Independent changes of inorganic pyrophosphate and the AT/ADP or UTP/UDP ratios in plant cell suspension cultures

Dancer, Jane E.; Veith, Robert; Feil, Regina; Komor, Ewald; Stitt, Mark

doi:10.1016/0168-9452(90)90169-o

Cited by 66 publications

(34 citation statements)

References 12 publications

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“…Combining this K eq value with literature data on UDP-glucose/ UDP ratios in plant cytosol allows calculation of the cytosolic pABA-Glc/pABA-Glc ratio. Cytosolic UDP-glucose/UDP ratios in plant tissues are generally 10 -20, the value reported for leaves being 13 (32)(33)(34). Using the value of 13 and a K eq value of 3 ϫ 10 Ϫ3 gives a pABA-Glc/pABA ratio of 0.04, so that 96% of the total pABA in the cytosol is predicted to be free and only 4% esterified.…”

Section: Discussionmentioning

confidence: 99%

Metabolism of the Folate Precursor p-Aminobenzoate in Plants

Eudes

Bozzo

Waller

et al. 2008

Journal of Biological Chemistry

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Plants produce p-aminobenzoate (pABA) in chloroplasts and use it for folate synthesis in mitochondria. In plant tissues, however, pABA is known to occur predominantly as its glucose ester (pABA-Glc), and the role of this metabolite in folate synthesis has not been defined. In this study, the UDP-glucose:pABA acylglucosyltransferase (pAGT) activity in Arabidopsis extracts was found to reside principally (95%) in one isoform with an apparent K m for pABA of 0.12 mM. Screening of recombinant Arabidopsis UDP-glycosyltransferases identified only three that recognized pABA. One of these (UGT75B1) exhibited a far higher k cat /K m value than the others and a far lower apparent K m for pABA (0.12 mM), suggesting its identity with the principal enzyme in vivo. Supporting this possibility, ablation of UGT75B1 reduced extractable pAGT activity by 95%, in vivo [ 14 C]pABA glucosylation by 77%, and the endogenous pABAGlc/pABA ratio by 9-fold. The K eq for the pABA esterification reaction was found to be 3 ؋ 10 ؊3 . Taken with literature data on the cytosolic location of pAGT activity and on cytosolic UDPglucose/UDP ratios, this K eq value allowed estimation that only 4% of cytosolic pABA is esterified. That pABA-Glc predominates in planta therefore implies that it is sequestered away from the cytosol and, consistent with this possibility, vacuoles isolated from [ 14 C]pABA-fed pea leaves were estimated to contain >88% of the [ 14 C]pABA-Glc formed. In total, these data and the fact that isolated mitochondria did not take up [ 3 H]pABAGlc, suggest that the glucose ester represents a storage form of pABA that does not contribute directly to folate synthesis.

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Section: Discussionmentioning

confidence: 99%

Metabolism of the Folate Precursor p-Aminobenzoate in Plants

Eudes

Bozzo

Waller

et al. 2008

Journal of Biological Chemistry

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“…By thermodynamics, PPi could work as an energy donor if present at relatively high concentration (Weiner et al, 1987). High PPi levels have been found in the cytosolic compartment of leaves (Weiner et al, 1987), in plant cell cultures (Dancer et al, 1990;Kubota and Ashihara, 1990), as well as in developing seeds of Arabidopsis (Gibon et al, 2002) and pea (Pisum sativum; Edwards et al, 1984). Therefore, specifically in the cytosol, PPi might be used as an energy metabolite and couple anabolism to catabolism.…”

Section: Genetic Control Of Tradeoff By Transcription Factorsmentioning

confidence: 99%

Predictive Modeling of Biomass Component Tradeoffs in Brassica napus Developing Oilseeds Based on in Silico Manipulation of Storage Metabolism

