Transcriptional Regulation of Nitrate Reductase mRNA Levels by Cytokinin-Abscisic Acid Interactions in Etiolated Barley Leaves

Abstract

To investigate the mechanisms by which the expression of a specific gene may be modulated by multiple hormones, the regulation of nitrate reductase (NR) mRNA levels by cytokinin and abscisic acid (ABA) was studied in etiolated barley (Hordeum vulgare L. cv Robust) leaves using a barley NR cDNA as a probe. Northem blot analyses of the levels of NR mRNA indicate that accumulation of the mRNA was enhanced by the cytokinin, benzyladenine (BA), and suppressed by ABA and that the hormonal effects on NR gene expression were responsive to dose concentrations (10-' to 10-4 molar). The NR mRNA levels were influenced by the BA/ABA concentration ratios, and the inhibition of the NR transcript levels by ABA was not totally reversed by addition of equal concentrations (10-to 10-4 molar) of BA. Nuclear runoff transcription studies showed that the level of transcription was suppressed by ABA, and addition of an equal concentration of BA partially reversed the ABA action. Thus, the interaction of cytokinin and ABA on NR gene expression is, at least in part, at the level of transcription.is, at least in part, at the level of transcription ( 13,21). Genes having related 5'-regulatory region DNA sequences are similarly regulated by either ABA (13, 29), GA3 ( 15), ethylene (2), or auxin (20), but the regulatory elements of genes responsive to these hormones have not been fully identified.The genetics and molecular biology of NR3 have been studied extensively (3, 5, 8,17,23), and the NR activity has been shown to be enhanced by cytokinins (16,18,19) (27). However, the mechanisms by which specific genes are regulated by multiple hormones are not well understood.The primary actions of cytokinins and ABA in the plant cells have not been fully elucidated at the molecular level. Cytokinins enhance and/or suppress the levels of specific mRNAs (4, 7, 9,19,26), and the enhancement has been shown to be mainly at the level of transcription (19) or posttranscription (9). ABA, like other phytohormones, induces, promotes, or suppresses the levels of specific mRNAs (1 1, 12, 21, 24), and the influence of ABA on gene expression Growth of PlantsBarley (Hordeum vulgare L. cv Robust ) seeds were soaked in water for 8 h and grown on sterile vermiculite at 25°C in the dark for 4 d. The seedlings (7-9 cm in length) were gently removed from the vermiculite and rinsed with water under green light. About 60 seedlings were placed into each 400-mL beaker containing 100 mL of 50 mm KNO3. The leaves were sprayed with 8 mL of the treatment solution (water or hormones dissolved in water) and placed at 25°C in darkness or cool-white fluorescent light (about 16 klx) for various time intervals. Excised leaves were frozen immediately in liquid nitrogen and stored at -700C. NR AssayThe following protocol was used to isolate and assay for NR in vitro. Barley leaves (3 g) were crushed in a mortar at 0 to 4°C in 3 mL of 25 mm potassium phosphate buffer (pH 7.5) containing 5 mm cysteine and 5 mM EDTA. The extract was strained through two layers of cheesecloth and...

Posttranscriptional and Posttranslational Control of Enolase Expression in the Facultative Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L

“…Pgkl mRNAs accumulate in leaves in response to cytokinin (6-BAP) treatment. Although cytokinins have been implicated in a wide variety of plant responses, including transcriptional activation of nitrate reductase (Lu et al, 1990(Lu et al, , 1992 and PEPC (Suzuki et al, 1994) and increases in PEPC and NADP-malic enzyme accumulation and activity in the ice plant (Dai et al, 1994), their role in controlling gene expression in response to osmotic stress is still not fully understood. One possibility is that cytokinin response elements interact with cytokininactivated transcription factors (Thomas et al, 1995) to mediate transcriptional activation events.…”

Transcriptional and Posttranscriptional Regulation of Nitrogen-Responding Expression of Phosphoenolpyruvate Carboxylase Gene in Maize

“…For example, the enhancement of the levels of NR mRNAs by cytokinins has been shown to be regulated transcriptionally (Lu et al, 1990(Lu et al, , 1992, but the cytokinin enhancement of the levels of LHCP mRNAs has been shown to be regulated posttranscriptionally Tobin, 1986, 1989). The products of these genes could be categorized as appendages, of which gene expression is inducible in nature in responding to environmental stimuli.…”

“…It has been shown that cytokinins also regulate the expression of various genes. In the cases of Rubisco small subunit (Lerbs et al, 1984;Funckes-Shippy and Levine, 1985;Flores and Tobin, 1989), LHCP Tobin, 1986, 1989), and NR (Lu et al, 1990(Lu et al, , 1992, up-regulation of the gene expression by cytokinins is considered to be due to an increase in rate of gene-specific transcription and/or an increase in mRNA stability. It is well known that changes in N availability strongly affect the level of cytokinins, which are thought to be synthesized in roots at least at the vegetative stage of higher plants (Feldman, 1975).…”

5[prime] Proximal Regions of Arabidopsis Nitrate Reductase Genes Direct Nitrate-Induced Transcription in Transgenic Tobacco

“…Light signals at red/far-red (Rajasekhar et al, 1988;Melzer et al, 1989) and at blue (Melzer et al, 1989) regions of the spectrum both increase the NR mRNA levels of etiolated seedlings. Cytokinin enhances, whereas ABA suppresses, the light induction of NR transcription in etiolated barley leaves (Lu et al, 1990(Lu et al, , 1992. The biological significance of light signal and hormonal regulation is unclear.…”