The phytochrome response of the Lemna gibba NPR1 gene is mediated primarily through changes in abscisic acid levels

Abstract

Two important signaling systems involved in the growth and development of plants, those triggered by the photoreceptor phytochrome and the hormone abscisic acid (ABA), are involved in the regulation of expression of the NPR1 gene of Lemna gibba. We previously demonstrated that phytochrome action mediates changes in ABA levels in L. gibba, correlating with changes in gene expression evoked by stimulation of the phytochrome system. We have now further characterized phytochrome- and ABA-mediated regulation of L. gibba NPR1 gene expression using a transient particle bombardment assay, demonstrating that regulatory elements controlling responses to both stimuli reside within 156 nucleotides upstream of the transcription start. Linker scan (LS) analysis of the region from -156 to -70 was used to identify two specific requisite and nonredundant cis-acting promoter elements between -143 to -135 (LS2) and -113 to -101 (LS5). Mutation of either of these elements resulted in a coordinate loss of regulation by phytochrome and ABA. This suggests that, unlike the L. gibba Lhcb2*1 promoter, in which phytochrome and ABA regulatory elements are separable, the phytochrome response of the L. gibba NPR1 gene can be attributed to alterations in ABA levels.

Figure 1

Sequences downstream of −156 from the transcription start confer a response to both ABA and phytochrome action. Following bombardment with the NPR1–354 or NPR1–156 constructs, intermittent R-grown L. gibba were treated with D (black bars), 2 min of R (white bars), or 2 min of R plus 10 μm ABA (cross-hatched bars); D and R samples received water as a control. Bombarded plants were returned to D for 16 to 18 h before reporter gene activity was assayed. The normalized ratios of reporter LUC to internal standard GUS activities are reported as relative activities; se values are shown.

Figure 2

LS constructs in the L. gibba NPR1 promoter. The wild-type sequence of the L. gibba NPR1 promoter between −156 and −70 from the transcriptional start site is given. The range of each LS construct is shown directly below the corresponding sequence, with mutated residues given in lowercase. Black boxes indicate the positions of ACGT motifs; the gray box indicates the position of a CE3-like motif. WT, Wild type.

Figure 3

Phytochrome responsiveness of L. gibba NPR1 LS constructs. The range of each LS construct is diagrammed on a schematic of the NPR1 promoter between −156 and −70 from the transcriptional start. Black boxes indicate the positions of ACGT motifs; the gray box indicates the position of a CE3-like motif. Following bombardment with the designated LS mutant or wild-type NPR1–156 construct, plants were treated with D (black bars) or 2 min of R (white bars) and returned to D for 16 to 18 h before reporter gene activity was assayed. The normalized ratios of reporter LUC to internal standard GUS activities are reported as relative activities; se values are shown. WT, Wild type.

Figure 4

ABA responsiveness of L. gibba NPR1 LS constructs. The range of each LS construct is diagrammed on a schematic of the NPR1 promoter between −156 and −70 from the transcriptional start. Black boxes indicate the positions of ACGT motifs; the gray box indicates the position of a CE3-like motif. Following bombardment with the designated LS mutant or wild-type NPR1–156 construct, plants were treated with 2 min of R plus water (white bars) or 10 μm ABA (cross-hatched bars) and returned to D for 16 to 18 h before reporter gene activity was assayed. The normalized ratios of reporter LUC to internal standard GUS activities are reported as relative activities; se values are shown. WT, Wild type.

Figure 5

Phytochrome and ABA regulatory elements converge in the L. gibba NPR1 promoter. Following bombardment with NPR1-LS2 (LS2), NPR1-LS5 (LS5), or the wild-type NPR1–156 (WT) construct, plants were treated with D (black bars), 2 min of R (white bars), or 2 min of R plus 10 μm ABA (cross-hatched bars); D and R samples received water as a control. Bombarded plants were returned to D for 16 to 18 h before reporter gene activity was assayed. The normalized ratios of reporter LUC to internal standard GUS activities are reported as relative activities; se values are shown.