Reducing phosphorylation of nitrate reductase improves nitrate assimilation in rice

AbstractGenes and genomes can evolve through interchanging genetic material, this leading to reticular evolutionary patterns. However, the importance of reticulate evolution in eukaryotes, and in particular of horizontal gene transfer (HGT), remains controversial. Given that metabolic pathways with taxonomically-patchy distributions can be indicative of HGT events, the eukaryotic nitrate assimilation pathway is an ideal object of investigation, as previous results revealed a patchy distribution and suggested one crucial HGT event. We studied the evolution of this pathway through both multi-scale bioinformatic and experimental approaches. Our taxon-rich genomic screening shows this pathway to be present in more lineages than previously proposed and that nitrate assimilation is restricted to autotrophs and to distinct osmotrophic groups. Our phylogenies show a pervasive role of HGT, with three bacterial transfers contributing to the pathway origin, and at least seven well-supported transfers between eukaryotes. Our results, based on a larger dataset, differ from the previously proposed transfer of a nitrate assimilation cluster from Oomycota (Stramenopiles) to Dikarya (Fungi, Opisthokonta). We propose a complex HGT path involving at least two cluster transfers between Stramenopiles and Opisthokonta. We also found that gene fusion played an essential role in this evolutionary history, underlying the origin of the canonical eukaryotic nitrate reductase, and of a novel nitrate reductase in Ichthyosporea (Opisthokonta). We show that the ichthyosporean pathway, including this novel nitrate reductase, is physiologically active and transcriptionally co-regulated, responding to different nitrogen sources; similarly to distant eukaryotes with independent HGT-acquisitions of the pathway. This indicates that this pattern of transcriptional control evolved convergently in eukaryotes, favoring the proper integration of the pathway in the metabolic landscape. Our results highlight the importance of reticulate evolution in eukaryotes, by showing the crucial contribution of HGT and gene fusion in the evolutionary history of the nitrate assimilation pathway.

Download Full-text

Inactivation of gltB Abolishes Expression of the Assimilatory Nitrate Reductase Gene (nasB) in Pseudomonas putida KT2442

Journal of Bacteriology ◽

10.1128/jb.182.12.3368-3376.2000 ◽

2000 ◽

Vol 182 (12) ◽

pp. 3368-3376 ◽

Cited By ~ 8

Author(s):

Leo Eberl ◽

Aldo Ammendola ◽

Michael H. Rothballer ◽

Michael Givskov ◽

Claus Sternberg ◽

...

Keyword(s):

Nitrate Reductase ◽

Pseudomonas Putida ◽

Nitrogen Starvation ◽

Nitrate Assimilation ◽

Glutamate Synthase ◽

Nitrogen Sources ◽

Nucleotide Sequence Analysis ◽

Nitrate Reductase Gene ◽

Physiological Characterization ◽

Reductase Gene

ABSTRACT By using mini-Tn5 transposon mutagenesis, random transcriptional fusions of promoterless bacterial luciferase,luxAB, to genes of Pseudomonas putida KT2442 were generated. Insertion mutants that responded to ammonium deficiency by induction of bioluminescence were selected. The mutant that responded most strongly was genetically analyzed and is demonstrated to bear the transposon within the assimilatory nitrate reductase gene (nasB) of P. putida KT2442. Genetic evidence as well as sequence analyses of the DNA regions flanking nasBsuggest that the genes required for nitrate assimilation are not clustered. We isolated three second-site mutants in which induction ofnasB expression was completely abolished under nitrogen-limiting conditions. Nucleotide sequence analysis of the chromosomal junctions revealed that in all three mutants the secondary transposon had inserted at different sites in the gltB gene of P. putida KT2442 encoding the major subunit of the glutamate synthase. A detailed physiological characterization of thegltB mutants revealed that they are unable to utilize a number of potential nitrogen sources, are defective in the ability to express nitrogen starvation proteins, display an aberrant cell morphology under nitrogen-limiting conditions, and are impaired in the capacity to survive prolonged nitrogen starvation periods.

