, and the activities of associated enzymes. Whole plant measurements in vivo have demonstrated that carbon flow rates are additionally dependent on the properties of sink, as well as those of whole transport system. In this article, some issues of the adjustment of this interaction through the complex of cellular responses to abiotic stress are reviewed. The aspects of pathway of stress signaling and its influence on the genetic program of starch expression are also elucidated. Additionally, the pathway, by which a plant senses and responds to environmental cues and endogenous signals through changes in carbon partitioning and increases in soluble sugar cell concentration, and other adaptive physiological strategies are considered.
We studied the influence of alien cytoplasm of spring goatgrass Aegilops ovata L. on some physiological parameters in winter wheat (Triticum aestivum L.), Mironovskaya 808, under normal conditions and in the case of modified source-sink relations. Measurements of relative
rates of plant dry matter growth and its distribution among organs, CO2 exchange (photosynthesis upon light saturation and dark respiration), content of sugars (sucrose + glucose + fructose) and
their ratio in leaves, frost hardiness, and indices of membrane stability and damage of leaves by frost have shown that, on
average, alloplasmic hybrid differed from the initial cultivar by almost all parameters. Reduced frost hardiness, increased
index of leaf damage by frost, lowered leaf content of sugars, and reduced sucrose/(glucose + fructose) ratio in the alloplasmic
hybrid were combined with higher roots/leaves ratio, relative rate of dry matter growth, and photosynthesis and respiration
rates. The alloplasmic hybrid was more tolerant to decreased source strength in source-sink relations as compared to the initial
cultivar.
Effect of suppression of the source activity on some physiological characteristics of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) was studied on plants grown in water culture. The plants were examined at the mixotrophic stage
of growth period, during their transition from vegetative state to relative dormancy in autumn. The average temperature over
10 days of the experiment was 6°C at 9-h photoperiod and illuminance of 8–20 klx. The source strength was suppressed successively
with a series of treatments: intact control plants (V1); plants with the seed endosperm removed (V2); plants with photosynthesis
inhibited (V3); plants with the seed endosperm removed and photosynthesis inhibited (V4); plants with the seed endosperm removed,
photosynthesis inhibited, and the root nutrient medium replaced with distilled water (V5). On the 6th–10th day of the experiment,
the relative growth rate (RGR) was determined from dry weight increments. At the same time, the distribution of biomass among
organs, the CO2 exchange rates (photosynthesis and dark respiration), the content and proportions of sugars (sucrose, glucose, and fructose),
the total content of phenolic compounds and flavonoids, the index of membrane stability (IMS) in leaves, and frost hardiness
of plants were measured. Frost hardiness of vegetating plants was shown to be inversely related to RGR (R = −0.906), dark respiration rate (R = −0.789), the percentage of sucrose in total sugar content (R = −0.737), leaf IMS (R = −0.390), and the rate of apparent photosynthesis (R = −0.288); it was directly proportional to the content of flavonoids (R =0.973), total phenols (R = 0.743), and sugars (R = 0.385). The role of modified source-sink relations in frost hardiness of vegetating plants at the stage of their transition
to cold hardening is discussed. The differences between plants undergoing this transition and cold-hardened plants are considered,
as well as the importance of phenolic compounds for the development of frost hardiness.
A cDNA clone encoding a soluble inorganic pyrophosphatase (EC 3.6.1.1) of potato (Solanum tuberosum L.) was isolated by screening a developing tuber library with a heterologous probe. The central domain of the encoded polypeptide is nearly identical at the sequence level with its Arabidopsis homolog (J.J. Kieber and E.R. Signer [1991] Plant Mol Biol 16: 345–348). Computer-assisted analysis of the potato, Arabidopsis, and Escherichia coli soluble pyrophosphatases indicated a remarkably conserved organization of the hydrophobic protein domains. The enzymatic function of the potato protein could be deduced from the presence of amino acid residues highly conserved in soluble pyrophosphatases and was confirmed by its capacity to complement a thermosensitive pyrophosphatase mutation in E. coli. The potato polypeptide was purified from complemented bacterial cells and its pyrophosphatase activity was shown to be strictly dependent on Mg2+ and strongly inhibited by Ca2+. The subcellular location of the potato pyrophosphatase is unknown. Structure analysis of the N-terminal protein domain failed to recognize typical transit peptides and the calculated molecular mass of the polypeptide (24 kD) is significantly inferior to the values reported for the plastidic (alkaline) or mitochondrial pyrophosphatases in plants (28–42 kD). Two unlinked loci could be mapped by restriction fragment length polymorphism analysis in the potato genome using the full-length cDNA as probe.
In order to determine the concentration of pyrophosphate (PPi) and its subcellular distribution in Chara corallina, a new method to concentrate PPi from cell extracts was developed. PPi was extracted and concentrated as Ca2P2O7 under alkaline conditions. The amount of PPi in the precipitate was measured using an enzyme system containing pyrophosphate:fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) coupled to NADH oxidation in the presence of [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid.
The subcellular localization of PPi and inorganic phosphate (Pi) was studied using the intracellular perfusion technique. The relative volumes of the cytoplasm (6.4%) and the vacuole (93.6%) were determined by perfusing Lucifer Yellow CH into the vacuole and by assuming that the Lucifer Yellow CH dead space represented the cytoplasmic volume. The volume of the chloroplast layer was determined microscopically, and it was found that it occupied 10% of the Chara cytoplasm. PPi was present predominantly in the cytosol at a level of 193 µM, while it existed in the vacuole at a level of only 2.20 µM and less than 1 µM in chloroplasts. By contrast, Pi was distributed almost equally in the cytosol (12.0 mM), chloroplasts (16.2 mM), and the vacuole (6.70 mM). The electrochemical potential gradient across the tonoplast for H⁺ (Δμ¯H+ = −11.6 to −18.0 KJ/mol) was nearly equal to the free energy release from the hydrolysis of PPi in cytoplasm (ΔGpp =−18.9 KJ/mol), indicating that the H⁺-translocating inorganic pyrophosphatase can work as a H⁺ pump in C. corallina.
