Influence of Elevated Fructose-2,6-Bisphosphate Levels on Starch Mobilization in Transgenic Tobacco Leaves in the Dark

Scott, Peter C.; Kruger, Nicholas J.

doi:10.1104/pp.108.4.1569

Cited by 36 publications

(19 citation statements)

References 21 publications

(25 reference statements)

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“…In the dark, the transformants with the largest decrease in PFP expression contained 2-to 3-fold more Fru2,6bisP than wild-type plants, suggesting that the reaction catalyzed by PFP is going in the glycolytic direction. A role for PFP in glycolysis in leaves in the dark is also indicated by the observation that leaves of transgenic tobacco plants with increased levels of Fru-2,6-bisP retained a higher level of starch during the night period (Scott and Kruger 1995), and it is in agreement with recent data suggesting that carbon is exported from the chloroplast as hexose units rather than triose-phosphates during the night (Schleucher et al 1998;Weber et al 2000). However, when labelled glucose was supplied in the dark to the leaf tips, the transformants only showed a slight decrease in the labelling of sucrose and starch, and a slight increase in amino acids, and acidic compounds, indicating that glycolysis has not been inhibited.…”

Section: Developmental and Metabolic Gradients In Young Tobacco Leavessupporting

confidence: 51%

Tobacco transformants with strongly decreased expression of pyrophosphate:fructose-6-phosphate expression in the base of their young growing leaves contain much higher levels of fructose-2,6-bisphosphate but no major changes in fluxes

Nielsen¹,

Stitt²

2001

Planta

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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.

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Section: Developmental and Metabolic Gradients In Young Tobacco Leavessupporting

confidence: 51%

Tobacco transformants with strongly decreased expression of pyrophosphate:fructose-6-phosphate expression in the base of their young growing leaves contain much higher levels of fructose-2,6-bisphosphate but no major changes in fluxes

Nielsen¹,

Stitt²

2001

Planta

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“…Important regulatory mechanisms such as the fructose 2, 6-bisphosphatemediated feedforward and feedback regulation of sucrose synthesis have been uncovered (see Stitt 1990;Winter and Huber 2000). This regulation was firmly established by the accumulation of data ranging from correlative evidence through in vitro kinetic studies of the constitutive enzymes of the metabolic network (see Stitt 1990 for a review), to direct analysis of these enzymes in vivo via the use of transgenics (Scott and Kruger 1995;Scott et al 2000;Draborg et al 2001). A similar range of experimental approaches has also been employed in the study of diurnal regulation of carbonnitrogen interactions (Scheible et al 2000;MasclauxDaubresse et al 2002;Carrari et al 2003a).…”

Section: Introductionmentioning

confidence: 98%

Profiling of diurnal patterns of metabolite and transcript abundance in potato (Solanum tuberosum) leaves

Urbańczyk-Wochniak

Baxter

Kolbe

et al. 2005

Planta

118

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Diurnal changes in carbohydrates and a broad range of primary metabolites were analysed through a diurnal period in potato leaves (Solanum tuberosum cv. Desiree) using an established gas chromatography-mass spectrometry based metabolic profiling protocol alongside conventional spectrophotometric technologies. In tandem, we profiled transcript levels using both a custom array containing approximately 2,500 cDNA clones predominantly representing transcripts involved in primary metabolism and commercially available arrays containing approximately 12,000 cDNA clones that gave coverage of transcript levels over a broader functional range. The levels of many metabolites and transcripts varied during the diurnal period with 56 significant differences observed in the metabolite contents and 832 significant differences recorded in transcript levels. Whilst a large number of the differences would be expected from what has been known previously, several novel changes were observed in these experiments. Notably, qualitative comparison of the combined data sets obtained from the parallel analysis of transcripts and metabolites suggests relatively few changes in gene expression strongly correlate with changes in metabolite levels during a diurnal cycle. Furthermore, these changes appear to be confined to the central metabolic pathways. However, principal component analysis of the metabolic profiles obtained here revealed that metabolite patterns change progressively through a diurnal period suggesting the operation of mechanisms for tight temporal regulation of metabolite composition.

