Functional analysis of the Arabidopsis PLDZ2 promoter reveals an evolutionarily conserved low-Pi-responsive transcriptional enhancer element

Abstract

Plants have evolved a plethora of responses to cope with phosphate (Pi) deficiency, including the transcriptional activation of a large set of genes. Among Pi-responsive genes, the expression of the Arabidopsis phospholipase DZ2 (PLDZ2) is activated to participate in the degradation of phospholipids in roots in order to release Pi to support other cellular activities. A deletion analysis was performed to identify the regions determining the strength, tissue-specific expression, and Pi responsiveness of this regulatory region. This study also reports the identification and characterization of a transcriptional enhancer element that is present in the PLDZ2 promoter and able to confer Pi responsiveness to a minimal, inactive 35S promoter. This enhancer also shares the cytokinin and sucrose responsive properties observed for the intact PLDZ2 promoter. The EZ2 element contains two P1BS motifs, each of which is the DNA binding site of transcription factor PHR1. Mutation analysis showed that the P1BS motifs present in EZ2 are necessary but not sufficient for the enhancer function, revealing the importance of adjacent sequences. The structural organization of EZ2 is conserved in the orthologous genes of at least eight families of rosids, suggesting that architectural features such as the distance between the two P1BS motifs are also important for the regulatory properties of this enhancer element.

Fig. 1.

Deletion analysis of the PLDZ2 promoter. (A) Structure of the 1,232 bp PLDZ2 promoter showing potential transcription binding DNA motifs. (B) PLDZ2 full-length promoter and end-points of progressive promoter deletions. Promoter lengths are indicated with respect to the start codon. Positions of putative P1BS elements are shown as black vertical lines. (C) GUS specific activity determined by fluorometric assays for a representative line of each promoter deletion construct. Values indicate the mean activity obtained from four independent replicate reactions. SD for each value is shown. (This figure is available in colour at JXB online.)

Fig. 2.

Effect of Pi availability on the tissue-specific expression of the PLDZ2 promoter and deletion derivatives. Arabidopsis seedlings of a representative line harbouring PLDZ2::GUS and truncated constructs grown for 10 days in media supplemented with 1 mM (P+) or 0 mM Pi (P–), subjected to histochemical GUS assays, and photographed using Nomarsky optics. The name of each construct is indicated at the top of each set of three images, which show the expression pattern for cotyledons, lateral roots, and root apical meristems from top to bottom, respectively. Bars, 100 μm. (This figure is available in colour at JXB online.)

Fig. 3.

Expression of PLDZ2::GUS in Arabidopsis Col0 and phr1. (A) Tissue-specific expression of PLDZ2::GUS in 10-day-old seedlings grown for 10 days in media supplemented with 1 mM (P+) or 0 mM Pi (P–), subjected to histochemical GUS assays, and photographed using Nomarsky optics. (B) GUS specific activity of PLDZ2::GUS in Col0 and phr1 seedlings grown under P-sufficient and P-limited conditions. (C) Quantitative reverse-transcription PCR analysis of PLDZ2, IPS1, and ATPT2 in Col0 and phr1. Expression levels are reported as relative expression of the corresponding gene in Col0 grown under Pi-sufficient conditions. UBQ 11 was used as a non-responsive control. (This figure is available in colour at JXB online.)

Fig. 4.

Pi-starvation responsiveness of EZ2, an enhancer element present in the PLDZ2 promoter. (A) Structure of a chimeric promoter composed of a PLDZ2 Pi-responsive enhancer inserted upstream of the –46 35S minimal promoter to direct expression of GUS (EZ2::GUS). (B) Arabidopsis seedlings of a representative line harbouring PLDZ2::GUS and EZ2::GUS constructs grown for 10 days in media supplemented with 1 mM (P+) or 0 mM (P–), subjected to histochemical GUS assays, and photographed using Nomarsky optics. The name of each construct is indicated at the top of each set of three images, which show the expression pattern for cotyledons, lateral roots, and root apical meristems from top to bottom, respectively. Bars, 100 μm. (This figure is available in colour at JXB online.)

Fig. 5.

Effect of sucrose and benzilaminopurine (BAP) on the expression of PLDZ2::GUS and EZ2::GUS. Seven-day-old PLDZ2::GUS and EZ2::GUS seedlings grown in 1 mM (P+) or 0 mM Pi (P–) solid medium were transferred into liquid medium with the same concentration of Pi supplemented with different concentrations of sucrose (0, 0.5, 1, 3, and 5%) or BAP (10⁻⁷, 10⁻⁶, and 10⁻⁵ M). Seedlings were harvested after 48 h of treatment for fluorometric GUS assay. Error bars represent SD of four replicates. Asterisks indicate significant differences with respect to 0.5% sucrose group (P < 0.05; ANOVA and Tukey analyses).

Fig. 6.

Two P1BS motifs are necessary for the function of the EZ2 enhancer. (A) Diagram showing the structure of the EZ2 enhancer element and its derivatives with mutations in P1BS4 (EZ2mP1BS4), P1BS3 (EZ2mP1BS3), or both (EZ2mP1BS4–3). (B) Seedlings grown for 10 days in media supplemented with 1 mM (P+) or 0 mM Pi (P–), subjected to histochemical GUS assays, and photographed using Nomarsky optics. The name of each construct is indicated at the top of each set of three images, which show the expression pattern for cotyledons, lateral roots, and root apical meristems from top to bottom, respectively. Bars, 100 μm. (This figure is available in colour at JXB online.)

Fig. 7.

P1BS motifs in EZ2 have different properties. (A) Diagram showing the structure of the EZ2 enhancer element and its derivatives containing either two copies of P1BS4 [EZP1BS4(2X)] or P1BS3 [EZP1BS3(2X)] motifs. (B) Seedlings grown for 10 days in media supplemented with 1 mM (P+) or 0 mM Pi (P–), subjected to histochemical GUS assays, and photographed using Nomarsky optics. The name of each construct is indicated at the top of each set of three images, which show the expression pattern for cotyledons, lateral roots, and root apical meristems from top to bottom, respectively. Bars, 100 μm. (This figure is available in colour at JXB online.)

Fig. 8.

Role of the sequences adjacent to the P1BS motifs in the function of the EZ2 enhancer. (A) Diagram showing the structure of the EZ2 enhancer element and its mutant derivatives where deletions and substitutions were introduced. The region upstream of P1SB4 (m5'EZ2), the spacer sequence between P1BS4 and P1BS3 (EZ2mMR), or both the upstream and spacer sequences (m5'EZ2mMR) were replaced by random DNA sequences and the distance between the P1BS sites was reduced by 5 bp (EZ2-5b). (B) Seedlings grown for 10 days in media supplemented with 1 mM (P+) or 0 mM Pi (P–), subjected to histochemical GUS assays, and photographed using Nomarsky optics. The name of each construct is indicated at the top of each set of three images, which show the expression pattern for cotyledons, lateral roots, and root apical meristems from top to bottom, respectively. Bars, 100 μm. (This figure is available in colour at JXB online.)

Fig. 9.

Sequence comparison of the PLDZ2 promoter with orthologous promoter regions. (A) Schematic representation of the promoter region of orthologues for PLDZ2 showing the location of the P1BS motifs (vertical lines) and the region corresponding to the EZ2 element (horizontal lines). Scale represents location in bp relative to the start codon. (B) Sequence comparison of the EZ2 element with similar regions found in orthologous promoter sequences. P1BS motif and other conserved sequences are shaded. Gaps (–) were included for clarity of the alignment. (This figure is available in colour at JXB online.)