Constitutive expression of the Escherichia coli ppa gene encoding inorganic pyrophosphatase resulted in sugar accumulation in source leaves and stunted growth of transgenic tobacco plants. The reason for this phenotype was hypothesized to be reduced sucrose utilization and loading into the phloem. To study the role of PPi in phloem cells, a chimeric gene was constructed using the phloem-specific rolC promoter of Agrobacterium rhizogenes to drive the expression of the ppa gene. Removal of cytosolic PPi in those cells resulted in photoassimilate accumulation in source leaves, chlorophyll loss, and reduced plant growth. From these data, it was postulated that sucrose hydrolysis via sucrose synthase is essential for assimilate partitioning. To bypass the PPi-dependent sucrose synthase step, transgenic plants were produced that express various levels of the yeast suc2 gene, which encodes cytosolic invertase, in their phloem cells. To combine the phloem-specific expression of the ppa gene and the suc2 gene, crosses between invertase- and pyrophosphatase-containing transgenic plants were performed. Analysis of their offspring revealed that invertase can complement the phenotypic effects caused by the removal of PPi in phloem cells.
A cDNA clone encoding a soluble inorganic pyrophosphatase (EC 3.6.1.1) of potato (Solanum tuberosum L.) was isolated by screening a developing tuber library with a heterologous probe. The central domain of the encoded polypeptide is nearly identical at the sequence level with its Arabidopsis homolog (J.J. Kieber and E.R. Signer [1991] Plant Mol Biol 16: 345-348). Computer-assisted analysis of the potato, Arabidopsis, and Escherichia coli soluble pyrophosphatases indicated a remarkably conserved organization of the hydrophobic protein domains. The enzymatic function of the potato protein could be deduced from the presence of amino acid residues highly conserved in soluble pyrophosphatases and was confirmed by its capacity to complement a thermosensitive pyrophosphatase mutation in E. coli. The potato polypeptide was purified from complemented bacterial cells and its pyrophosphatase activity was shown to be strictly dependent on Mg2+ and strongly inhibited by Ca2+. The subcellular location of the potato pyrophosphatase is unknown. Structure analysis of the N-terminal protein domain failed to recognize typical transit peptides and the calculated molecular mass of the polypeptide (24 kD) is significantly inferior to the values reported for the plastidic (alkaline) or mitochondrial pyrophosphatases in plants (28-42 kD). Two unlinked loci could be mapped by restriction fragment length polymorphism analysis in the potato genome using the full-length cDNA as probe.
Expression of four Arabidopsis (thale cress) genes corresponding to the small (ApS) and large subunits (ApL1, ApL2, ApL3) of ADP-glucose pyrophosphorylase (AGPase), a key enzyme of starch biosynthesis, was found to be profoundly and differentially regulated by sugar and light/dark exposures. Transcript levels of both ApL2 and ApL3, and to a lesser extent ApS, increased severalfold upon feeding sucrose or glucose to the detached leaves in the dark, whereas the mRNA content for ApL1 decreased under the same conditions. Glucose was, in general, less effective than sucrose in inducing regulation of AGPase genes, possibly due to observed limitations in its uptake when compared with sucrose uptake by detached leaves. Osmotic agents [sorbitol, poly(ethylene glycol)] had no effect on ApS, ApL2 and ApL3 transcript level, but they did mimic the effect of sucrose on ApL1 gene, suggesting that the latter is regulated by osmotic pressure rather than any particular sugar. For all the genes the sugar effect was closely mimicked by an exposure of the dark-pre-adapted leaves to the light. Under both dark and light conditions, sucrose fed to the detached leaves was found to be rapidly metabolized to hexoses and, to some extent, starch. Starch production reflected most probably an increase in substrate availability for AGPase reaction rather than being due to changes in AGPase protein content, since both the sugar feeding and light exposure had little or no effect on the activity of AGPase or on the levels of its small and large subunit proteins in leaf extracts. The data suggest tight translational or post-translational control, but they may also reflect spatial control of AGPase gene expression within a leaf. The sugar/light-dependent regulation of AGPase gene expression may represent a part of a general cellular response to the availability/allocation of carbohydrates during photosynthesis.
Potato is a globally important crop. Unfortunately, potato farming is plagued with problems associated with the sprouting behavior of seed tubers. The data presented here demonstrate that using transgenic technology can influence this behavior. Transgenic tubers cytosolically expressing an inorganic pyrophosphatase gene derived from Escherichia coli under the control of the tuber-specific patatin promoter display significantly accelerated sprouting. The period of presprouting dormancy for transgenic tubers planted immediately after harvest is reduced by six to seven weeks when compared to wild-type tubers. This study demonstrates a method with which to regulate dormancy, an important aspect of potato crop management.