Download Full-text

Nitrate Reductase from a Mutant Strain of Chlamydomonas reinhardii Incapable of Nitrate Assimilation

Zeitschrift für Naturforschung C ◽

10.1515/znc-1983-5-618 ◽

1983 ◽

Vol 38 (5-6) ◽

pp. 439-445 ◽

Cited By ~ 8

Author(s):

Emilio Fernández ◽

Jacobo Cárdenas

Keyword(s):

Nitrate Reductase ◽

Nitrate Reductase Activity ◽

Reductase Activity ◽

Wild Strain ◽

Nitrate Assimilation ◽

Pyridine Nucleotide ◽

Spectral Studies ◽

Binding Region ◽

Chlamydomonas Reinhardii ◽

Blue Sepharose

Nitrate reductase from mutant 305 of Chlamydomonas reinhardii has been purified about 90-fold and biochemically characterized. The enzyme can use reduced flavins and viologens as electron donors to reduce nitrate but, unlike the nitrate reductase complex from its parental wild strain, lacks NAD(P)H-nitrate reductase and NAD(P)H-cytochrome c reductase activities, does not bind to Blue-Agarose or Blue-Sepharose and exhibits a significantly lower molecular weight (177.000 vs. 241.000), whereas its kinetic characteristics and its sensitivity against several inhibitors and treatments are very similar to those of the terminal nitrate reductase activity of the wild strain complex. Spectral studies and antagonistic experiments with tungstate show the presence of cytochrome b557 and molybdenum. These facts lead us to propose that nitrate reductase from mutant 305 has a protein deletion which affects the pyridine nucleotide binding region of the diaphorase protein but without any effect on the terminal nitrate reductase activity.

Download Full-text

The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple

Horticulture Research ◽

10.1038/hortres.2017.23 ◽

2017 ◽

Vol 4 (1) ◽

Cited By ~ 57

Author(s):

Jian-Ping An ◽

Feng-Jia Qu ◽

Ji-Fang Yao ◽

Xiao-Na Wang ◽

Chun-Xiang You ◽

...

Keyword(s):

Transcription Factor ◽

Nitrate Reductase ◽

Leucine Zipper ◽

Anthocyanin Biosynthesis ◽

Nitrate Assimilation ◽

Bzip Transcription Factor ◽

Anthocyanin Accumulation ◽

Mobility Shift ◽

Basic Leucine Zipper ◽

Transgenic Apple

Abstract The basic leucine zipper (bZIP) transcription factor HY5 plays a multifaceted role in plant growth and development. Here the apple MdHY5 gene was cloned based on its homology with Arabidopsis HY5. Expression analysis demonstrated that MdHY5 transcription was induced by light and abscisic acid treatments. Electrophoretic mobility shift assays and transient expression assays subsequently showed that MdHY5 positively regulated both its own transcription and that of MdMYB10 by binding to E-box and G-box motifs, respectively. Furthermore, we obtained transgenic apple calli that overexpressed the MdHY5 gene, and apple calli coloration assays showed that MdHY5 promoted anthocyanin accumulation by regulating expression of the MdMYB10 gene and downstream anthocyanin biosynthesis genes. In addition, the transcript levels of a series of nitrate reductase genes and nitrate uptake genes in both wild-type and transgenic apple calli were detected. In association with increased nitrate reductase activities and nitrate contents, the results indicated that MdHY5 might be an important regulator in nutrient assimilation. Taken together, these results indicate that MdHY5 plays a vital role in anthocyanin accumulation and nitrate assimilation in apple.

