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

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.

The interaction of light and abscisic acid in the regulation of plant gene expression.

Extended dark treatments of light-grown plants of both Lemna gibba and Arabidopsis thaliana resulted in substantial increases in abscisic acid (ABA) concentrations. The concentration of ABA could be negatively regulated by phytochrome action in Lemna. As has been noted in other species, ABA treatment reduced Lemna rbcS and Lhcb RNA levels, which are positively regulated by phytochrome in many species. In view of these observations, the possibility that phytochrome effects on gene expression may be mediated primarily by changes in ABA was tested using a transient assay in intact plants. The phytochrome responsiveness of the Lemna Lhcb2*1 promoter was still apparent in the presence of exogenous ABA. Additionally, when 2-bp mutations were introduced into this promoter so that phytochrome responsiveness was lost, a response to exogenous ABA was still present. We conclude that phytochrome- and ABA-response elements are separable in the Lhcb2*1 promoter. We tested whether the effects of ABA on RNA abundance could be inhibited by treatment with gibberellin and found no evidence for such an inhibition. We have also found that the ABA-responsive Em promoter of wheat can be negatively regulated by phytochrome action. It is likely that this regulation is mediated at least in part by phytochrome-induced changes in ABA levels. Our results demonstrate that it is essential to take into account that dark treatments and the phytochrome system can affect ABA levels when interpreting studies of light-regulated genes.

NPR genes, which are negatively regulated by phytochrome action in Lemna gibba L. G-3, can also be positively regulated by abscisic acid.

We have found that NPR1 and NPR2, two genes from Lemna gibba L. G-3 that can be negatively regulated by phytochrome action, can also be positively regulated by the plant hormone abscisic acid (ABA). Both genes were responsive to low concentrations of exogenous ABA; an increase in NPR1 RNA could be detected in response to concentrations as low as 10 nM. We have also tested phytochrome responsiveness of 5' promoter-deletion constructs of one of these genes, NPR1, in transient assays utilizing particle bombardment. This analysis demonstrated that DNA sequences important for phytochrome regulation are present downstream of -198 from the transcription start site. A response to ABA treatment could also be observed in the transient assay system. When intact plants were placed in darkness, there was an increase in ABA levels as well as increased levels of NPR1 and NPR2 RNA.