A Soluble Guanylate Cyclase Mediates Negative Signaling by Ammonium on Expression of Nitrate Reductase in Chlamydomonas  

Abstract

Nitrate assimilation in plants and related organisms is a highly regulated and conserved pathway in which the enzyme nitrate reductase (NR) occupies a central position. Although some progress has been made in understanding the regulation of the protein, transcriptional regulation of the NR gene (NIA1) is poorly understood. This work describes a mechanism for the ammonium-mediated repression of NIA1. We report the characterization of a mutant defective in the repression of NIA1 and NR in response to ammonium and show that a gene (CYG56) coding for a nitric oxide (NO)-dependent guanylate cyclase (GC) was interrupted in this mutant. NO donors, cGMP analogs, a phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), and a calcium ionophore (A23187) repress the expression of NIA1 in Chlamydomonas reinhardtii wild-type cells and also repress the expression of other ammonium-sensitive genes. In addition, the GC inhibitors LY83,583 (6-anilino-5,8-quinolinedione) and ODQ (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one) release cells from ammonium repression. Intracellular NO and cGMP levels were increased in the presence of ammonium in wild-type cells. In the cyg56 mutant, NIA1 transcription was less sensitive to NO donors and A23187, but responded like the wild type to IBMX. Results presented here suggest that CYG56 participates in ammonium-mediated NIA1 repression through a pathway that involves NO, cGMP, and calcium and that similar mechanisms might be occurring in plants.

Hypoxic survival requires a 2-on-2 hemoglobin in a process involving nitric oxide

“…Nitrite-dependent NO production, absent in a nitrate reductase mutant (34), suggests a nitrate reductase-dependent process like that in higher plants (32,35). NO synthesis in C. reinhardtii supplied with ammonium versus nitrate is sensitive to the Arg analog N ω -Nitro-L-arginine methyl ester (L-NAME) (28), an inhibitor of mammalian NO synthases. Because the C. reinhardtii wild type used in this study is a natural nitrate reductase mutant, the former reaction cannot be responsible for hypoxic NO generation in this strain.…”

“…In plants, a variety of NO-producing reactions, including the respiratory chain, have been described and probably differ depending on the organ and the environmental stimulus (35). In Chlamydomonas, NO has been shown to be also involved in the acclimation of the alga to growth on ammonium versus nitrate (28) or to copper stress (36), indicating that NObased signaling pathways in Chlamydomonas are as versatile as in higher plants (35) or mammals (37).…”

“…More pronounced differences of transcript fold-changes in amiTHB8-strains versus wild type were observed for CYG12. The CYG12 gene encodes a soluble guanylyl cyclase (sGC) (27,28). sGCs of mammals are heme-containing heterodimeric NO sensors that are activated upon binding of NO to the heme group (29).…”

Nitric Oxide–Triggered Remodeling of Chloroplast Bioenergetics and Thylakoid Proteins upon Nitrogen Starvation inChlamydomonas reinhardtii

“…THB2 and CYG11 code for a truncated hemoglobin and for a NO-responsive guanylate cyclase, respectively, whose transcription is induced during nitrogen starvation. (de Montaigu et al, 2010;Sanz-Luque et al, 2013). Here, the coregulation of cytochrome b 6 f degradation and nucleus-encoded LAO1 induction upon nitrogen starvation, both being slowed in nit1 mutants and accelerated in nit2 mutants, suggests that NO acts as a signaling molecule at least for the transcriptional induction of LAO1.…”

“…Furthermore L-Arg plays little role in NO production under nitrogen replete conditions, whether in C. reinhardtii (Sakihama et al, 2002) or in the closely related alga Scenedesmus obliquus (Mallick et al, 2000b). Nevertheless, the NO-dependent repression of the nitrate assimilation pathway by ammonium is partially prevented upon addition of the L-Arg analog L-NAME, a known NOS inhibitor, suggesting some NOS-like activity in C. reinhardtii (de Montaigu et al, 2010). In any event, we could not test the effect of L-NAME on the loss of the cytochrome b 6 f complex during nitrogen starvation because the drug was efficiently used as a source of nitrogen by C. reinhardtii (data not shown).…”