To change the hexose-to-sucrose ratio within phloem cells, yeast-derived cytosolic invertase was expressed in transgenic potato (Solanum tuberosum cv. Desirée) plants under control of the rolC promoter. Vascular tissue specific expression of the transgene was verified by histochemical detection of invertase activity in tuber cross-sections. Vegetative growth and tuber yield of transgenic plants was unaltered as compared to wild-type plants. However, the sprout growth of stored tubers was much delayed, indicating impaired phloem-transport of sucrose towards the developing bud. Biochemical analysis of growing tubers revealed that, in contrast to sucrose levels, which rapidly declined in growing invertase-expressing tubers, hexose and starch levels remained unchanged as compared to wild-type controls. During storage, sucrose and starch content declined in wild-type tubers, whereas glucose and fructose levels remained unchanged. A similar response was found in transgenic tubers with the exception that starch degradation was accelerated and fructose levels increased slightly. Furthermore, changes in carbohydrate metabolism were accompanied by an elevated level of phosphorylated intermediates, and a stimulated rate of respiration. Considering that sucrose breakdown was restricted to phloem cells it is concluded that, in response to phloem-associated sucrose depletion or hexose elevation, starch degradation and respiration is triggered in parenchyma cells. To study further whether elevated hexose and/or hexose-phosphates or decreased sucrose levels are responsible for the metabolic changes observed, sucrose content was decreased by tuber-specific expression of a bacterial sucrose isomerase. Sucrose isomerase catalyses the reversible conversion of sucrose into palatinose, which is not further metabolizable by plant cells. Tubers harvested from these plants were found to accumulate high levels of palatinose at the expense of sucrose. In addition, starch content decreased slightly, while hexose levels remained unaltered, compared with the wild-type controls. Similar to low sucrose-containing invertase tubers, respiration and starch breakdown were found to be accelerated during storage in palatinose-accumulating potato tubers. In contrast to invertase transgenics, however, no accumulation of phosphorylated intermediates was observed. Therefore, it is concluded that sucrose depletion rather than increased hexose metabolism triggers reserve mobilization and respiration in stored potato tubers.
During the last 15 years, much progress has been made in discovering genes encoding solute transporters of the inner plastid
envelope membrane. For example, genes encoding transporters for phosphorylated intermediates, dicarboxylates, adenine nucleotides,
inorganic anions, and monosaccharides have been cloned. In many cases, the corresponding proteins have been expressed in recombinant
host systems for further functional studies, thus allowing detailed in vitro characterization of transporter properties. Knowledge of the gene sequences encoding these transporters have allowed reverse‐genetic
approaches to study transporter function in vivo. Antisense repression and T‐DNA insertion mutagenesis have provided a range of transgenic and mutant plants in which the
activity of specific plastid envelope transporters are massively decreased or abolished. Plants with altered transporter activities
represent excellent tools to probe the in vivo function of these transporters. Moreover, changing the permeability of the plastid envelope membrane permits the targeted
manipulation of subcellular metabolite pools.
The chloroplast envelope triose-phosphate/phosphate translocator (TPT) is responsible for carbohydrate export during photosynthesis. Using measurements of carbohydrates, partitioning of assimilated 14CO2, photosynthetic gas exchange, and chlorophyll fluorescence, we show that a mutant of Arabidopsis lacking the TPT increases synthesis of starch compared to the wild type, thereby compensating for a deficiency in its ability to export triose-phosphate from the chloroplast. However, during growth under high light, the capacity for starch synthesis becomes limiting so that the chloroplastic phosphate pool is depleted, resulting in a restriction on electron transport, a reduction in the rate of photosynthesis, and slowed plant growth. Under the same conditions but not under low light, we observe release of 14C label from starch, indicating that its synthesis and degradation occur simultaneously in the light. The induction of starch turnover in the mutant specifically under high light conditions leads us to conclude that it is regulated by one or more metabolic signals, which arise as a result of phosphate limitation of photosynthesis.
A series of experiments is presented investigating short term and long term changes of the nature of the response of rate of CO2 assimilation to intercellular p(CO2). The relationships between CO2 assimilation rate and biochemical components of leaf photosynthesis, such as ribulose-bisphosphate (RuP2) carboxylase-oxygenase activity and electron transport capacity are examined and related to current theory of CO2 assimilation in leaves of C3 species. It was found that the response of the rate of CO2 assimilation to irradiance, partial pressure of O2, p(O2), and temperature was different at low and high intercellular p(CO2), suggesting that CO2 assimilation rate is governed by different processes at low and high intercellular p(CO2). In longer term changes in CO2 assimilation rate, induced by different growth conditions, the initial slope of the response of CO2 assimilation rate to intercellular p(CO2) could be correlated to in vitro measurements of RuP2 carboxylase activity. Also, CO2 assimilation rate at high p(CO2) could be correlated to in vitro measurements of electron transport rate. These results are consistent with the hypothesis that CO2 assimilation rate is limited by the RuP2 saturated rate of the RuP2 carboxylase-oxygenase at low intercellular p(CO2) and by the rate allowed by RuP2 regeneration capacity at high intercellular p(CO2).
The aim of this work was to see if amyloplasts contained inorganic pyrophosphatase. Alkaline pyrophosphatase activity, largely dependant upon MgCl2 but not affected by 100 μM ammonium molybdate or 60-100 mM KCl, was demonstrated in exracts of developing and mature clubs of the spadix of Arum maculatum L. and of suspension cultures of Glycine max L., but not in extracts of the developing bulb of Allium cepa L. The maximum catalytic activity of alkaline pyrophosphatase in the above tissues showed a positive correlation with starch synthesis, and in the first two tissues was shown to exceed the activity of ADPglucose pyrophosphorylase. Of the alkaline pyrophosphatase activity in lysates of protoplasts of suspension cultures of Glycine max, 57% was latent. Density-gradient centrifugation of these lysates showed a close correlation between the distribution of alkaline pyrophosphatase and the plastid marker, nitrite reductase. It is suggested that much, if not all, of the alkaline pyrophosphatase in suspension cultures of Glycine max is located in the plastids.
