Role Of Phosphate Research Articles

Fibroblast Growth Factor 23 and Its Receptors

Fibroblast growth factor 23 (FGF23) is a circulating factor that plays critical roles in phosphate and vitamin D metabolism, as evidenced by the fact that FGF23 missense mutations cause autosomal dominant hypophosphatemic rickets (ADHR). Autosomal dominant hypophosphatemic rickets is characterized by hypophosphatemia with inappropriately normal 1,25-dihydroxyvitamin D concentrations, as well as bone pain, fracture and rickets. This phenotype parallels that of patients with tumor induced osteomalacia (TIO), X-linked hypophosphatemic rickets (XLH), and fibrous dysplasia (FD), in whom elevated serum FGF23 levels are often observed. The fibroblast growth factor receptors (FGFR1-4) play key roles in skeletal development, as well as in normal metabolic processes. Several FGFR isoforms that potentially mediate the activity of FGF23 have been implicated. In the short term, these findings will lead to further understanding of FGF23 function, and potentially in the long term, to targeted therapies in disorders of hypo- and hyperphosphatemia that involve FGF23.

Multivalent Activation in Temporary Phosphate Tethers: A New Tether for Small Molecule Synthesis

[reaction: see text]. A new tether for small molecule synthesis is reported. This functionally active tether mediates the desymmetrization of a pseudo-C(2)-symmetric tris-allylic phosphate triester to generate a P-chiral bicyclo[4.3.1]phosphate containing ample steric and stereoelectronic differentiation for investigating chemo-, regio-, and stereoselective transformations. Overall, the method reported herein demonstrates a fundamentally new role of phosphates in synthesis and provides differentiated polyol building blocks for use in natural product synthesis.

Genetic dissection of phosphate- and vitamin D-mediated regulation of circulating Fgf23 concentrations

Fibroblast growth factor-23 (FGF23) is a circulating factor that plays critical roles in phosphate and vitamin D metabolism. The goal of our studies was to dissect the pathways directing the vitamin D–phosphate–FGF23 homeostatic axis. To test the role of diet in the regulation of Fgf23, wild-type (WT) mice were fed either a standard (0.44% phosphorus) or a low-phosphate (0.02%) diet. WT mice on standard diet had a serum phosphate of 9.5 ± 0.3 mg/dl and an Fgf23 concentration of 99.0 ± 10.6 pg/ml; mice on the low-phosphate diet had a phosphate of 5.0 ± 0.2 mg/dl ( P < 0.01) and an Fgf23 of 10.6 ± 3.7 pg/ml ( P < 0.01). To genetically separate the effects of phosphate and vitamin D on Fgf23, we examined vitamin D receptor null (VDR −/−) mice, which are hypocalcemic and hypophosphatemic secondary to hyperparathyroidism. On standard diets, WT and VDR +/− mice had Fgf23 levels of 106.0 ± 30.7 and 90.6 ± 17.3 pg/ml, respectively, whereas Fgf23 was undetectable in the VDR −/−. Animals were then placed on a diet that normalizes serum calcium and phosphorus. This ‘rescue’ increased Fgf23 in WT to 192.3 ± 32.5 pg/ml and in VDR +/− to 388.2 ± 89.6pg/ml, and importantly, in VDR −/− to 476.9 ± 60.1 pg/ml ( P < 0.01 vs. WT). In addition, renal vitamin D 1-alpha hydroxylase (1α-OHase) mRNA levels were corrected to WT levels in the VDR −/− mice. In summary, Fgf23 is suppressed in diet-induced hypophosphatemia and in hypophosphatemia associated with secondary hyperparathyroidism. Normalization of serum phosphate by diet in VDR −/− mice increases Fgf23. Thus, our results demonstrate that Fgf23 is independently regulated by phosphate and by vitamin D.

Role of phosphates in the promotion of silver catalysts for partial oxidation: I. Structure and properties of phosphates on the surface of polycrystalline silver

The chemical composition, structure, and properties of an H3PO4-promoted polycrystalline silver foil are studied by XPS. The surface of the doped sample contains oxygen in two states with binding energies of 531.1 and 533.0 eV. Heat treatment results in silver clusters distributed in the polyphosphate matrix. It follows from TDS data that the surface of the promoted catalyst contains a strongly bound oxygen species that desorbs at ~900 K. It is believed that the silver clusters stabilized by the phosphate matrix are active sites on the surface of the phosphorus-promoted catalyst.

