Two secreted acid phosphatases (SAP1 and SAP2) were markedly up-regulated during P i -starvation of tomato suspension cells. SAP1 and SAP2 were resolved during cation-exchange FPLC of culture media proteins from 8-dayold P i -starved cells, and purified to homogeneity and final p-nitrophenylphosphate hydrolyzing specific activities of 246 and 940 lmol P i producedAEmin, respectively. SDS/PAGE, periodic acid-Schiff staining and analytical gel filtration demonstrated that SAP1 and SAP2, respectively, exist as 84 and 57 kDa glycosylated monomers. SAP1 and SAP2 are purple acid phosphatases (PAPs) as they displayed an absorption maximum at 518 and 538 nm, respectively, and were not inhibited by L-tartrate. The respective sequence of a SAP1 and SAP2 tryptic peptide was very similar to a portion of the deduced sequence of several putative Arabidopsis thaliana PAPs. CNBr peptide mapping indicated that SAP1 and SAP2 are structurally distinct. Both isozymes displayed a pH optimum of approximately pH 5.3 and were heat stable. Although they exhibited wide substrate specificities, the V max of SAP2 with various phosphate-esters was significantly greater than that of SAP1. SAP1 and SAP2 were activated by up to 80% by 5 mM Mg 2+, and demonstrated potent competitive inhibition by molybdate, but mixed and competitive inhibition by P i , respectively. Interestingly, both SAPs exhibited significant peroxidase activity, which was optimal at approximately pH 8.4 and insensitive to Mg 2+ or molybdate. This suggests that SAP1 and SAP2 may be multifunctional proteins that operate: (a) PAPs that scavenge P i from extracellular phosphate-esters during P i deprivation, or (b) alkaline peroxidases that participate in the production of extracellular reactive oxygen species during the oxidative burst associated with the defense response of plants to pathogen infection.Keywords: phosphate starvation (plants); acid phosphatase (purple); peroxidase; Lycopersicon esculentum.Acid phosphatases (APs; orthophosphoric-monoester phosphohydrolase) catalyze the hydrolysis of a broad and overlapping range of P-monoesters with a pH optimum below pH 7.0 [1]. APs are ubiquitous and abundant in plants, animals, fungi, and bacteria. They are believed to function in the production, transport and recycling of P i , which is crucial for cellular metabolism and energy transduction processes. Eukaryotic APs exist as tissue-and/or cellular compartment-specific isozymes that display variation in subunit M r , substrate specificity, localization and sensitivity to inhibition by various divalent cations and metabolites [1,2].Control of plant AP expression is mediated by a variety of environmental and developmental factors [2]. APs are induced under various stresses, such as water deficiency, salinity stress, and nutritional P i -deficiency [2,3]. Plant AP activity is also abundant in storage tubers, ripening fruit, and germinating seeds [2,4,5]. APs can be distinguished based on relative substrate specificities, and are categorized as one of the following: (a) nonspec...

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An intracellular acid phosphatase (IAP) from P(i)-starved (-P(i)) tomato ( Lycopersicon esculentum ) suspension cells has been purified to homogeneity. IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (lambda(max)=546 nm) and was insensitive to L-tartrate. PAGE, periodic acid-Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (alpha-subunit) and 57 kDa (beta-subunit) polypeptides. However, the nine N-terminal amino acids of the alpha- and beta-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs. Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to P(i)-deprivation was correlated with similar increases in the amount of antigenic IAP alpha- and beta-subunits. IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg(2+), but was potently inhibited by Zn(2+), Cu(2+), Fe(3+), molybdate, vanadate, fluoride and P(i). Although IAP demonstrated broad substrate selectivity, its specificity constant ( V (max)/ K (m)) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate. IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg(2+) or molybdate. This IAP is proposed to scavenge P(i) from intracellular phosphate esters in -P(i) tomato. A possible secondary IAP role in the metabolism of reactive oxygen species is discussed. IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of -P(i) tomato cells [Bozzo, Raghothama and Plaxton (2002) Eur. J. Biochem. 269, 6278-6286].

Cyanobacterial and plant genomes encode proteins with some similarity to the folate and biopterin transporters of the trypanosomatid parasite Leishmania. The Synechocystis slr0642 gene product and its closest Arabidopsis homolog, the At2g32040 gene product, are representative examples. Both have 12 probable transmembrane domains, and the At2g32040 protein has a predicted chloroplast transit peptide. When expressed in Escherichia coli pabA pabB or folE, mutants, which are unable to produce or take up folates, the slr0642 protein and a modified At2g32040 protein (truncated and fused to the N terminus of slr0642) enabled growth on 5-formyltetrahydrofolate or folic acid but not on 5-formyltetrahydrofolate triglutamate, demonstrating that both proteins mediate folate monoglutamate transport. Both proteins also mediate transport of the antifolate analogs methotrexate and aminopterin, as evidenced by their ability to greatly increase the sensitivity of E. coli to these inhibitors. The full-length At2g32040 polypeptide was translocated into isolated pea chloroplasts and, when fused to green fluorescent protein, directed the passenger protein to the envelope of Arabidopsis chloroplasts in transient expression experiments. At2g32040 transcripts were present at similar levels in roots and aerial organs, indicating that the protein occurs in non-green plastids as well as chloroplasts. Insertional inactivation of At2g32040 significantly raised the total folate content of chloroplasts and lowered the proportion of 5-methyltetrahydrofolate but did not discernibly affect growth. These findings establish conservation of function among folate and biopterin transporter family proteins from three kingdoms of life.

Our results confirm recent transcript profiling studies suggesting that PSI proteins are subject to both temporal and tissue-specific syntheses in-Pi plants.

SummaryFolates in vivo undergo oxidative cleavage, giving pterin and p-aminobenzoylglutamate (pABAGlu) moieties. These breakdown products are excreted in animals, but their fate is unclear in microorganisms and unknown in plants. As indirect evidence from this and previous studies strongly suggests that plants can have high folatebreakdown rates (approximately 10% per day), salvage of the cleavage products seems likely. Four sets of observations support this possibility. First, cleavage products do not normally accumulate: pools of pABAGlu (including its polyglutamyl forms) are equivalent to, at most, 4-14% of typical total folate pools in Arabidopsis thaliana, Lycopersicon esculentum and Pisum sativum tissues. Pools of the pterin oxidation end-product pterin-6-carboxylate are, likewise, fairly small (3-37%) relative to total folate pools. Second, little pABAGlu built up in A. thaliana plantlets when net folate breakdown was induced by blocking folate synthesis with sulfanilamide. Third, A. thaliana and L. esculentum tissues readily converted supplied breakdown products to folate synthesis precursors: pABAGlu was hydrolysed to p-aminobenzoate and glutamate, and dihydropterin-6-aldehyde was reduced to 6-hydroxymethyldihydropterin. Fourth, both these reactions were detected in vitro; the reduction used NADPH as cofactor. An alternative salvage route for pABAGlu, direct reincorporation into dihydrofolate via the action of dihydropteroate synthase, appears implausible from the properties of this enzyme. We conclude that plants are excellent organisms in which to explore the biochemistry of folate salvage.

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