This work provides further evidence that plants contain appreciable amounts of inorganic pyrophosphate (PPi), and that breakdown of phosphoribosyl pyrophosphate (PPRibP) does not contribute significantly to the PPi detected in plant extracts. Inorganic pyrophosphate in extracts of the roots of Pisum sativum L., clubs of the spadices of Arum maculatum L., and the developing endosperm of Zea mays L. was assayed with pyrophosphate fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90), and with sulphate adenyltransferase (EC 2.7.7.4). The two different assays gave the same value for PPi content, and for recovery of added PPi. It was shown that PPRibP is converted to PPi during the extraction of PPi. However, the amounts of PPRibP in clubs of A. maculatum and the developing endosperm of Z. mays were negligible in comparison with the contents of PPi.
Metabolite levels and carbohydrates were investigated in the leaves of tobacco (Nicotiana tabacum L.) and leaves and tubers of potato (Solanum tuberosum L.) plants which had been transformed with pyrophosphatase from Escherichia coli. In tobacco the leaves contained two- to threefold less pyrophosphate than controls and showed a large increase in UDP-glucose, relative to hexose phosphate. There was a large accumulation of sucrose, hexoses and starch, but the soluble sugars increased more than starch. Growth of the stem and roots was inhibited and starch, sucrose and hexoses accumulated. In potato, the leaves contained two- to threefold less pyrophosphate and an increased UDP-glucose/ hexose-phosphate ratio. Sucrose increased and starch decreased. The plants produced a larger number of smaller tubers which contained more sucrose and less starch. The tubers contained threefold higher UDP-glucose, threefold lower hexose-phosphates, glycerate-3-phosphate and phosphoenolpyruvate, and up to sixfold more fructose-2,6-bisphosphatase than the wild-type tubers. It is concluded that removal of pyrophosphate from the cytosol inhibits plant growth. It is discussed how these results provide evidence that sucrose mobilisation via sucrose synthase provides one key site at which pyrophosphate is needed for plant growth, but is certainly not the only site at which pyrophosphate plays a crucial role.
Previous studies provided evidence that the carbohydrate status triggers developmental processes in the growing cotyledons of Vicia faba. We describe here the high-resolution mapping of glucose concentrations in tissue sections of developing faba bean cotyledons by quantitative bioluminescence and single-photon imaging. Patterns of local glucose distributions are compared with tissue cell type, mitotic index and the distribution pattern of starch. During cotyledon differentiation, gradients in the glucose concentration emerge which are related to the particular cell type. Higher concentrations are found in non-differentiated premature regions of the cotyledon whereas mature starch-accumulating regions contain particularly low concentrations of glucose. In addition, glucose concentration is correlated to mitotic activity. The glucose distribution pattern is therefore related to the developmental gradient. Our data provide for the first time evidence for steep glucose gradients across developing plant embryos and favour the idea that sugar gradients may have morphogenic functions in developing cotyledons.
The subcellular localisation of pyrophosphate and alkaline pyrophosphatase in leaves has been studied using non-aqueous density gradient centrifugation of spinach leaves, and membrane filtration of wheat mesophyll protoplasts. The pyrophosphate was measured in extracts prepared in trichloroacetic acid, and could be quantitatively recovered from the leaf material. It was located predominantly in the cytosol, with a concentration of 0.2–0.3 mM. In contrast, the alkaline pyrophosphatase was largely, if not, exclusively, located in the chloroplast. By comparing the pyrophosphate levels in the cytosol with previously published data on the cytosolic levels of phosphate and metabolic intermediates, it is shown that the reactions catalysed by pyrophosphate: fructose-6-phosphate phosphotransferase and UDP-glucose pyrophosphorylase are close to the thermodynamic equilibrium and, thus, freely reversible in vivo. Comparison of the pyrophosphate levels with the reported electrical and pH gradient across the tonoplast membrane shows the free energy released during pyrophosphate hydrolysis is similar to that required to move a proton across the tonoplast membrane. It is suggested that pyrophosphate could operate as a secondary energy donor in the cytosol of plant cells.
Publisher Summary This chapter focuses on the metabolite levels in specific cells and the subcellular compartments of plant leaves. Leaf material is difficult to fractionate because a typical plant cell contains several different, mechanically fragile subcellular organelles and is surrounded by a mechanically strong plant cell wall. Rather than attempting to isolate whole organelles or cells, leaves are frozen in liquid N 2 and then broken to small fragments that are enriched in material from a given compartment. These fragments are physically separated under conditions when the metabolic activity or redistribution of metabolites is prevented and subsequently their metabolism is quenched. The exclusion of water, or the use of extremely low temperatures, prevents metabolic activity during the fractionation procedures. The chapter describes the silicone oil centrifugation that allows chloroplasts to be separated from the remainder of the protoplast and to be quenched within 2–3 sec of disrupting the protoplast.
The vacuolar membrane (tonoplast) of plant cells contains two functionally and physically distinct phosphohydrolases, which catalyse electrogenic H+ -translocation: An ATPase (tp-ATPase; EC 3.6.1.3) and an inorganic pyrophosphatase (tp-PPase; 3.6.1.1). Neither enzyme belongs to the F0F1– or E1E2-categories of primary cation pumps, but instead belong to a third and fourth category of enzyme, respectively. Research priorities for the tp-ATPase are studies directed at understanding the roles of the 70 and 60 kDa subunits in catalysis and regulation; the involvement of the 16 kDa subunit in transmembrane H+ conduction; and investigations of F0F1- like structure/function partitioning. In the longer term, comparisons of sequence homology between the N,N′- dicyclohexylcarbodiimide -binding (16 kDa) proteins from different sources may enable elucidation of the evolutionary relationship of the tp-ATPase with other putative third-category H+– translocases. The tp-PPase, on the other hand, represents an exciting but largely unexplored biochemical entity, which necessitates a reconsideration of accepted views concerning the involvement of inorganic pyrophosphate (PPi) in transmembrane energy conservation. Just why the tonoplast should be endowed with two H+-translocases is a problem that can only be approached once consideration is given to the paramount question of H+/PPi stoichiometry. Once the stoichiometry is known, it should be possible to establish the physiological poise of the tp-PPase, and hence to speculate on its role in the metabolism of plant cells.
