Dual positive and negative control of Chlamydomonas PII signal transduction protein expression by nitrate/nitrite and NO via the components of nitric oxide cycle

Abstract

Background: The PII proteins constitute a large superfamily, present in all domains of life. Until now, PII proteins research in Chloroplastida (green algae and land plants) has mainly focused on post-translation regulation of these signal transductors. Emerging evidence suggests that PII level is tightly controlled with regard to the nitrogen source and the physiological state of cells.

Result: Here we identify that a balance of positive (nitrate and nitrite) and negative (nitric oxide) signals regulates Chlamydomonas GLB1. We found that PII expression is downregulated by ammonium through a nitric oxide (NO)-dependent mechanism. We show that nitrate reductase (NR) and its partner, truncated hemoglobin 1 (THB1), participate in a signaling pathway for dual control of GLB1 expression. Moreover, NO dependent guanilate cyclase appeared to be involved in the negative control of GLB1 transcription.

Conclusion: This study has revealed the existence of the complex GLB1 control at transcription level, which is dependent on nitrogen source. Importantly, we found that GLB1 gene expression pattern is very similar to that observed for nitrate assimilation genes, suggesting interconnecting/coordinating PII-dependent and nitrate assimilation pathways.

Keywords: Chlamydomonas reinhardtii; NO signaling; Nitrate; Nitrite; PII signal transduction protein; Truncated hemoglobin.

Fig. 1

PII expression is increased by nitrate. a Time course of the GLB1 transcripts accumulation during incubation of illuminated cells in nitrate-containing medium. Chlamydomonas cells of cw15–325 strain were grown in ammonium-containing medium (Con, control) and transferred to media containing 4 mM, 100 μM, or 10 μM NO₃⁻ in the light for 0.5 h, 1 h, 2 h or 4 h. Values are means ± SE of three biological replicates and three technical replicates and are given as expression level relative to a house-keeping gene RACK1 that has a value of 1.The increase in NIT1 transcript abundance was used as a positive control (insertion). b Changes in nitrate levels in the media. Chlamydomonas cells of cw15–325 strain were grown in ammonium-containing medium and transferred to media containing 4 mM, 100 μM, or 10 μM NO₃⁻ in the light. At the indicated times, the amounts of nitrate present in the media were quantified. ND, not determined. Values are means ± SE of three biological replicates. cTime course of the PII protein accumulation during incubation of illuminated cells in nitrate-containing medium. PII levels were analyzed by western blotting in the same conditions as in (a). Each line corresponds to 20 μg of soluble proteins extracted from samples taken from cultures at the time points indicated. Quantitation of protein blots is given as proportion of signal in test variant to control variant. HSP70B signal served as a loading control

Fig. 2

GLB1 transcription depends on the balance of ammonium and nitrate in the medium. a GLB1 transcript levels were determined in Chlamydomonas cells of cw15–325 strain grown in ammonium-containing medium and transferred to media containing 4 mM NO₃⁻, 4 mM NO₃⁻ + 7.5 mM NH₄⁺ or 4 mM NO₃⁻ + 1 mM NH₄⁺. Values are means ± SE of three biological replicates and three technical replicates and are given as expression level relative to a house-keeping gene RACK1 that has a value of 1. Expression level at 0.5 h is considered as a control. The increase in NIT1 transcript abundance was used as a positive control (insertion). b GLB1 transcript levels were determined in Chlamydomonas cells of cw15–325 strain grown in ammonium-containing medium and transferred to media containing the indicated concentrations of NO₃ ⁻ and 1 mM NH₄⁺ for 2 h. Values are means ± SE of three biological replicates and three technical replicates and are given as expression level relative to a house-keeping gene RACK1 that has a value of 1