Role of phosphates in promotion of silver catalysts for partial oxidation: II. Formation of active sites in the structure of silver phosphate under the action of a reductive medium

The morphology and chemical composition of the surface of silver phosphate are studied. The qualitative and quantitative compositions of silver phosphate are quite the same as those of the surface of the phosphorus-promoted silver catalyst. Silver particles surrounded by silver phosphate form in the subsurface region upon treatment of the sample with hydrogen. As a result, a complex catalytic system is formed in which silver phosphate is both the precursor of the reactive component (metallic silver particles) and the stabilizing matrix for this component. The system obtained is stable toward high temperature and redox reaction media.

A possible role for phosphate in complexing the arginines of S4 in voltage gated channels

Phosphate ions are known to complex guanidinium groups, which are the side chains of arginine. Voltage gated channels that allow passage of ions through cell membranes, producing, for example the nerve impulse, are in many cases composed of four domains, each with six transmembrane segments. The S4 transmembrane segments of these channels have arginines placed in such a way that they would be expected to complex phosphate. Known phosphate–arginine complexes are reasonably strong. Here, an ab initio calculation reinforces the expectation that a strong complex could form. As a consequence, if the S4 moved, it would carry either no charge, or at most half of what is expected from fully charged arginines. This suggests that it may be necessary to rethink voltage gating models in which the gating current is produced by physical motion of the S4 transmembrane segments.

What have we learnt about the regulation of phosphate metabolism?

The search for hormones which specifically regulate phosphate metabolism has fuelled recent tantalizing studies. These studies have been motivated by diseases involving renal phosphate wasting, including tumor-induced osteomalacia, X-linked hypophosphatemic rickets, and autosomal dominant hypophosphatemia. This review focuses on likely candidate 'phosphatonins' and their possible physiological significance. Candidate phosphatonins include fibroblast growth factor 23, matrix extracellular phosphoglycoprotein, stanniocalcin, and Frizzled-related protein 4. Fibroblast growth factor 23 has emerged as the prime candidate explaining pathophysiology of these diseases. FGF-23 is expressed in most tumors in tumor-induced osteomalacia. Serum fibroblast growth factor 23 is increased in most patients with X-linked hypophosphatemic rickets and tumor-induced osteomalacia. Injection of recombinant fibroblast growth factor 23 induces phosphaturia, hypophosphatemia, and suppression of 1,25-dihydroxyvitamin D in animals. Many unanswered questions remain, including the relationship between PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) mutations and elevated fibroblast growth factor 23. It is also not clear whether these candidate phosphatonins play a role in phosphate or vitamin D metabolism in healthy humans, or that this role is endocrine. The most compelling evidence derives from the fibroblast growth factor 23-knockout mouse which shows hyperphosphatemia and increased serum 1,25-dihydroxyvitamin D. A physiologically relevant phosphatonin should explain renal adaptation to variable dietary phosphate intake. The tissue source and determinants of serum fibroblast growth factor 23 are unknown. Pathophysiological and animal studies serve as a logical foundation on which to base further questions of human physiology. The definition of what is or is not a phosphatonin may need to be refined. There is a need to return to 'old-fashioned' human physiology studies to place recent findings in perspective.

Specificity of the Synergistic Anion for Iron Binding by Ferric Binding Protein from Neisseria gonorrhoeae

Ferric binding protein in Neisseria gonorrhoeae (nFbpA) transports iron from outer membrane receptors for host proteins across the periplasm to a permease in an alternative pathway to the use of siderophores in some pathogenic bacteria. Phosphate and nitrilotriacetate, both at pH 8, and vanadate at pH 9 are shown to be synergistic in promoting ferric binding to nFbpA, in contrast to carbonate and sulfate. Interestingly, only phosphate produces the fully closed conformation of nFbpA as defined by native electrophoresis. The role of phosphate was probed by constructing three mutants: Q58E, Q58R, and G140H. The anion and iron binding properties of the Q58E mutant are similar to the wild-type protein, implying that one phosphate oxygen is a hydrogen bond donor and may in part define the specificity of nFbpA for phosphate over sulfate. Phosphate is a weakly synergistic anion in the Q58R and G140H mutants, and these mutants do not form completely closed structures. Ferric binding was investigated by both isothermal titration and differential scanning calorimetry. The apparent affinity of nFbpA for iron in a solution of 30 mM citrate is 1 order of magnitude larger in the presence (K(app)= 1.7 x 10(5) M(-1)) of phosphate than in its absence (K(app) = 1.6 x 10(4) M(-1)) at pH 7. Similar results were obtained at pH 8. This increase in affinity with phosphate as well as the formation of closed structure allows nFbpA to compete for free ferric ions in solution and suggests that ferric binding to nFbpA is regulated by the synergistic phosphate anion at sites of iron uptake.

