Okadaic Acid Mimics Nitrogen-Stimulated Transcription of the NADH-Glutamate Synthase Gene in Rice Cell Cultures

Abstract

Okadaic acid (OKA), a potent and specific inhibitor of protein serine/threonine phosphatases 1 and 2A, induced the accumulation of NADH-glutamate synthase (GOGAT) mRNA within 4 h in rice (Oryza sativa L.) cell cultures. In contrast to the transient accumulation of NADH-GOGAT mRNA by NH(4)(+), OKA caused a continuous accumulation for at least 24 h. The induction of NADH-GOGAT mRNA by OKA was not inhibited in the presence of methionine sulfoximine, which inhibited the NH(4)(+)-induced accumulation of mRNA. These results suggest that the OKA-sensitive protein phosphatase is involved in the regulation of NADH-GOGAT gene expression and probably plays a role in the signal transduction pathway downstream from NH(4)(+), although a signal transduction pathway other than that of nitrogen sensing could be responsible. Nuclear run-on assays demonstrated that the accumulation of NADH-GOGAT mRNA induced by the supply of either NH(4)(+) or OKA was mainly regulated at the transcription level. OKA effects were synergistic to the NH(4)(+)-induced expression of the NADH-GOGAT gene. In the presence of K-252a, a protein kinase inhibitor, the accumulation of NADH-GOGAT mRNA induced by either NH(4)(+) or OKA was reduced. The possible roles of protein phosphatases in the regulation of NADH-GOGAT gene expression are discussed.

Figure 1

Time-course studies of NADH-GOGAT mRNA accumulation induced by 1 μm OKA (A), 1 μm calyculin A (B), or 20 mm NH₄Cl (C). RNA gel-blot analyses were performed with digoxigenin-labeled cDNA probes for NADH-GOGAT, tubulin, or glyceraldehyde 3-P dehydrogenase (GAPDH). The ethidium-bromide-stained ribosomal bands are shown as a loading control.

Figure 2

Effects of MSX on the induction of NADH-GOGAT mRNA accumulation by NH₄⁺ and OKA. Suspension-cultured rice cells were pretreated with 10 μm MSX for 30 min, and then treated with 20 mm NH₄Cl or 1 μm OKA for 6 or 12 h, respectively. Total RNA was isolated and subjected to RNA gel-blot analysis using digoxigenin-labeled cDNA probes for NADH-GOGAT or tubulin. The ethidium-bromide-stained ribosomal bands are shown as a loading control.

Figure 3

Comparison of the rate of nuclear run-on transcription with mRNA accumulation. A, Suspension-cultured rice cells were treated with 20 mm NH₄Cl (N), 1 μm OKA, their combination (N + OKA), or 0.1% (v/v) DMSO (control) for 6 h. Nuclei were prepared and subjected to transcriptional run-on assays as described in “Materials and Methods.” Nylon membrane containing the indicated plasmids were hybridized with the in vitro-labeled RNA and autoradiographed. B, RNA gel-blot analysis was performed using total RNA isolated from the same cells as in A. Hybridization was performed with [α-³²P]dCTP-labeled NADH-GOGAT cDNA probes or digoxigenin-labeled tubulin cDNA probes. The ethidium-bromide-stained ribosomal bands are shown as a loading control. C, Signals from run-on transcription and RNA gel-blot analyses were quantified with a bioimaging analyzer and plotted as the increase (-fold) in signal relative to that of zero time. White bars, rDNA gene transcription; hatched bars, tubulin gene transcription; black bars, NADH-GOGAT gene transcription; cross-hatched bars, NADH-GOGAT mRNA accumulation. Error bars represent ±sd for three independent experiments.

Figure 4

A, Dose dependence of NADH-GOGAT mRNA accumulation induced by OKA. Suspension-cultured rice cells were treated with the indicated concentrations of OKA for 12 h. B, Suspension-cultured rice cells were treated with 0.5 μm OKA (lane 1), 1 μm OKA (lane 2), 0.5 μm 1-norokadaone (lane 3), 1 μm 1-norokadaone (lane 4), or 0.1% (v/v) DMSO (lane 5) for 12 h. RNA gel-blot analyses were performed using the digoxigenin-labeled cDNA probes for NADH-GOGAT or tubulin. The ethidium bromide-stained ribosomal bands are shown as a loading control.

Figure 5

Effects of protein kinase inhibitors on the induction of NADH-GOGAT mRNA by NH₄⁺ and OKA. Suspension-cultured rice cells were pretreated with 1 μm staurosporine (STA) or 1 μm K-252a for 1 h, and then treated with 20 mm NH₄Cl or 1 μm OKA for 6 or 12 h, respectively. Total RNA was isolated and subjected to RNA gel-blot analysis using the digoxigenin-labeled cDNA probes for NADH-GOGAT or tubulin. The ethidium-bromide-stained ribosomal bands are shown as a loading control.