Fig. 3

Effects of DEA-NONOate on the nitrate-induced expression of GLB1 gene and the levels of intracellular nitric oxide. a The effect of DEA-NONOate (10 μM, 50 μM or 100 μM) on the GLB1 transcripts accumulation was determined in 4 mM nitrate-induced C. reinhardtii cells of cw15–325 strain for 0.5 and 1 h. Nitrate-induced cells represent controls (set to 100%) without the added DEA-NONOate. Values are means ± SE of three biological replicates and three technical replicates and are given as expression level relative to a house-keeping gene RACK1 that has a value of 1. The increase in NIT1 transcript abundance was used as a positive control (insertion). b Fluorescence intensity due to intracellular NO was determined using 1 μM DAF-FM DA and was expressed as arbitrary units per μg protein. Cell autofluorescence was subtracted from the total fluorescence obtained. Insertion shows NO levels in the media (see Materials and Methods). Data are the means±SE from three independent experiments

Fig. 4

Nitrate reductase promotes NO-dependent GLB1 repression. a Time course of the GLB1 transcripts accumulation during incubation of illuminated cells in nitrite-containing medium. Chlamydomonas cells of the wild strain 6145c and its derivative mutant 305, affected in NAD(P) H-NR activity and without diaphorase-NR activity, were grown in ammonium-containing medium and transferred to medium containing 10 mM NO₂⁻ in the light for 1, 2, 3 or 4 h. Values are means ± SE of three biological replicates and three technical replicates and are given as expression level relative to a house-keeping gene RACK1 that has a value of 1. b Time course of the PII protein accumulation during incubation of illuminated cells of the strains 6145c and 305, in nitrite-containing medium. PII levels were analyzed by Western blotting in the same conditions as in (a). Each line corresponds to 10 μg of soluble proteins extracted from samples taken from cultures at the time points indicated. Quantitation of protein blots is given as proportion of signal in test variant to control variant. HSP70B signal served as a loading control. c NO production in nitrite-induced cells. Chlamydomonas cells of the strains 6145c and 305 were grown in TAP medium and transferred to nitrite-containing medium in the light for 15 min. Fluorescence intensity due to intracellular NO was determined using DAF-FM DA and was expressed as arbitrary units per μg chlorophyll . Cell autofluorescence was subtracted from the total fluorescence obtained. Fluorescence in the strain 305 is considered as control (set to 100%). Data are the means±SE from three independent experiments. Production of NO was measured by the microplate reader CLARIOstar (BMG). d NO visualization by confocal microscopy. Images of cells grown in TAP (TAP) or incubated in nitrite-containing medium (10 mM NO₂⁻) for 15 min. The left-hand panels show DAF-FM fluorescence (green color) while the right-hand panels show Chl autofluorescence (red color). Scale bar equals 10 μm

Fig. 5

Effects of reduced THB1 levels on GLB1 expression and NO generation. a Time course of the GLB1 transcripts accumulation during incubation of cw15–325 and amiRNA-THB1 cells in 4 mM nitrite-containing medium with or without DEA-NONOate (100 μM). Values are means ± SE of three biological replicates and three technical replicates and are given as expression level relative to a house-keeping gene RACK1 that has a value of 1. b Fluorescence increase was measured in cw15–325 and amiRNA-THB1 cells following the incubation in 4 mM nitrate with or without of DEA-NONOate (100 μM) for 15 min. Fluorescence intensity due to intracellular NO was determined using DAF-FM DA and was expressed as arbitrary units per μg protein. Cell autofluorescence was subtracted from the total fluorescence obtained. Data are the means±SE from three technical replicates of a representative experiment. Production of NO was measured by the microplate reader CLARIOstar (BMG)

Fig. 6

Effects of guanylate cyclase inhibitor on GLB1 repression in medium containing nitrate and ammonium. GLB1 transcript levels were determined in Chlamydomonas cells of cw15–325 strain grown in ammonium-containing medium and transferred to media containing 4 mM NO₃⁻ + 7.5 mM NH₄⁺ with or without 1 μM, 2.5 μM, 5 μM or 7.7 μM 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ). Values are means ± SE of three biological replicates and three technical replicates and are given as expression level relative to a house-keeping gene RACK1 that has a value of 1

Fig. 7

Model for a nitrogen-dependent control of GLB1 expression. Nitrate and nitrite act as positive regulators of GLB1 transcription (dotted arrows). Conversely, NO that is mediated via the components of nitric oxide cycle or via a rise in intracellular ammonium, represses GLB1 transcription (thick T-like line). GC may also control GLB1 expression in NO-GC-dependent manner