Characterization of Arabidopsis acid phosphatase promoter and regulation of acid phosphatase expression

Abstract

The expression and secretion of acid phosphatase (APase) was investigated in Indian mustard (Brassica juncea L. Czern.) plants using sensitive in vitro and activity gel assays. Phosphorus (P) starvation induced two APases in Indian mustard roots, only one of which was secreted. Northern-blot analysis indicated transcriptional regulation of APase expression. Polymerase chain reaction and Southern-blot analyses revealed two APase homologs in Indian mustard, whereas in Arabidopsis, only one APase homolog was detected. The Arabidopsis APase promoter region was cloned and fused to the beta-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS expression was first evident in leaves of the P-starved Arabidopsis plants. In P-starved roots, the expression of GUS initiated in lateral root meristems followed by generalized expression throughout the root. GUS expression diminished with the addition of P to the medium. Expression of GFP in P-starved roots also initiated in the lateral root meristems and the recombinant GFP with the APase signal peptide was secreted by the roots into the medium. The APase promoter was specifically activated by low P levels. The removal of other essential elements or the addition of salicylic or jasmonic acids, known inducers of gene expression, did not activate the APase promoter. This novel APase promoter may be used as a plant-inducible gene expression system for the production of recombinant proteins and as a tool to study P metabolism in plants.

Figure 1

Time course of APase activity exuded from Indian mustard roots grown in sterile media containing 3, 1, 0.25, and 0.01 mm P. Each point is a mean of six replicates ± se. The experiment was repeated three times with similar results.

Figure 2

Activity-gel detection of APase exuded from Indian mustard roots. Lanes 1 and 3 contained 50 μg of root-extracted proteins; lanes 2 and 4 contained 5 μg of root-secreted proteins, renatured following their separation by SDS-PAGE. Lanes 1 and 2, Proteins produced after 9 d of normal fertilization conditions (1 mm P); lanes 3 and 4, proteins produced after 9 d of P starvation (0.01 mm P).

Figure 3

Southern-blot analysis of EcoRI-digested DNA using the Arabidopsis APase gene fragment as a probe. Lane 1, 5 μg of EcoRI digested Arabidopsis DNA. Lane 2, 10 μg of EcoRI-digested Indian mustard DNA.

Figure 4

Northern-blot analysis of APase expression in P-starved Indian mustard roots using the Indian mustard 900-bp APase gene fragment as a probe. The size of the expressed APase mRNA was 1.4 kb (upper panel). The lower panel shows amounts of total RNA loaded on each lane. Lane C0, A control plant at d 0 grown in standard (1 mm) P concentration. Lanes 0 to 5, Plants grown in low (0.01 mm) P for 0 to 5 d, respectively. Lane C5,_, Control plant at d 5 grown in standard (1 mm P) concentration.

Figure 5

Arabidopsis APase promoter sequence. The putative TATA box is indicated by a dotted line, the ATG start codon is indicated by an arrow, and the position of the 562L primer (used in cloning the promoter) is underlined.

Figure 6

GUS expression in Arabidopsis plants transformed with the Pr-GUS construct. Plants were grown for 14 d under normal fertilization and then transferred to a hydroponic system lacking P for 14 (A–D) or 17 d (E). Plants were germinated in sand cultures fertilized with Hoagland solutions, with or without P, and stained after 10 d (F–G). A, Initiation of a lateral root primordium with localized expression (×200). B, Lateral root primordium (×100) showing expression in several cell layers. C, Emerging LRM (×200). D, Portion of root showing LRM and an ARM (×40). E, Root after 17 d in hydroponic system without P (×40). F, Roots after germination in sand cultures fertilized without P (×40). G, Ten-day-old seedling from a normally fertilized sand culture. H, Ten-day-old seedling from sand culture lacking P.

Figure 7

Expression of GFP in Arabidopsis plants transformed with the PS-GFP construct, which contains the APase promoter and signal peptide, fused to the GFP gene. Plants were grown for 14 d with P and then transferred to a hydroponic solution lacking P for 14 d. Wild-type plants grown under the same conditions served as controls. A, Light microscopy of PS-GFP transformed roots showing emerging LRM and ARM (×100). B, Fluorescence microscopy image of the roots shown in A (×100). C, Light microscopy of an emerging lateral meristem of the wild-type root (×200). D, Fluorescence microscopy image of the wild-type root shown in C (×200). E, Arabidopsis wild-type plant (left) and T2 PS-GFP transformed plant (right), grown in a hydroponic system. Photograph taken with normal light. F, Visualization of GFP secretion into the medium from the roots of transgenic Arabidopsis plant (right) and wild type (left) under UV illumination (same plants as in Fig. 7E photographed under UV light illumination). Seedlings were aseptically transferred into 20-mL glass vials containing 2 mL of hydroponic medium with or without P (see “Materials and Methods”). Photograph was taken after 5 d of incubation.