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“…The aqueous basic fraction, consisting of amino acids, was freeze dried, resuspended in 1 mL of water, and fractionated using an anion-exchange column to separate acidic amino acids from neutral/basic amino acids (Cossins and Beevers, 1963). Each neutral fraction was freeze dried, resuspended in 1 mL of water, and separated into Suc, Glc, and Fru using thin-layer chromatography (Scott and Kruger, 1995).…”

Section: Fractionation Of Methanolic Cell Extractsmentioning

confidence: 99%

Subcellular Flux Analysis of Central Metabolism in a Heterotrophic Arabidopsis Cell Suspension Using Steady-State Stable Isotope Labeling

et al. 2009

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The presence of cytosolic and plastidic pathways of carbohydrate oxidation is a characteristic feature of plant cell metabolism. Ideally, steady-state metabolic flux analysis, an emerging tool for creating flux maps of heterotrophic plant metabolism, would capture this feature of the metabolic phenotype, but the extent to which this can be achieved is uncertain. To address this question, fluxes through the pathways of central metabolism in a heterotrophic Arabidopsis (Arabidopsis thaliana) cell suspension culture were deduced from the redistribution of label in steady-state 13 C-labeling experiments using [1-13 C]-, [2-13 C]-, and [U- 13C 6 ]glucose. Focusing on the pentose phosphate pathway (PPP), multiple data sets were fitted simultaneously to models in which the subcellular compartmentation of the PPP was altered. The observed redistribution of the label could be explained by any one of three models of the subcellular compartmentation of the oxidative PPP, but other biochemical evidence favored the model in which the oxidative steps of the PPP were duplicated in the cytosol and plastids, with flux through these reactions occurring largely in the cytosol. The analysis emphasizes the inherent difficulty of analyzing the PPP without predefining the extent of its compartmentation and the importance of obtaining high-quality data that report directly on specific subcellular processes. The Arabidopsis flux map also shows that the potential ATP yield of respiration in heterotrophic plant cells can greatly exceed the direct metabolic requirements for biosynthesis, highlighting the need for caution when predicting flux through metabolic networks using assumptions based on the energetics of resource utilization.Extensive subcellular compartmentation, with unique locations for many steps and pathways, as well as the duplication of other steps and pathways in different compartments, adds greatly to the structural complexity of the plant metabolic network (Lunn, 2007;. The need to consider discrete pools of metabolites in specific compartments, and the transporters that link them, complicates the quest for a detailed, predictive understanding of the regulation of plant metabolism; as a result, it remains difficult to manipulate flows of material through the central metabolic network in a predictable way (Carrari et al., 2003;Sweetlove et al., 2008).The emergence of steady-state metabolic flux analysis (MFA) as a practicable systems biology tool for generating flux maps of the central metabolic pathways in plants offers new opportunities for analyzing plant metabolic phenotypes (Ratcliffe and ShacharHill, 2006;Libourel and Shachar-Hill, 2008;Schwender, 2008;Kruger and Ratcliffe, 2009). In this approach, substrates labeled with stable isotopes are introduced into the network, and fluxes are determined by measuring the labeling of the system after it has reached an isotopic and metabolic steady state. Subcellular compartmentation of steps and pathways can be incorporated into the model that describes the redistribution of t...

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Influence of Elevated Fructose-2,6-Bisphosphate Levels on Starch Mobilization in Transgenic Tobacco Leaves in the Dark

Cited by 36 publications

References 21 publications

Tobacco transformants with strongly decreased expression of pyrophosphate:fructose-6-phosphate expression in the base of their young growing leaves contain much higher levels of fructose-2,6-bisphosphate but no major changes in fluxes

Tobacco transformants with strongly decreased expression of pyrophosphate:fructose-6-phosphate expression in the base of their young growing leaves contain much higher levels of fructose-2,6-bisphosphate but no major changes in fluxes

Profiling of diurnal patterns of metabolite and transcript abundance in potato (Solanum tuberosum) leaves

Subcellular Flux Analysis of Central Metabolism in a Heterotrophic Arabidopsis Cell Suspension Using Steady-State Stable Isotope Labeling

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