Kinetics and mechanism of iron(III) complexation by ferric binding protein: the role of phosphate.

Iron transport across the periplasmic space to the cytoplasmic membrane of certain Gram-negative bacteria is mediated by a ferric binding protein (Fbp). This requires Fe(3+) loading of Fbp at the inner leaflet of the outer membrane. A synergistic anion is required for tight Fe(3+) sequestration by Fbp. Although phosphate fills this role in the protein isolated from bacterial cell lysates, nitrilotriacetate anion (NTA) can also satisfy this requirement in vitro. Here, we report the kinetics and mechanism of Fe(3+) loading of Fbp from Fe(NTA)(aq) in the presence of phosphate at pH 6.5. The reaction proceeds in four kinetically distinguishable steps to produce Fe(3+)Fbp(PO(4)) as a final product. The first three steps exhibit half-lives ranging from ca. 20 ms to 0.5 min, depending on the concentrations, and produce Fe(3+)Fbp(NTA) as an intermediate product of significant stability. The rate for the first step is accelerated with an increasing phosphate concentration, while that of the third step is retarded by phosphate. Conversion of Fe(3+)Fbp(NTA) to Fe(3+)Fbp(PO(4)) in the fourth step is a slow process (half-life approximately 2 h) and is facilitated by free phosphate. A mechanism for the Fe(3+)-loading process is proposed in which the synergistic anions, phosphate and NTA, play key roles. These data suggest that not only is a synergistic anion required for tight Fe(3+) sequestration by Fbp, but also the synergistic anion plays a critical role in the process of inserting Fe(3+) into the Fbp binding site.

Role of phosphate and carboxylate ions in maillard browning.

The Maillard reaction of carbohydrates and amino acids is the underlying chemical basis for flavor and color formation in many processed foods. Phosphate and other polyatomic anions will accelerate the rate of Maillard browning, and this effect has been explained by invoking enhanced proton abstraction from intermediate Amadori compounds. In this work, the effect of phosphate and carboxylate ions on browning was measured for a series of reducing sugars with and without the presence of beta-alanine. Significant browning was observed for sugars alone suggesting that polyatomic anions contribute to Maillard browning by providing reactive intermediates directly from sugars. A mechanism is proposed for decomposition of sugars by polyatomic anions and efforts to trap reactive species using o-phenylenediamine (OPD) are described. The results of this study suggest how complications may arise from the popular usage of phosphate buffers in the study of Maillard reaction kinetics. In addition, the results imply how phosphates may be useful for enhancing browning during food processing.

Low temperature effects on grapevine photosynthesis: the role of inorganic phosphate.

The photosynthetic response of grapevine leaves (Vitis vinifera L. cv. Riesling) to low temperature was studied to determine the role of end-product limitation and orthophosphate (Pi) recycling to the chloroplast under these conditions. As reported previously, the response of photosynthesis in air to stomatal conductance declined at temperatures below 15°C, suggesting that at low temperatures inhibition of photosynthesis in grapevine has a strong non-stomatal component. Stimulation of carbon assimilation at ambient CO2 by reducing O2 from 21 to 2 kPa, O2 declined to zero below 15°C, a phenomenon often associated with a restriction in photosynthesis due to end-product-synthesis limitation. This stimulation could be restored by feeding Pi. Photosynthesis in leaf disks at both high and low irradiances in non-photorespiratory conditions (1% CO2) was highly sensitive to reductions in temperature. Below 15°C, feeding Pi caused a large stimulation of photosynthetic O2 evolution. Metabolite measurements indicated that despite a decline in Rubisco carbamylation state, ribulose 1,5-bisphosphate (RuBP) levels dropped at low temperature and the ratio of 3-phosphoglycerate (3-PGA) to triose phosphate (TP) remained largely unchanged. These results suggest that grapevine-leaf photosynthesis is severely restricted at low temperature by non-stomatal mechanisms. The return of Pi to the chloroplast plays an important role in this limitation but a coordinated set of regulatory processes maintain a homeostasis of phosphorylated sugar levels.

