Identification of a cis-regulatory element involved in phytochrome down-regulated expression of the pea small GTPase gene pra2

Abstract

The pra2 gene encodes a pea (Pisum sativum) small GTPase belonging to the YPT/rab family, and its expression is down-regulated by light, mediated by phytochrome. We have isolated and characterized a genomic clone of this gene and constructed a fusion DNA of its 5'-upstream region in front of the gene for firefly luciferase. Using this construct in a transient assay, we determined a pra2 cis-regulatory region sufficient to direct the light down-regulation of the luciferase reporter gene. Both 5'- and internal deletion analyses revealed that the 93-bp sequence between -734 and -642 from the transcriptional start site was important for phytochrome down-regulation. Gain-of-function analysis showed that this 93-bp region could confer light down-regulation when fused to the cauliflower mosaic virus 35S promoter. Furthermore, linker-scanning analysis showed that a 12-bp sequence within the 93-bp region mediated phytochrome down-regulation. Gel-retardation analysis showed the presence of a nuclear factor that was specifically bound to the 12-bp sequence in vitro. These results indicate that this element is a cis-regulatory element involved in phytochrome down-regulated expression.

Figure 1

Nucleotide sequence of the pea pra2 gene. Nucleotides are numbered on the right side, with the transcriptional start site designated +1. Amino acids are numbered on the left side, with the first residue of the protein designated +1. Arrowheads show exon-intron boundaries. The 113-bp inverted repeat element is underlined. Arrows show inverted repeats. The location of the 93-bp region is boxed.

Figure 2

Determination of the experimental conditions. a, Effect of cutting the stem on pra2 protein levels. Total proteins from the stem (1.0 cm from the top of the hook) were extracted, separated by SDS-PAGE, and probed with anti-pra2 protein IgG. The same amount of protein (30 μg) was put in each lane. Six-day-old seedlings grown in darkness (lane 1) were irradiated with white light for 12 h (lane 2). Stem sections (1.0 cm from the top of the hook) of the 6-d-old seedlings were cut and kept in darkness for 12 h on wet cotton (lane 3). The growing zone of etiolated 6-d-old seedlings were bombarded with gold particles and kept in darkness for 12 h (lane 4). b, Effect of pra2 upstream fragment on the reporter-enzyme activity in stems of intact plants and sections. The PL1 construct was introduced into the growing zone of intact etiolated stem (left) or stem section (1 cm from the top of the hook; right) by particle bombardment with the 35S-GUS construct as the internal standard. Reporter-enzyme activity was measured after 12 h of darkness (D) or 12 h of white-light irradiation (L). Relative activity was defined in “Materials and Methods,” and the average of PL1 in darkness was taken to be 100. Values are the means of at least four independently bombarded samples with error bars representing se (n ≥ 4). c, Comparison of reporter-gene expression in different parts of intact stem. PL1 construct was bombarded into the indicated parts, and the reporter-enzyme activity was measured as described.

Figure 3

5′-Deletion analysis of the pra2 upstream region. The 5′-deletion constructs containing the pra2 upstream region were fused to the promoter-less pBI221-LUC+ plasmid. The numbers refer to the 5′ end of the pra2 upstream fragments from the transcriptional start site in Figure 1. The names of the resulting plasmids are indicated. Equivalent amounts of pBI221-LUC+ plasmid DNA fused to different pra2 upstream regions were introduced into the growing zone of etiolated pea stem by particle bombardment, with the 35S-GUS construct as the internal standard. After bombardment, samples were kept in darkness (D) or under continuous white light (L) for 12 h. Relative activity is defined in Figure 2b. Values are the means of at least four independently bombarded samples with error bars representing se (n ≥ 4). UTR, Untranslated region; NOS, nopaline synthase terminator.

Figure 4

Gain-of-function analysis. A, CaMV 35S promoter of 90 bp from the transcriptional start site fused to the LUC reporter gene was used as the control construct. To the control construct, several 3′-deleted fragments of the pra2 upstream region were fused. The names of the resulting plasmids are indicated on the right. White bold line, 93-bp region; thin line, deleted region. Control or GF1 to GF5 constructs were introduced into the growing zone of etiolated, intact stem by particle bombardment, with the 35S-GUS construct as the internal standard. After bombardments, the plants were kept in darkness (D) or irradiated with white light (L) for 12 h. Values are the means of at least four independently bombarded samples, with error bars representing se (n ≥ 4). Relative activity is defined in Figure 2b. NOS, Nopaline synthase terminator.

Figure 5

Red/far-red reversibility of the change in the reporter-enzyme activity. Different kinds of constructs containing the pra2 upstream region were prepared as described in Methods. The names of the resulting plasmids are indicated on the right. White bold line, 93-bp region; thin line, deleted region. Equivalent amounts of each plasmid were introduced into 5- or 6-d-old etiolated seedlings as described in Methods. After bombardment, the plants were exposed to red light for 2 min (R) or far-red light for 5 min immediately after red light for 2 min (R/FR) and then were returned to darkness for 12 h. Dark and far-red controls are indicated as D and F, respectively. Values are the means of 5 to 11 independently bombarded samples, with error bars representing se. Relative activity is defined in Figure 2b. A fine-scale figure of PL4C is shown in the inset. NOS, Nopaline synthase terminator.

Figure 6

LS analysis to define the 12-bp phytochrome down-regulated element. LS constructs in the pra2 5′-upstream region are shown. The positions of mutated nucleotides are indicated in lowercase and underlined. The WT sequence corresponds to the PL4A construct in Figure 5. The 12-bp phytochrome-responsive element is boxed. Equivalent amounts of each plasmid were introduced into 5- or 6-d-old etiolated seedlings as described. After bombardment, the plants were exposed to red light for 2 min (R) and then returned to darkness for 12 h. D, Dark control. Values are the means of at least five independently bombarded samples, with error bars representing se. Relative activity is defined in Figure 2b.

Figure 7

Gel-mobility shift assays with the synthetic oligonucleotides. a, Sequences of synthetic oligonucleotides used in this experiment are shown. The 12-bp sequence is boxed. Nucleotides in the MT oligonucleotide that are different from the WT oligonucleotide are indicated in lowercase with arrows. b, Binding of nuclear proteins from pea stems to the WT oligonucleotide and competition with the WT and MT oligonucleotides. The 12-bp-specific bound complex is indicated by the arrow. D and L indicate the nuclear extracts of dark-grown plants and plants exposed to light for 6 h, respectively.