Transformation of tobacco with the potato gene encoding the subunit of pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) in the antisense orientation under the control of the constitutive CaMV 35S promoter, followed by selfing and crossing of the transformants, generated a line of tobacco (5–37) with up to an 85% reduction in PFP activity in the shoot. Transformants containing a sense construct (4-40-91) contained only 1–3% of wild-type PFP, presumably due to co-suppression. Rates of photosynthesis and partitioning between sucrose and starch in source leaves were identical in 4-40-91 transformants and the wild type. In the dark in sink leaves of 4-40-91 transformants, levels of hexose phosphates were up to 50% higher, glycerate-3-phosphate 30% lower and fructose-2,6-bisphosphate threefold higher than in the wild type; inorganic pyrophosphate, pyruvate and the ATP/ADP ratio were unaltered. Low -PFP and wild-type plants did not differ significantly in their rate of growth at 25 C and 200 mol quanta m–2 s–1 on full nutrient medium. Growth on limiting phosphate and limiting nitrogen was inhibited identically in the wild type and transformants, and transformants adjusted their shoot/root ratio in an identical manner to the wild type. Differences in fructose-2,6-bisphosphate and glycolytic metabolites between the wild type and transformants were no larger in these suboptimal nutrient conditions, than in optimal conditions. Growth of the wild type and 4-40-91 transformants was inhibited identically at 12 C compared to 25 C. Differences in fructose-2,6-bisphosphate were smaller when the genotypes were compared at 12 C than at 25 C. We conclude that PFP does not play an essential role in photosynthate partitioning in source leaves. During respiratory metabolism in sink leaves it catalyzes a net glycolytic flux, as in potato tubers. However, tobacco seedlings are able to compensate for a large decrease in expression of PFP without loss of growth, or the ability to cope with suboptimal phosphate, nitrogen or temperature.
Potato (Solanum tuberosum L.) plants were transformed with antisense constructs to the genes encoding the -and -subunits of pyrophosphate: fructose-6-phosphate phosphotransferase (PEP), their expression being driven by the constitutive CaMV 35S promotor. (i) In several independent transformant lines, PFP expression was decreased by 70–90% in growing tubers and by 88–99% in stored tubers. (ii) The plants did not show any visual phenotype, reduction of growth or decrease in total tuber yield. However, the tubers contained 20–40% less starch than the wild type. Sucrose levels were slightly increased in growing tubers, but not at other stages. The rates of accumulation of sucrose and free hexoses when tubers were stored at 4 C and the final amount accumulated were the same in antisense and wild-type tubers. (iii) Metabolites were investigated at four different stages in tuber life history; growing (sink) tubers, mature tubers, cold-sweetening tubers and sprouting (source) tubers. At all stages, compared to the wild type, antisense tubers contained slightly more hexose-phosphates, two- to threefold less glycerate-3-phosphate and phosphoenolpyruvate and up to four-to fivefold more fructose-2,6-bisphosphate. (iv) There was no accumulation or depletion of inorganic pyrophosphate (PPi), or of UDP-glucose relative to the hexose-phosphates. (v) The pyruvate content was unaltered or only marginally decreased, and the ATP/ADP ratio did not change. (vi) Labelling experiments on intact tubers did not reveal any significant decrease in the unidirectional rate of metabolism of [U-14C]sucrose to starch, organic acids or amino acids. Stored tubers with an extreme (90%) reduction of PFP showed a 25% decrease in the metabolism of [U14-C] sucrose. (vii) Metabolism (cycling) of [U-14C]glucose to surcrose increased 15-fold in discs from growing antisense tubers, compared with growing wild-type tubers. Resynthesis of sucrose was increased by 10–20% when discs from antisense and wild-type tubers stored at 4 C (cold sweetening) were compared. The conversion of [U-14C]glucose to starch was decreased by about 30% and 50%, respectively. (viii) The randomisation of [1-13C]glucose in the glucosyl and fructosyl moieties of sucrose was decreased from 13.8 and 15.7% in the wild type to 3.6 and 3.9% in an antisense transformant. Simultaneously, randomisation in glucosyl residues isolated from starch was reduced from 14.4 to 4.1%. (ix) These results provide evidence that PFP catalyses a readily reversible reaction in tubers, which is responsible for the recycling of label from triose-phosphates to hexose-phosphates, but with the net reaction in the glycolytic direction. The results do not support the notion that PFP is involved in regulating the cytosolic PPi concentration. They also demonstrate that PFP does not control the rate of glycolysis, and that tubers contain exessive capacity to phosphorylate fructose-6-phosphate. The decreased concentration of phosphoenolpyruvate and glycerate-3-phosphate compensates for the decrease of PFP protein by stimulating ATP-dependent phosphofructokinase, and by stimulating fructose-6-phosphate,2-kinase to increase the fructose-2,6-bisphosphate concentration and activate the residual PFP. The decreased starch accumulation is explained as an indirect effect, caused by the increased rate of resynthesis (cycling) of sucrose in the antisense tubers.