Proliferation in hyperplastic human and normal rat parathyroid glands: Role of phosphate, calcitriol, and gender

Parathyroid gland hyperplasia develops in azotemic patients. A phosphate excess and calcitriol deficiency play critical roles in its development. Our goals were to determine whether differences in serum phosphate values at parathyroidectomy (PTX) in hemodialysis patients with refractory hyperparathyroidism: (1) correlated with parathyroid cell proliferation; and (2) affected the antiproliferative response to in vitro calcitriol. Studies were also performed to determine whether the phosphate concentration in the medium affected the antiproliferative response to calcitriol, and whether a high phosphate diet and calcitriol treatment affected parathyroid cell proliferation and parathyroid hormone (PTH) levels in normal rats. Forty-seven parathyroid glands from 19 hemodialysis patients were obtained at PTX. Flow cytometry was used to determine cell proliferation (percent cells in S phase) in excised parathyroid glands. Similarly, cell proliferation was determined in parathyroid tissue incubated for 24 hours in medium with or without 10(-7) mol/L calcitriol and with 1 or 4 mmol/L phosphate. In normal rats, the effect of 3 days of a high phosphate diet (1.2% P) and calcitriol treatment (100 pmol/kg) on PTH values and cell proliferation was evaluated. In cells from freshly removed parathyroid glands obtained at PTX from hemodialysis patients, there were no significant correlations between the percent cells in S phase and age, gender, and serum phosphate, calcium, and PTH. While incubation of parathyroid tissue with 10(-7) mol/L calcitriol did reduce cell proliferation (P < 0.001), both the pre-PTX serum phosphate value (P= 0.003) and female gender (P=0.003) were associated with a decreased response to calcitriol. Incubation of parathyroid tissue in medium containing 4 mmol/L phosphate did not increase cell proliferation. In normal rats, a high phosphate diet for 3 days increased cell proliferation (P < 0.05) and PTH levels (P < 0.05), and calcitriol treatment was without effect. Our findings suggest a high phosphate burden, as well as female gender, favor parathyroid cell proliferation and both may reduce the inhibition of parathyroid function by calcitriol.

Phosphate is a specific signal for ATDC5 chondrocyte maturation and apoptosis-associated mineralization: possible implication of apoptosis in the regulation of endochondral ossification.

Involvement of Pi and Ca in chondrocyte maturation was studied because their levels increase in cartilage growth plate. In vitro results showed that Pi increases type X collagen expression, and together with Ca, induces apoptosis-associated mineralization, which is similar to that analyzed in vivo, thus suggesting a role for both ions and apoptosis during endochondral ossification. During endochondral ossification, regulation of chondrocyte maturation governs the growth of the cartilage plate. The role of inorganic phosphate (Pi), whose levels strongly increase in the hypertrophic zone of the growth plate both in intra- and extracellular compartments, on chondrocyte maturation and mineralization of the extracellular matrix has not yet been deciphered. The murine chondrogenic cell line ATDC5 was used. Various Pi and calcium concentrations were obtained by adding NaH2PO4/Na2HPO4 and CaCl2, respectively. Mineralization was investigated by measuring calcium content in cell layer by atomic absorption spectroscopy and by analyzing crystals with transmission electron microscopy and Fourier transform infrared microspectroscopy. Cell differentiation was investigated at the mRNA level (reverse transcriptase-polymerase chain reaction [RT-PCR] analysis). Cell viability was assessed by methyl tetrazolium salt (MTS) assay and staining with cell tracker green (CTG) and ethidium homodimer-(EthD-1). Apoptosis was evidenced by DNA fragmentation and caspase activation observed in confocal microscopy, as well as Bcl-2/Bax mRNA ratio (RT-PCR analysis). We showed that Pi increases expression of the hypertrophic marker, type X collagen. When calcium concentration is slightly increased (like in cartilage growth plate), Pi also induces matrix mineralization that seems identical to that observed in murine growth plate cartilage and stimulates apoptosis of differentiated ATDC5 cells, with a decrease in Bcl-2/Bax mRNA ratio, DNA fragmentation, characteristic morphological features, and caspase-3 activation. In addition, the use of a competitive inhibitor of phosphate transport showed that these effects are likely dependent on Pi entry into cells through phosphate transporters. Finally, inhibition of apoptosis with ZVAD-fmk reduces pi-induced mineralization. These findings suggest that Pi regulates chondrocyte maturation and apoptosis-associated mineralization, highlighting a possible role for Pi in the control of skeletal development.

