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Evidence for PII with NAGK interaction that regulates Arg synthesis in the microalga Myrmecia incisa in response to nitrogen starvation.

ABSTRACT: To understand why most eukaryotic microalgae accumulate lipids during nitrogen starvation stress, a gene, MiglnB, encoding PII, a signal transduction protein, was cloned from the arachidonic acid-rich microalga Myrmecia incisa Reisigl. Similarly to its homologues, MiPII contains three conserved T-, B-, and C-loops. In the presence of abundant Mg2+, ATP, and Gln, MiPII upregulates Arg biosynthesis by interacting with the rate-limiting enzyme, MiNAGK, as evidenced by yeast two-hybrid, co-immunoprecipitation assays, and kinetics analysis of enzyme-catalyzed reactions. However, this interaction of MiPII with MiNAGK is reversed by addition of 2-oxoglutarate (2-OG). Moreover, this interaction is present in the chloroplasts of M. incisa, as illustrated cytologically by both immunoelectron microscopy and agroinfiltration of Nicotiana benthamiana leaves to determine the subcellular localization of MiPII with MiNAGK. During the process of nitrogen starvation, soluble Arg levels in M. incisa are modulated by a change in MiNAGK enzymatic activity, both of which are significantly correlated (r?=?0.854). A model for the manipulation of Arg biosynthesis via MiPII in M. incisa chloroplasts in response to nitrogen starvation is proposed. The ATP and 2-OG saved from Arg biosynthesis is thus suggested to facilitate the accumulation of fatty acids and triacylglycerol in M. incisa during exposure to nitrogen starvation.

SUBMITTER: Li Y

PROVIDER: S-EPMC5701185 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Evidence for PII with NAGK interaction that regulates Arg synthesis in the microalga Myrmecia incisa in response to nitrogen starvation.

Li Yan Y Liu Wei W Sun Li-Ping LP Zhou Zhi-Gang ZG

Scientific reports 20171124 1

To understand why most eukaryotic microalgae accumulate lipids during nitrogen starvation stress, a gene, MiglnB, encoding PII, a signal transduction protein, was cloned from the arachidonic acid-rich microalga Myrmecia incisa Reisigl. Similarly to its homologues, MiPII contains three conserved T-, B-, and C-loops. In the presence of abundant Mg<sup>2+</sup>, ATP, and Gln, MiPII upregulates Arg biosynthesis by interacting with the rate-limiting enzyme, MiNAGK, as evidenced by yeast two-hybrid, c ...[more]

PMID: 29176648

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Project description:The green microalga Lobosphaera incisa accumulates triacylglycerols (TAGs) with exceptionally high levels of long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid (ARA) under nitrogen (N) deprivation. Phosphorous (P) deprivation induces milder changes in fatty acid composition, cell ultrastructure, and growth performance. We hypothesized that the resource-demanding biosynthesis and sequestration of ARA-rich TAG in lipid droplets (LDs) are associated with the enhancement of catabolic processes, including membrane lipid turnover and autophagic activity. Although this work focuses mainly on N deprivation, a comparative analysis of N and P deprivation responses is included. The results of lipidomic profiling showed a differential impact of N and P deprivation on the reorganization of glycerolipids. The formation of TAG under N deprivation was associated with the enhanced breakdown of chloroplast glycerolipids and the formation of lyso-lipids. N-deprived cells displayed a profound reorganization of cell ultrastructure, including internalization of cellular material into autophagic vacuoles, concomitant with the formation of LDs, while P-deprived cells showed better cellular ultrastructural integrity. The expression of the hallmark autophagy protein ATG8 and the major lipid droplet protein (MLDP) genes were coordinately upregulated, but to different extents under either N or P deprivation. The expression of the ?5-desaturase gene, involved in the final step of ARA biosynthesis, was coordinated with ATG8 and MLDP, exclusively under N deprivation. Concanamycin A, the inhibitor of vacuolar proteolysis and autophagic flux, suppressed growth and enhanced levels of ATG8 and TAG in N-replete cells. The proportions of ARA in TAG decreased with a concomitant increase in oleic acid under both N-replete and N-deprived conditions. The photosynthetic apparatus's recovery from N deprivation was impaired in the presence of the inhibitor, along with the delayed LD degradation. The GFP-ATG8 processing assay showed the release of free GFP in N-replete and N-deprived cells, supporting the existence of autophagic flux. This study provides the first insight into the homeostatic role of autophagy in L. incisa and points to a possible metabolic link between autophagy and ARA-rich TAG biosynthesis.

Dataset Information

Evidence for PII with NAGK interaction that regulates Arg synthesis in the microalga Myrmecia incisa in response to nitrogen starvation.

Publications

Evidence for PII with NAGK interaction that regulates Arg synthesis in the microalga Myrmecia incisa in response to nitrogen starvation.

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