Tobacco (Nicotiana tabacum L.) plants were transformed with an antisense construct of the chloroplast triose phosphate/phosphate translocator (TPT). Three transformant lines of the T4 progeny, which showed a large decrease in the transcript level of the TPT were used for further biochemical and physiological characterisation. In all antisense lines tested, TPT transport activity was diminished by 50-70% compared with the wild type (WT). Despite this high reduction in the transport capacity, alpha TPT plants lacked any visible phenotype. Hexokinase and alpha-amylase activities were increased in alpha TPT plants compared with the WT, whereas activities of ribulose-1,5-bisphosphate carboxylase/oxygenase and ADP-glucose pyrophosphorylase (AGPase) were not affected. At the end of a 14-h light period, leaf starch contents in alpha TPT lines were similar to those of the WT and controls, indicating that a decrease in the TPT had no effect on starch accumulation. Sucrose contents were diminished by more than 50% in alpha TPT lines compared with control plants. The time course of starch accumulation revealed a transient increase in the starch content in a selected alpha TPT line after 6 h in the light, followed by a decrease towards the end of the light period. Labelling with 14C indicated that during the dark and light (late afternoon) periods starch is mobilised at higher rates in alpha TPT lines than in the controls. Glucose/fructose ratios at the end of the dark period were increased from 1.2 in control plants to 2 in alpha TPT lines indicating increased amylolytic starch degradation. Initial rates of [14C] glucose transport in isolated chloroplasts were increased by a factor of 2-3 in alpha TPT plants compared with the WT. Rates of CO2 assimilation were substantially diminished in the alpha TPT lines in high CO2 and low O2, but remained unaffected in ambient CO2. The rate of photosynthetic electron transport during the induction of photosynthesis in saturating CO2 exhibited pronounced oscillations only in WT and control plants. Oscillations were less pronounced in alpha TPT plants, indicating that phosphate limitation of photosynthesis is lowered in alpha TPT plants compared with the WT. It is proposed that photoassimilates are more readily directed into starch biosynthesis in alpha TPT plants. This is supported by determinations of 3-phosphoglycerate levels (an activator of AGPase) during the transition from dark to light in high CO2.
Transgenic plants were constructed expressing a novel cytosolic inorganic pyrophosphatase in order to reduce the cytosolic pyrophosphate content. To this end the Escherichia coli gene ppa encoding inorganic pyrophosphatase was cloned between the 35S CaMV promoter and the poly(A) site of the octopine synthase gene and transferred into tobacco and potato plants by Agrobacterium-mediated gene transfer. Regenerated plants were tested for the expression of the ppa gene by Northern blots and activity gels. Plants expressing active inorganic pyrophosphatase showed a dramatic change in photoassimilate partitioning. In both transgenic tobacco and potato plants the ratio between soluble sugars and starch was increased by about 3–4-fold in source leaves as compared with the wild-type. However, whereas source leaves of transgenic tobacco plants accumulated much higher levels of glucose (up to 68-fold), fructose (up to 24-fold), sucrose (up to 12-fold) and starch (up to 8-fold) this was not observed in potato plants where the change in assimilate partitioning in source leaves was due to an increase of about 2-fold in sucrose and a reduction in starch content.
Expression of the cytosolic inorganic pyrophosphatase in tobacco results in stunted growth of vegetatively growing plants due to a reduced internode distance. Upon flowering the transgenic plants increase their growth rate, reaching almost the same height as control plants at the end of the growth period. Old source leaves accumulate up to 100-fold more soluble sugars than control leaves. This increase in soluble sugars is accompanied by a reduction in chlorophyll content (up to 85%).
Transgenic potato plants showed a less dramatic change in their growth behaviour. Plants were slightly reduced in size, with stems more highly branched. Tuber number increased 2–3-fold, but tuber weight was lower resulting in no net increase in fresh weight.
High-voltage electroporation was used to transform Agrobacterium tumefaciens strains A136 and A348, reaching the efficiency of 1-3 x 10(8) transformants/micrograms DNA. Transformation frequency was dependent on the electrical field strength and the pulse length. No significant reduction in transformation efficiency was observed when the transforming DNA contained sites sensitive to endonuclease AtuCI of A. tumefaciens.
A new and convenient method for the determination of Pi was developed. Phosphomolybdate is measured colorimetrically, without reduction to molybdenum blue, by dissolving the whole assay mixture in acetone, where phosphomolybdate is bright yellow. The hydrolysis of acid-labile phosphates (e.g., creatine phosphate) causes no problems, because extra molybdate is complexed with citrate immediately after the color has been developed. Strong reductants and SH compounds which interfere, if present in high concentrations, are eliminated by adding H2O2. Detergents, organic bases, protein, and sucrose do not interfere. The assay is as sensitive as most modifications of the Fiske-SubbaRow method. In the routine procedure the useful range is 50–1500 nmol of Pi. The application of the method to the assay of inorganic pyrophosphatase in the cells of Escherichia coli is described.
The expression of the enzyme UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) from potato (Solanum tuberosum L.) was analysed with respect to sink-source interactions and potato tuber storage. The highest level of expression was found in developing tubers, the strongest sink tissue. Storage of mature tubers at low temperatures led to an increase of the steady-state level of UGPase mRNA, implicating a role of this enzyme in the process of "cold-sweetening". Transgenic plants were created expressing UGPase antisensee RNA under the control of the 35S promoter of the Cauliflower Mosaic Virus with the polyadenylation signal of the octopine-synthase gene. Regenerated plants were tested for reduction of UGPase at the RNA, protein and activity levels. Plants with a 95%-96% reduction of UGPase activity in growing tubers showed no change in growth and development. Also, carbohydrate metabolism in tubers of these plants was not substantially affected, indicating that only 4% of the wild-type UGPase activity is sufficient for the enzyme to function in plant growth and development.