Happily at Work

Happily at Work

Anatomical and histochemical differentiation in lobes of the lichen Lobaria pulmonaria

The growth pattern of the thallus of Lobaria pulmonaria, a foliose lichen, has been described as differentiated into upwards and downwards growing lobes. The former show meristematic properties, whereas the latter, owing to the formation of soralia inactivating apical meristems, become senile lobes. A simple and sensitive histochemical procedure, the TBO test performed in this study, shows co-distribution of zones of active growth and polyphosphates accumulation in L. pulmonaria and gives evidence of the central role of phosphate in lichen metabolism. Upwards and downwards growing lobes mainly differentiate in pattern of accumulation of histochemically detectable polyphosphates. In the former, actively growing zones correspond to the algae, cortical and medullary hyphal cells adjacent to the algal layer of the pseudomeristematic marginal rim and of the adjacent elongation zone. In the downwards sorediate lobes, lacking apical growth, the actively growing zones correspond to medullary hyphae and algae occurring in areas where soredia are formed.

Parathyroid Hormone Suppression by Intravenous Calcitriol: Role of Phosphate, Calcium, Race and Diabetes

Parathyroid hormone (PTH) suppression in patients with end-stage renal disease (ESRD) undergoing maintenance hemodialysis is achieved largely by the use of intravenous calcitriol. Aspects of the utility and efficacy of this therapy remain controversial. It is debated whether oral versus intravenous therapy is more effective. Most existing studies examine the effect of calcitriol in isolation, without adjusting for other factors that might influence PTH levels. Thus, the simultaneous role of factors such as dosing, control of serum calcium and phosphorus, and demographic variables such as age, sex, race, and duration of ESRD is not well understood. We examined the relationship between the administration of calcitriol and PTH suppression in a cohort of hemodialysis patients at a large urban dialysis facility over a period of 30 months. Hemodialysis patients (n = 155) who received at least 3 months of treatment in this facility were included. Using a time sensitive multiple linear regression modeling technique, we found that second and subsequent PTH levels were positively correlated with black race (P < 0.0001) and serum phosphate (P < 0.03) and strongly negatively correlated with serum calcium (P< 0.0001) and diabetes (P< 0.0039). Drug dose (in micrograms per kilogram per month) was weakly negatively correlated (P < 0.04). Unlike previous studies, we adjusted for the simultaneous confounding influence of demographic and laboratory variables, as well as for drug dose normalized for body weight. This analysis suggests that calcitriol therapy in hemodialysis patients is adversely affected by higher phosphate levels and needs to account for such patient characteristics as race and diabetes and such laboratory variables as calcium and phosphate control. Finally, as has been recently suggested by others, the patient's race may require us to aim for different PTH target levels with therapy.

Role of phosphate and calcium stores in muscle fatigue.

Intensive activity of muscles causes a decline in performance, known as fatigue, that is thought to be caused by the effects of metabolic changes on either the contractile machinery or the activation processes. The concentration of inorganic phosphate (P(i)) in the myoplasm ([P(i)](myo)) increases substantially during fatigue and affects both the myofibrillar proteins and the activation processes. It is known that a failure of sarcoplasmic reticulum (SR) Ca(2+) release contributes to fatigue and in this review we consider how raised [P(i)](myo) contributes to this process. Initial evidence came from the observation that increasing [P(i)](myo) causes reduced SR Ca(2+) release in both skinned and intact fibres. In fatigued muscles the store of releasable Ca(2+) in the SR declines mirroring the decline in SR Ca(2+) release. In muscle fibres with inoperative creatine kinase the rise of [P(i)](myo) is absent during fatigue and the failure of SR Ca(2+) release is delayed. These results can all be explained if inorganic phosphate can move from the myoplasm into the SR during fatigue and cause precipitation of CaP(i) within the SR. The relevance of this mechanism in different types of fatigue in humans is considered.