Schwender

Hay

2012

Plant Physiology

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Seed oil content is a key agronomical trait, while the control of carbon allocation into different seed storage compounds is still poorly understood and hard to manipulate. Using bna572, a large-scale model of cellular metabolism in developing embryos of rapeseed (Brassica napus) oilseeds, we present an in silico approach for the analysis of carbon allocation into seed storage products. Optimal metabolic flux states were obtained by flux variability analysis based on minimization of the uptakes of substrates in the natural environment of the embryo. For a typical embryo biomass composition, flux sensitivities to changes in different storage components were derived. Upper and lower flux bounds of each reaction were categorized as oil or protein responsive. Among the most oil-responsive reactions were glycolytic reactions, while reactions related to mitochondrial ATP production were most protein responsive. To assess different biomass compositions, a tradeoff between the fractions of oil and protein was simulated. Based on flux-bound discontinuities and shadow prices along the tradeoff, three main metabolic phases with distinct pathway usage were identified. Transitions between the phases can be related to changing modes of the tricarboxylic acid cycle, reorganizing the usage of organic carbon and nitrogen sources for protein synthesis and acetylcoenzyme A for cytosol-localized fatty acid elongation. The phase close to equal oil and protein fractions included an unexpected pathway bypassing a-ketoglutarate-oxidizing steps in the tricarboxylic acid cycle. The in vivo relevance of the findings is discussed based on literature on seed storage metabolism.

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“…These results suggested that ATP synthesis may be required to maintain the highly activated form of SPS. The inactivation that occurred in the presence of an uncoupler would not be expected to be an ATP-dependent process, as uncouplers would likely interfere with ATP production in the dark (5). It is possible that under these extreme conditions, spontaneous inactivation (i.e.…”

Section: Effect Of Uncouplers On the Activation State Of Sps In Situmentioning

confidence: 99%

Inactivation of Highly Activated Spinach Leaf Sucrose-Phosphate Synthase by Dephosphorylation

Huber

Hite²,

Outlaw³

1991

Plant Physiol.

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Spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) can be phosphorylated and inactivated in vitro with ['-32P]ATP Arch Biochem Biophys 270: 681-690). Thus, it was surprising to find that SPS, extracted from leaves fed mannose in the light to highly activate the enzyme, could be inactivated in an ATP-independent manner when desalted crude extracts were preincubated at 250C before assay. The "spontaneous" inactivation involved a loss in activity measured with limiting substrate concentrations in the presence of the inhibitor, Pi, without affecting maximum catalytic activity. The spontaneous inactivation was unaffected by exogenous carrier proteins and protease inhibitors, but was inhibited by inorganic phosphate, fluoride, and molybdate, suggesting that a phosphatase may be involved. Okadaic acid, a potent inhibitor of mammalian type 1 and 2A protein phosphatases, had no effect up to 5 micromolar. Inactivation was stimulated about twofold by exogenous Mg2+ and was relatively insensitive to Ca2+ and to pH over the range pH 6.5 to 8.5. Radioactive phosphate incorporated into SPS during labeling of excised leaves with [32PJPi (initially in the dark and then in the light with mannose) was lost with time when desalted crude extracts were incubated at 250C, and the loss in radiolabel was substantially reduced by fluoride. These results provide direct evidence for action of an endogenous phosphatase(s) using SPS as substrate. We postulate that highly activated SPS contains phosphorylated residue(s) that increase activation state, and that spontaneous inactivation occurs by removal of these phosphate group(s). Inactivation of SPS in vivo caused by feeding uncouplers to darkened leaf tissue that had previously been fed mannose in the dark, may occur by this mechanism. However, there is no evidence that this mechanism is involved in light-dark regulation of SPS in vivo.Regulation of SPS2 activity is thought to be one of the components that controls the flux of carbon into sucrose, and hence translocation, in situ (for review see ref. 19 (9). Thus, phosphorylation of SPS occurred both in vivo and in vitro, and in both systems, the more highly phosphorylated enzyme had a lower activation state. Attempts to study the biochemical mechanism involved have been successful because the spinach leaf enzyme is often relatively stable (for short periods of time) at room temperature. The work we report here focused on the highly activated enzyme extracted from illuminated leaves that were fed mannose. We compared this SPS form with the relatively inactive (extracted from darkened leaves) and intermediate enzymeforms (extracted from leaves illuminated without mannose). We observed that highly activated enzyme was not stable when desalted crude extracts were preincubated at 25°C. Our study of this loss in activation state indicates that the "spontaneous" inactivation is mediated by an endogenous phosphatase(s), which constitutes evidence for a novel regulatory covalent modification of SPS.

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Independent changes of inorganic pyrophosphate and the AT/ADP or UTP/UDP ratios in plant cell suspension cultures

Cited by 66 publications

References 12 publications

Metabolism of the Folate Precursor p-Aminobenzoate in Plants

Metabolism of the Folate Precursor p-Aminobenzoate in Plants

Predictive Modeling of Biomass Component Tradeoffs in Brassica napus Developing Oilseeds Based on in Silico Manipulation of Storage Metabolism

Inactivation of Highly Activated Spinach Leaf Sucrose-Phosphate Synthase by Dephosphorylation

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