The new pPZP Agrobacterium binary vectors are versatile, relatively small, stable and are fully sequenced. The vectors utilize the pTiT37 T-DNA border regions, the pBR322 bom site for mobilization from Escherichia coli to Agrobacterium, and the ColE1 and pVS1 plasmid origins for replication in E. coli and in Agrobacterium, respectively. Bacterial marker genes in the vectors confer resistance to chloramphenicol (pPZP100 series) or spectinomycin (pPZP200 series), allowing their use in Agrobacterium strains with different drug resistance markers. Plant marker genes in the binary vectors confer resistance to kanamycin or to gentamycin, and are adjacent to the left border (LB) of the transferred region. A lacZ alpha-peptide, with the pUC18 multiple cloning site (MCS), lies between the plant marker gene and the right border (RB). Since the RB is transferred first, drug resistance is obtained only if the passenger gene is present in the transgenic plants.
A cDNA clone with sequence homology to soluble inorganic pyrophosphatase (IPPase) was isolated from a library of developing barley grains. The protein encoded by this clone was produced in transgenic Escherichia coli, and showed IPPase activity. In nondormant barley grains, the gene appeared to be expressed in metabolically active tissue such as root, shoot, embryo and aleurone. During inhibition, a continuous increase of the steady state mRNA level of IPPase was observed in embryos of non-dormant grains. In the embryos of dormant grains its production declined, after an initial increase. With isolated dormant and nondormant embryos, addition of recombinant IPPase, produced by E. coli, enhanced the germination rate. On the other hand, addition of pyrophosphate (PPi), substrate for this enzyme, appeared to reduce the germination rate. A role for this IPPase in germination is discussed.
Overexpression of inorganic pyrophosphatase (PPase) from Escherichia coli in the cytosol of plants (ppa 1 plants) leads to a decrease of inorganic pyrophosphate (PPi; U. Sonnewald, 1992, Plant J 2: 571-581). The consequences for sucrose-starch interconversions have now been studied in growing potato (Solanum tuberosum L. cv. Desirée) tubers. Sucrose is degraded via sucrose synthase and UDP-glucose pyrophosphorylase in growing tubers, and it was expected that the low PPi in the ppa 1 transformants would restrict the mobilisation of sucrose and conversion to starch. Over-expression of PPase resulted in an accumulation of sucrose and UDP-glucose, and decreased concentrations of hexose phosphates and glycerate-3-phosphate in growing ppa 1 tubers. Unexpectedly, the rate of degradation of [14C] sucrose was increased by up to 30%, the rate of starch synthesis was increased, and the starch content was increased by 20-30% in ppa 1 tubers compared to wild-type tubers. Reasons for this unexpectedly efficient conversion of sucrose to starch in the ppa 1 tubers were investigated. (i) The transformed tubers contained increased activities of several enzymes required for sucrose-starch interconversions including two- to three-fold more sucrose synthase and 60% more ADP-glucose pyrophosphorylase. They also contained 30-100% increased activities of several glycolytic enzymes and amylase, increased protein, and unaltered or slightly decreased starch phosphorylase, acid invertase and mannosidase. (ii) The transformants contained higher pools of uridine nucleotides. As a result, although the UDP-glucose pool is increased two- to threefold, this does not lead to a decrease of UTP or UDP. (iii) The transformants contained twofold larger pools of ATP and ADP, and ADP-glucose was increased by up to threefold. In stored ppa 1 tubers, there were no changes in the activities of glycolytic enzymes, and nucleotides did not increase. It is concluded that in growing tubers PPi has a wider-significance than just being an energy donor for specific reactions in the cytosol. Increased rates of PPi hydrolysis also affect general aspects of cell activity including the levels of nucleotides and protein. Possible ways in which PPi hydrolysis could affect these processes are discussed.
The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
The role of pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) in developing leaves was studied using wild-type tobacco (Nicotiana tabacum L.) and transformants with decreased expression of PFP. (i) The leaf base, which is the youngest and most actively growing area of the leaf, had 2.5-fold higher PFP activity than the leaf tip. T3 transformants, with a 56-95% decrease in PFP activity in the leaf base and an 87-97% decrease in PFP activity in the leaf tip, were obtained by selfing and re-selfing individuals from two independent transformant lines. (ii) Other enzyme activities also showed a gradient from the leaf base to the leaf tip. There was a decrease in PFK and an increase in fructose-6-phosphate,2-kinase and plastidic fructose-1, 6-bisphosphatase, whereas cytosolic fructose-1,6-bisphosphatase activity was constant. None of these gradients was altered in the transformants. (iii) Fructose-2,6-bisphosphate (Fru2,6bisP) levels were similar at the base and tip of wild-type leaves in the dark. Illumination lead to a decrease in Fru2,6bisP at the leaf tip and an increase in Fru2,6bisP at the leaf base. Compared to wild-type plants, transformants with decreased expression of PFP had up to 2-fold higher Fru2,6bisP at the leaf tip in the dark, similar levels at the leaf tip in the light, 15-fold higher levels at the leaf base in the dark, and up to 4-fold higher levels at the leaf base in the light. (iv) To investigate metabolic fluxes, leaf discs were supplied with 14CO2 in the light or [14C]glucose in the light or the dark. Discs from the leaf tip had higher rates of photosynthesis than discs from the leaf base, whereas the rate of glucose uptake and metabolism was similar in both tissues. Significantly less label was incorporated into neutral sugars, and more into anionic compounds, cell wall and protein, and amino acids in discs from the leaf base. Metabolism of 14CO2 and [14C]glucose in transformants with low PFP was similar to that in wild-type plants, except that synthesis of neutral sugars from 14CO2 was slightly reduced in discs from the base of the leaf. (v) These results reveal that the role of PFP in the growing cells in the base of the leaf differs from that in mature leaf tissue. The increase in Fru2,6bisP in the light and the high activity of PFP relative to cytosolic fructose-1,6-bisphosphatase in the base of the leaf implicate PFP in the synthesis of sucrose in the light, as well as in glycolysis. The large increase in Fru2,6bisP at the base of the leaf of transformants implies that PFP plays a more important role in metabolism at the leaf base than in mature leaf tissue. Nevertheless, there were no major changes in carbon fluxes, or leaf or plant growth in transformants with below 10% of the wild-type PFP activity at the leaf base, implying that large changes in expression can be compensated by changes in Fru2,6-bisP, even in growing tissues.
