24.13. Reversible Inactivation of NR (higher plants)

        In higher plants nitrate reduction is highly regulated; NR is controlled by light, temperature, pH, CO₂, O₂, water potential and N source. Drought causes increased NR protein turnover, and accelerated mRNA turnover (Foyer et al, 1998; Ferrario-Mery et al, 1998). Plants rapidly inactivate NR in response to loss of light, a decrease of CO₂ levels, or an increase in cytosolic pH. Nitrate and light are also required for maximum synthesis of NiR.

        Recent studies indicate that leaf NR in spinach undergoes a reversible phosphorylation in response to light/dark transitions (Huber et al, 1992).

        The low-activity, phosphorylated form of nitrate reductase (NR) from dark-treated leaves of spinach becomes activated during purification. This activation resulted from separation of NR from an approx. 110-kDa nitrate reductase inhibitory protein (NIP). Readdition of NIP inactivated the purified phosphorylated NR, but not the active dephosphorylated form of NR, indicating that the inactivation of NR requires its interaction with NIP as well as phosphorylation. Consistent with this, NR that had been inactivated in vitro in the presence of NR kinase, ATP-Mg, and NIP could be reactivated either by dephosphorylation with protein phosphatase 2A or by dissociation of NIP from NR (MacKintosh et al, 1995).

        NIP has been shown to be a 14-3-3 protein. 14-3-3 proteins are chaperone proteins that modulate interactions between components of signal transduction pathways (Aitken, 1996; Wu et al, 1997). An increase in ionic strength disrupts the binding of 14-3-3 to NR (Athwal et al, 1998). 5'-AMP also appears to disrupt the NR:14-3-3 complex (Athwal et al, 1998; 2000). The availability of 14-3-3s for binding to phosphorylated-NR controls the stability of NR via proteolysis Weiner and Kaiser, 1999).

        Plants carrying an NR with an N-terminal deletion show impairment of NR phosphorylation, and this may result in abolishment of post-transcriptional regulation of NR by light (Nussaume et al, 1995). This may be due to a difference in dissociation of the NR-NIP complex (Lillo et al, 1997), or a different way of binding for 14-3-3 in the truncated NR (Provan et al, 2000). An N-terminal acidic motif of tobacco NR is necessary for inactivation of the enzyme in the dark by phosphorylation and 14-3-3 binding (Pigaglio et al, 1999).

        Abolition of post-transcriptional regulation of NR prevents the decrease in leaf nitrate reduction when photosynthesis is inhibited by CO₂ deprivation, but not in darkness (Lejay et al, 1997).

        However, site-directed mutagenesis studies with Arabidopsis NIA2 nitrate reductase expressed in the yeast Pichia pastoris, indicate that N terminal deletions or substitutions conserved NR activity and ability to be inactivated in vitro by incubation with ATP (Su et al, 1997). Thus, the N terminus is not essential for enzyme activity or phosphorylation-dependent regulation in Arabidopsis (Su et al, 1997).

        The phosphorylation site (Ser-543) of NR is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of spinach NR -- the phosphorylation causes a block in electron flow. Two NR kinases (NRk's) have been resolved; both are calcium-dependent but are distinct immunochemically (Bachmann et al, 1996; Douglas et al, 1998). The 14-3-3 proteins that inactivates the phosphorylated form of spinach leaf NADH:nitrate reductase (NR) binds to the enzyme at the regulatory phosphorylation site (Ser-543), and can thus reduce dephosphorylation of Ser-543 by endogenous protein phosphatases (Bachmann et al, 1996).

        In Arabidopsis Ser-534 (located in the hinge 1 of NR) is an essential site for post-translational regulation (Su et al, 1996). Substitutions of other Ser residues with Ala in the MoCo-binding regions (Ser-216, Ser-261, Ser-266, Ser-324, Ser-365, Ser-395 and Ser-438) all retained NR activity and ability to be inactivated. Thus, none of these other Ser residues are essential for phosphorylation-dependent regulation (Su et al, 1997).

        14-3-3-binding proteins in plants include: nitrate reductase, glyceraldehyde-3-phosphate dehydrogenase, sucrose-phosphate synthase, trehalose-6-phosphate synthase, glutamine synthetases, glutamyl-tRNA synthetase, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium-dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14-3-3 proteins (Moorhead et al, 1999; Cotelle et al, 2000).

        Fusicoccin (FC) is a fungal toxin that activates the plant plasma membrane H⁺-ATPase by binding with 14-3-3 proteins, causing membrane hyperpolarization (Roberts and Bowles, 1999). Osmotic regulation of H⁺-ATPase in the plant plasma membrane is achieved via modulation of the coupling between H⁺ transport and ATP hydrolysis; this regulation involves 14-3-3 proteins (Babakov et al, 2000). A phosphothreonine residue (threonine-948) at the C-terminal end of the plasma membrane H⁺-ATPase is protected by fusicoccin-induced 14-3-3 binding (Olsson et al, 1998). Outward-rectifying K⁺ channels are also targets for modulation by 14-3-3 proteins (Booij et al, 1999).

|Nitrate and Nitrate Uptake and Reduction|Nitrate and Ammonium Transport|N Demand and Nitrate Uptake|Nitrate and Nitrite Reductase Structures|Three-Dimensional Structure of Maize NR|Chlorate as an Analog of Nitrate|Reduction of FMN and NAD(P)⁺|Tungstate as an Analog of Molybdate|Structural and Regulatory Genes of Nitrate Reduction (Aspergillus) (Neurospora) (algae) (higher plants)|Reversible Inactivation of NR (algae) (higher plants)|Regulation of Sucrose-phosphate Synthase by Reversible Phosphorylation||Role of Nitrate in the Control of Carbon Metabolism

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This document prepared by David Rhodes, Purdue University, and modified by Robert D. Locy  

Last updated on October 19, 2001.