Download Full-text

Seasonal variation in nitrate reductase activity in needles of high-elevation red spruce trees

Canadian Journal of Forest Research ◽

10.1139/x92-049 ◽

1992 ◽

Vol 22 (3) ◽

pp. 375-380 ◽

Cited By ~ 12

Author(s):

M.G. Tjoelker ◽

S.B. McLaughlin ◽

R.J. DiCosty ◽

S.E. Lindberg ◽

R.J. Norby

Keyword(s):

Nitrate Reductase ◽

Nitrate Reductase Activity ◽

Reductase Activity ◽

Nitrate Assimilation ◽

High Elevation ◽

Preliminary Evidence ◽

Air Pollutant ◽

Red Spruce ◽

Acid Vapor ◽

Assimilation Capacity

To assess seasonal and site variation in foliar nitrate reductase activity and its utility as a biochemical marker for the uptake of nitrogen oxide pollutants in high-elevation forests, we measured nitrate reductase activity in current-year needles of red spruce (Picearubens Sarg.) saplings at two high-elevation stands (1935 and 1720 m) in the Great Smoky Mountains, North Carolina. Measurements spanned two growing seasons between September 1987 and September 1988. Nitrate reductase activity peaked near 60 nmol•g−1•h−1 at both sites in September and October 1987 and August 1988 and declined 80% in November 1987 and 65% in September 1988. Although nitrate reductase activity was 30% greater in saplings at the higher site relative to the lower site in September and October 1987, activity dropped to approximately 10 nmol•g−1•h−1 at both sites in November 1987. No differences among sites were evident the following year. Comparing deposition of nitric acid vapor at a nearby site to nitrate reductase activity suggests that needle nitrate reductase activity is not an unequivocal marker for foliar uptake of nitrogen oxides during air pollutant episodes. The changes in soil nitrate levels in this system provide preliminary evidence that foliar nitrate assimilation may, in part, include nitrate taken up from the soil, as the highest activity occurred during periods of higher A-horizon nitrate concentrations in 1988. These measurements of nitrate reductase activity suggest that red spruce are capable of assimilating nitrate in foliage in the field and that the nitrate assimilation capacity varies throughout the year.

Download Full-text

Multiple forms of nitrate reductase and their role in nitrate assimilation in roots of wheat at low temperature or high salinity

Physiologia Plantarum ◽

10.1034/j.1399-3054.1995.930206.x ◽

1995 ◽

Vol 93 (2) ◽

pp. 249-252 ◽

Cited By ~ 1

Author(s):

Saule Kenjebaeva ◽

Natasha Rakova

Keyword(s):

Low Temperature ◽

Nitrate Reductase ◽

High Salinity ◽

Nitrate Assimilation ◽

Multiple Forms

Download Full-text

Molecular Components of Nitrate and Nitrite Efflux in Yeast

Eukaryotic Cell ◽

10.1128/ec.00268-13 ◽

2013 ◽

Vol 13 (2) ◽

pp. 267-278 ◽

Cited By ~ 16

Author(s):

Elisa Cabrera ◽

Rafaela González-Montelongo ◽

Teresa Giraldez ◽

Diego Alvarez de la Rosa ◽

José M. Siverio

Keyword(s):

Nitrate Reductase ◽

Nitrate Uptake ◽

Reductase Activity ◽

Hansenula Polymorpha ◽

Nitrate Assimilation ◽

Nitrate Reductase Gene ◽

Content Type ◽

Nitrite Toxicity ◽

Essential Components ◽

Nitrate And Nitrite

ABSTRACTSome eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeastHansenula polymorphawas used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking eitherSSU2orNAR1along with the nitrate reductase geneYNR1showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-knownSaccharomyces cerevisiaesulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation.

Download Full-text

Dephosphorylation of Nitrate Reductase Protein Regulates Growth of Rice and Adaptability to Low Temperature

10.21203/rs.3.rs-1183137/v1 ◽

2022 ◽

Author(s):

Ruicai Han ◽

Chenyan Li ◽

Huijie Li ◽

Yupeng Wang ◽

Xiaohua Pan ◽

...