Oxyanion-Mediated Protein Stabilization: Differential Roles of Phosphate for Preventing Inactivation of Bacterial α-Glucan Phosphorylases

Maltodextrin phosphorylase (MalP) from Escherichia coli and starch phosphorylase (StP) from Corynebacterium callunae are significantly stabilized in the presence of phosphate against inactivation by elevated temperature or urea. The stabilizing effect of phosphate was observed at ion concentrations below 50 mM. Therefore, it is probably due to preferential binding of phosphate to the folded conformations of the phosphorylases. For StP, phosphate binding inhibited the dissociation of the active-site cofactor pyridoxal 5′-phosphate. Phosphate-liganded StP was at least 500-fold more stable at 60d`C than the free enzyme at the same temperature. It showed an apparent transition midpoint of 5.2 M for irreversible denaturation by urea, and this midpoint was increased by a denaturant concentration of 4M relative to the corresponding transition midpoint of free StP in urea. The mechanisms of inactivation and denaturation of MalP at 45d`C and by urea involve formation of a cofactor-containing, insoluble protein aggregate. Under denaturing conditions, phosphate was shown to inhibit aggregation of the reversibly inactivated MalP dimer.

Roles of phosphate and an enoyl radical in ferritin iron mobilization by 5-aminolevulinic acid

5-Aminolevulinic acid (ALA), a heme precursor that accumulates in acute intermittent porphyria (AIP) and lead poisoning, undergoes enolization and subsequent iron-catalyzed oxidation at neutral pH. Iron is released from horse spleen ferritin (HoSF) by both ALA-generated O 2 •− and enoyl radical (ALA ), which amplifies the chain of ALA oxidation (autocatalysis). Iron chelators such as EDTA, ATP, but not citrate, and phosphate accelerate this process and ALA-promoted iron release from HoSF is faster in horse spleen isoferritins containing larger amounts of phosphate in the core. ALA (+0.377 V versus standard hydrogen electrode) is less effective in releasing iron from ferritin than are thioglycollic acid, 6-hydroxydopamine, and N,N,N′,N′-tetramethyl- p-phenylenediamine. During electrochemical one electron oxidation of ALA in a nitrogen atmosphere, spin trapping experiments with 3,5-dibromo-4-nitrosobenzenesulfonic acid demonstrated the formation of a spin adduct characterized by a six line signal, indicating a secondary carbon-centered radical and attributed to a resonant ALA radical. Iron is also released in such anaerobic electrochemical oxidations of ALA in the presence of ferritin, suggesting that, in addition to O 2 •−, ALA can promote iron mobilization from ferritin. Hence, ALA may amplify the metal-catalyzed oxidation of ALA, damaging ALA-accumulating cells and possibly contributing to the symptoms of porphyria.

The role of inorganic phosphate in the development of freezing tolerance and the acclimatization of photosynthesis to low temperature is revealed by the pho mutants of Arabidopsis thaliana.

Low temperature inhibits sucrose synthesis, leading to a phosphate-limitation of photosynthesis. We have used the Arabidopsis pho1-2 and pho2-1 mutants with decreased and increased shoot phosphate, respectively, to investigate whether low phosphate triggers cold acclimatization of photosynthetic carbon metabolism. Wild-type Arabidopsis, pho1-2 and pho2-1 were grown at 23 degrees C and transferred to 5 degrees C to investigate acclimatization in pre-existing leaves and in new leaves developing at 5 degrees C. The development of frost tolerance and the accumulation of proline and sugars was unaltered or improved in pho1-2, and impaired in pho2-1. Sucrose phosphate synthase and cytoplasmic fructose-1,6-bisphosphatase activity and protein increase after transfer to 5 degrees C. This increase was accentuated in pho1-2 and attenuated in pho2-1. RBCS and LHCB2 transcript levels decrease in pre-formed wild-type leaves after transfer to 5 degrees C and recover in new leaves that develop at 5 degrees C. The initial decrease was attenuated in pho1-2, and accentuated in pho2-1, where the recovery in new leaves was also suppressed. Rubisco activity increased in wild-type leaves that developed at 5 degrees C. This increase was accentuated in pho1-2 and absent in pho2-1. NADP-glyceraldehyde-3-phosphate dehydrogenase, plastidic fructose-1,6-bisphosphatase and aldolase activity increase relative to phosphoglycerate kinase, transketolase and phosphoribulokinase in wild-type leaves at 5 degrees C. This shift was accentuated in pho1-2 and reversed in pho2-1. Transcript levels for COR genes increase transiently 1 day after transfer to 5 degrees C but were very low in leaves that developed at 5 degrees C in wild-type Arabidopsis, pho1-2 and pho2-1. We conclude that low phosphate plays an important role in triggering cold acclimatization of leaves, leading in particular to an increase of Rubisco expression, changes in other Calvin cycle enzymes to minimize sequestration of phosphate in metabolites, and increased expression of sucrose biosynthesis enzymes.