Fig. 5. Signaling by inorganic vs. organic nitrogen can be distinguished by using MSX and Glu treatments. (a) Treatments include N±MSX±Glu. (b) A simplified diagram of the N-metabolic pathway from inorganic nitrate (NO₃) to organic Glu, and the block by MSX. Below this pathway are the predicted effects of the given treatments on nitrogen metabolism. Arrows indicate progression through the pathway. Line breaks, represented with a short perpendicular line, indicate the step in the pathway blocked by MSX. (c and d) The expected transcript levels for genes induced by inorganic nitrogen (c) vs. genes regulated by Glu or a Glu-derived metabolite (d).

Fig. 6. Analysis of the expression of asparagine synthetase genes. Shown is a comparison of ASN1 (a) and ASN2 (b) mRNA levels in control seedlings (transferred to 1 mM NO3) along with MSX control (treated with NO3 and 1 mM MSX) compared to seedlings treated with a stepwise combination of Nms, MSX, and Glu/Gln. (a) mRNA levels of ASN1 are increased in Nms, are sensitive to MSX treatment, and can be recovered with exogenous application of Glu or Gln. (b) mRNA levels of ASN2 are increased in Nms. However, this expression is insensitive to MSX treatment and is slightly repressed with exogenous application of Glu or Gln. mRNA levels were normalized to EIF4A (At3g13920).

Fig. 7. RT-qPCR confirmation of the regulation for two transcription factors TAZ and bZIP1. Shown is confirmation of TAZ (A) and bZIP1 (B) mRNA levels in control seedlings along with the MSX control and compared to seedlings treated with a stepwise combination of Nms, Nms+MSX, and Nms+MSX+Glu (or Gln). In both cases, although increased expression in the presence of N is blocked in the presence of MSX, this suppression can be overcome by exogenous application of Glu or Gln. Plants transferred to control media do not show mRNA levels different from treatments without MSX. Primers used for RT-qPCR are as follows: TAZ forward, 5'-TCCTCGTCTCGGTCTT-3'; reverse, 5'-CAACCACCAGGGATTC-3'; bZIP forward, 5'-TCAGGTTCCGACATAGATG-3'; reverse, 5'-CCACGGTGTACGTCTACA-3'.

Fig. 8. Analysis of the expression of bZIP1 in the CCA1-ox. To test some of the predictions of our network CCA1-ox and Col-0 plants were collected 3 h after dawn; three biological replicates were taken at each time point. RNA was extracted from whole seedlings (as described in Materials and Methods), and RT-qPCR was performed to measure mRNA levels for bZIP1 (At5g49450). Two technical replicates were carried out for each sample. mRNA levels were normalized to clathrin (At4g24550).

Fig. 9. Circadian regulation of the response of clock gene (CCA1) expression to N-assimilation inhibitors and inorganic and organic N. Mean ± SEM luciferase activity of CCA1::LUC in response to exogenous inorganic N, Glu, or Gln is presented. Seedlings were entrained for 8 days in a 16-h white light/8-h dark photoperiod on MS medium containing 1 mM KNO₃ before being transferred to continuous light and exposed for 4-h pulses of inorganic N (20 mM KNO₃/20 mM NH₄NO₃), 10 mM Glu, or 10 mM Gln presented at 3-h intervals over one circadian cycle before return to MS medium containing 1 mM KNO₃ in continuous light for luciferase measurements for 6 days. Luciferase activity values were normalized by the mean expression value for the treatment. The entraining photocycle is indicated by the vertical white (light) and gray (dark) bars.