Keyword(s):

Low Temperature ◽

Nitrate Reductase ◽

Chlorophyll Content ◽

Phosphorylation Site ◽

Nitrate Assimilation ◽

Normal Growth ◽

Growth Conditions ◽

Nitrogen Utilization ◽

Utilization Efficiency ◽

Transgenic Lines

Abstract Nitrate reductase (NR) is an important enzyme for nitrate assimilation in plants, and its activity is regulated by post-translational phosphorylation. To investigate the effect of NIA1 protein dephosphorylation on the growth of rice and its adaptability to low temperature, we analyzed phenotype, chlorophyll content, nitrogen utilization, and antioxidant capacity at low temperature in lines with a mutated NIA1 phosphorylation site (S532D and S532A), an OsNia1 over-expression line (OE), and wild-type Kitaake rice (WT). Plant height, dry matter weight, and chlorophyll content of S532D and S532A were lower than those of WT and OE under normal growth conditions but were higher than those of WT and OE at low temperature. Compared with WT and OE, the nitrite, H2O2, and MDA contents of S532D and S532A leaves were higher under normal growth conditions. The difference in leaf nitrite content between transgenic lines and WT was narrower at low temperature, especially in S532D and S532A, while H2O2 and MDA contents of S532D and S532A leaves were lower than those in WT and OE leaves. The NH4+-N and amino acid contents of S532D and S532A leaves were higher than those of WT and OE leaves under normal or low temperature. qRT-PCR results revealed that transcription levels of OsNrt2.4, OsNia2, and OsNADH-GOGAT were positively correlated with those of OsNia1, and the transcription levels of OsNrt2.4, OsNia2, and OsNADH-GOGAT were significantly higher in transgenic lines than in WT under both normal and low temperature. Phosphorylation of NR is a steady-state regulatory mechanism of nitrogen metabolism, and dephosphorylation of NIA1 protein improved NR activity and nitrogen utilization efficiency in rice. Excessive accumulation of nitrite under normal growth conditions inhibits the growth of rice; however, accumulation of nitrite is reduced at low temperature, enhancing the cold tolerance of rice.

Download Full-text

A recently evolved diflavin-containing monomeric nitrate reductase is responsible for highly efficient bacterial nitrate assimilation

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012859 ◽

2020 ◽

Vol 295 (15) ◽

pp. 5051-5066 ◽

Cited By ~ 2

Author(s):

Wei Tan ◽

Tian-Hua Liao ◽

Jin Wang ◽

Yu Ye ◽

Yu-Chen Wei ◽

...

Keyword(s):

Nitrate Reductase ◽

Biochemical Characterization ◽

Catalytic Domain ◽

Inorganic Nitrogen ◽

Nitrate Assimilation ◽

Nitrogen Sources ◽

Electron Transfer Mechanism ◽

Cofactor Binding ◽

Environmental Mycobacteria

Nitrate is one of the major inorganic nitrogen sources for microbes. Many bacterial and archaeal lineages have the capacity to express assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite. Although a nitrate assimilatory pathway in mycobacteria has been proposed and validated physiologically and genetically, the putative NAS enzyme has yet to be identified. Here, we report the characterization of a novel NAS encoded by Mycolicibacterium smegmatis Msmeg_4206, designated NasN, which differs from the canonical NASs in its structure, electron transfer mechanism, enzymatic properties, and phylogenetic distribution. Using sequence analysis and biochemical characterization, we found that NasN is an NADPH-dependent, diflavin-containing monomeric enzyme composed of a canonical molybdopterin cofactor-binding catalytic domain and an FMN–FAD/NAD-binding, electron-receiving/transferring domain, making it unique among all previously reported hetero-oligomeric NASs. Genetic studies revealed that NasN is essential for aerobic M. smegmatis growth on nitrate as the sole nitrogen source and that the global transcriptional regulator GlnR regulates nasN expression. Moreover, unlike the NADH-dependent heterodimeric NAS enzyme, NasN efficiently supports bacterial growth under nitrate-limiting conditions, likely due to its significantly greater catalytic activity and oxygen tolerance. Results from a phylogenetic analysis suggested that the nasN gene is more recently evolved than those encoding other NASs and that its distribution is limited mainly to Actinobacteria and Proteobacteria. We observed that among mycobacterial species, most fast-growing environmental mycobacteria carry nasN, but that it is largely lacking in slow-growing pathogenic mycobacteria because of multiple independent genomic deletion events along their evolution.

Download Full-text