LEPS2, a phosphorus starvation-induced novel acid phosphatase from tomato

Abstract

Phosphate (Pi) is one of the least available plant nutrients found in the soil. A significant amount of phosphate is bound in organic forms in the rhizosphere. Phosphatases produced by plants and microbes are presumed to convert organic phosphorus into available Pi, which is absorbed by plants. In this study we describe the isolation and characterization of a novel tomato (Lycopersicon esculentum) phosphate starvation-induced gene (LePS2) representing an acid phosphatase. LePS2 is a member of a small gene family in tomato. The cDNA is 942 bp long and contains an open reading frame encoding a 269-amino acid polypeptide. The amino acid sequence of LePS2 has a significant similarity with a phosphatase from chicken. Distinct regions of the peptide also share significant identity with the members of HAD and DDDD super families of phosphohydrolases. Many plant homologs of LePS2 are found in the databases. The LePS2 transcripts are induced rapidly in tomato plant and cell culture in the absence of Pi. However, the induction is repressible in the presence of Pi. Divided root studies indicate that internal Pi levels regulate the expression of LePS2. The enhanced expression of LePS2 is a specific response to Pi starvation, and it is not affected by starvation of other nutrients or abiotic stresses. The bacterially (Escherichia coli) expressed protein exhibits phosphatase activity against the synthetic substrate p-nitrophenyl phosphate. The pH optimum of the enzyme activity suggests that LePS2 is an acid phosphatase.

Figure 1

Comparative sequence analysis of LePS2 peptide. A, Alignment of deduced amino acid sequence of LePS2 with that of 3X11A, a phosphatase from chicken. B, Alignment of the N-terminal regions of deduced amino acid sequence of LePS2 with that of an Arabidopsis and rice homolog. Alignment utility used is CLUSTAL X (1.62b). Asterisk, Fully conserved residue; :, a “strong” group is conserved; ., a “weaker” group is conserved. C, Table showing the peptide similarity of LePS2 homologs from plants. Asterisk, Partial EST; double asterisk, bacteria artificial chromosome sequence. D, Alignment of amino acid sequences corresponding to two peptide motifs that are conserved in the active site of the HAD and DDDD superfamily of phosphotransferases. The Asp residue predicted to be transiently phosphorylated is marked by an arrow. Invariant residues are shown in bold and other conserved residues are highlighted.

Figure 2

Southern analysis of LePS2. Tomato genomic DNA (10 μg) was digested with BamHI (B), EcoRI (E), and XbaI (X). DNA size markers are indicated.

Figure 3

Northern-blot analysis of Pi starvation-induced LePS2 gene. Total RNA isolated from roots (A) and leaves (B) of aeroponically grown plants, supplied with one-half strength modified Hoagland solution containing 250 μm Pi (+) or no Pi (−) for the indicated time, were probed with labeled LePS2 cDNA. Expression of LePS2 gene was also analyzed using RNA isolated from cell suspension culture grown in the presence of 250 μm Pi (+) or absence of Pi (−) for indicated time (C). Ethidium bromide-stained gel in the bottom panel indicates RNA integrity and uniform loading. D, Expression of LePS2 in different plant parts under Pi starvation. Tomato plants were grown in the presence of 250 μm Pi (+) or absence of Pi (−) for 7 d. Plants were harvested and dissected into the following parts: S, stem; P, petioles; L, leaf; LL, leaf lamina; and LM, leaf midrib. Total RNA (10 μg/lane) extracted from different parts was used for northern-blot analysis.

Figure 4

A. LePS2 is induced specifically in response to Pi stress. Plants were provided with one-half strength Hoagland solution deficient in Pi (P−), nitrogen (N–), potassium (K–), or iron (Fe–), and a control solution with all nutrients (P+). Total RNA was isolated and analyzed by northern blots using ³²P-labeled LePS2 cDNA. RNA gel stained with ethidium bromide shows uniform loading. B, Abiotic stresses does not affect the expression of LePS2. Plants were grown in hydroponics for 4 d under 4°C or 37°C (root zone temperature), salt (S, 150 mm NaCl), and absence of Pi (P−). A set of plants was allowed to wilt at room temperature (D). Total RNA (10 μg/lane) isolated from these plants was subjected to northern-blot analysis.

Figure 5

A, Expression of LePS2 in plants supplied with different concentrations of Pi. Plants were supplied with indicated micromolar concentrations of Pi for 5 d. Total RNA (10 μg/lane) isolated from these plants was used for analyzing the expression of LePS2 by northern blots. B, Resupply of Pi suppresses the expression of LePS2. Plants were sprayed with 250 μm Pi (C+) or no Pi (C−) for 5 d. After 5 d a set of plants were continued to grow in the absence of Pi. Another set of Pi-deficient (C−) plants was provided with 250 μm Pi for the indicated time. Bottom, An ethidium bromide-stained gel showing uniform loading of RNA.

Figure 6

Internal signals may regulate the expression of LePS2 gene. In this divided-root study, roots of plants were exposed to 250 μm Pi (C+) or no Pi (C−). Roots of another set of plants were separated into two portions and each portion was placed in an aerated hydroponic solution containing 250 μm Pi (D+) or no Pi (D−) for 5 d. Total RNA was isolated and analyzed by northern blot. The L+ and L− represents the RNA isolated from leaves of Pi-sufficient (250 μm) or -deficient plants. Ethidium bromide-stained gel indicates RNA integrity and uniform loading.

Figure 7

A, Expression of LePS2 in E. coli. The coding sequence of LePS2 gene was cloned into a pET28a vector and transferred to BL21, pLysis S strain of bacteria. The cells were induced to produce recombinant protein by IPTG. Bacterial proteins were separated on a 10% (w/v) SDS-PAGE. M, Marker; BL21, control; 0i, induced BL21 carrying pET28a vector; Lei, induced BL21 carrying LePS2 coding sequence in a pET28a vector; LeP, Ni affinity column purified recombinant LePS2 protein. Arrow indicates 30-kD LePS2 protein. B, Bacterially expressed LePS2 has ACP activity. The synthetic substrate p-nitrophenol Pi was used to quantify the activity of the column-purified LePS2 protein. The assay was done with extracts from bacteria containing only vector (pET-0) as a control or recombinant protein (LePS2). The fractions 1 to 5 were eluted from the Ni affinity column and tested for phosphatase activity. Data suggest that LePS2 is an ACP with a pH optimum of 4 to 5 C, Error bars represent the sd of all samples tested.