Phosphorus deficiency is one of the major abiotic stresses affecting plant growth. Plants respond to the persistent deficiency of phosphate (Pi) by coordinating the expression of genes involved in alleviation of the stress. The high-affinity Pi transporters are among the major molecular determinants that are activated during Pi stress. In this study, using three reporter genes (green fluorescent protein, luciferase, and beta-glucuronidase) regulated by two Pi transporter promoters, we have carried out an extensive analysis of transcriptional and spatial regulation of gene expression. Activation of the genes was rapid, repressible, and specific in response to changes in Pi availability. The phytohormones auxin and cytokinin suppressed the expression of the reporter gene driven by the AtPT1 promoter, and that of the native gene, suggesting that hormones may be involved in regulation of some component(s) of Pi starvation response pathway. These studies also provide molecular evidence for a potential role of high-affinity Pi transporters in mobilizing Pi into reproductive organs. The results suggest that members of the Pi transporter family may have similar but nonredundant functions in plants.
Growing potato tubers have been used as a model system to investigate the regulation of starch synthesis. Results indicate that sucrose degradation and starch synthesis are controlled via regulatory signals in response to sucrose and oxygen availability. (i) Sucrose leads to a co-ordinated up-regulation of sucrose synthase and ADP-glucose phosphorylase at the transcriptional and post-transcriptional level. Transcriptional regulation of ADP-glucose phosphorylase leads to rapid changes in transcript levels, but relatively slow changes in protein levels. The rapid regulation of this enzyme in response to sucrose is mediated by a novel mechanism, involving redox-activation of ADPGlc pyrophosphorylase. Sucrose synthase is regulated via transcriptional regulation, but again the resulting changes in enzyme activity occur relatively slowly. More rapid changes in the flux of this enzyme follow due to rapid changes in the levels of uridine nucleotides. (ii) Internal oxygen concentrations fall to low levels in growing tubers, triggering a restriction of respiration, a decrease in the adenylate energy status, and a widespread decrease in metabolic and biosynthetic activity. These metabolic adaptations will allow oxygen consumption to be decreased and prevent the tissue from becoming anoxic. It will be discussed how these factors interact at different levels and different time-scales of control to regulate tuber metabolism in response to physiological and environmental inputs.
Analysis of the Arabidopsis genome revealed the complete set of plastidic phosphate translocator (pPT) genes. The Arabidopsis genome contains 16 pPT genes: single copies of genes coding for the triose phosphate/phosphate translocator and the xylulose phosphate/phosphate translocator, and two genes coding for each the phosphoenolpyruvate/phosphate translocator and the glucose-6-phosphate/phosphate translocator. A relatively high number of truncated phosphoenolpyruvate/phosphate translocator genes (six) and glucose-6-phosphate/phosphate translocator genes (four) could be detected with almost conserved intron/exon structures as compared with the functional genes. In addition, a variety of PT-homologous (PTh) genes could be identified in Arabidopsis and other organisms. They all belong to the drug/metabolite transporter superfamily showing significant similarities to nucleotide sugar transporters (NSTs). The pPT, PTh, and NST proteins all possess six to eight transmembrane helices. According to the analysis of conserved motifs in these proteins, the PTh proteins can be divided into (a) the lysine (Lys)/arginine group comprising only non-plant proteins, (b) the Lys-valine/alanine/glycine group of Arabidopsis proteins, (c) the Lys/asparagine group of Arabidopsis proteins, and (d) the Lys/threonine group of plant and non-plant proteins. None of these proteins have been characterized so far. The analysis of the putative substrate-binding sites of the pPT, PTh, and NST proteins led to the suggestion that all these proteins share common substrate-binding sites on either side of the membrane each of which contain a conserved Lys residue.
Metabolite translocators in the inner membrane of the plastid envelope are the interface between cytosolic and plastidial metabolism. Hence, they integrate plastidial pathways, such as photosynthesis, starch biosynthesis, the oxidative pentose phosphate pathway and the shikimate pathway, into the metabolic network of plant cells. Metabolite transporters not only catalyze the flux of metabolites between compartments but also represent information pathways that communicate the metabolic status of the various compartments within plant cells. Recently, a pentose-phosphate translocator was shown to be a novel member of the phosphate translocator protein family. Furthermore, a protein of previously unknown function was identified as a novel type of maltose transporter, and a glutamate/malate translocator that is involved in photorespiration was discovered. In addition, the pathway for maltose metabolism in the cytosol has been unraveled.
Decreased sucrose content triggers starch breakdown and respiration in stored potato tubers (Solanum tuberosum) Compen-sation of decreased triose phosphate/phosphate translocator activity by accelerated starch turnover and glucose transport in transgenic tobacco
- M F Bornke
- M Peisker
- Y Takahata
- J Lerchl
- A Kirakosyan
- Sonnewald
M, Bornke F, Peisker M, Takahata Y, Lerchl J, Kirakosyan A, Sonnewald U (2003) Decreased sucrose content triggers starch breakdown and respiration in stored potato tubers (Solanum tuberosum). J Exp Bot 4:477–488 Hä RE, Schlieben NH, Schulz B, Flü UI (1998) Compen-sation of decreased triose phosphate/phosphate translocator activity by accelerated starch turnover and glucose transport in transgenic tobacco. Planta 204:366–376