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, 1996).

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).

Aitken A 1996 14-3-3 and its possible role in co-ordinating multiple signalling pathways. Trends in Cell Biology 6: 341-347.

Athwal GS, Huber JL, Huber SC 1998 Phosphorylated nitrate reductase and 14-3-3 proteins. Site of interaction, effects of ions, and evidence for an AMP-binding site on 14-3-3 proteins. Plant Physiol. 118: 1041-1048.

Athwal GS, Lombardo CR, Huber JL, Masters SC, Fu H, Huber SC 2000 Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors. Plant Cell Physiol. 41: 523-33.

Babakov AV, Chelysheva VV, Klychnikov OI, Zorinyanz SE, Trofimova MS, De Boer AH 2000 Involvement of 14-3-3 proteins in the osmotic regulation of H⁺-ATPase in plant plasma membranes. Planta 211: 446-448.

Bachmann M, Huber JL, Athwal GS, Wu K, Ferl RJ, Huber SC 1996 14-3-3 proteins associate with the regulatory phosphorylation site of spinach leaf nitrate reductase in an isoform-specific manner and reduce dephosphorylation of Ser-543 by endogenous protein phosphatases. FEBS Lett. 398: 26-30.

Booij PP, Roberts MR, Vogelzang SA, Kraayenhof R, De Boer AH 1999 14-3-3 proteins double the number of outward-rectifying K⁺ channels available for activation in tomato cells. Plant J. 20: 673-683.

Cotelle V, Meek SE, Provan F, Milne FC, Morrice N, MacKintosh C 2000 14-3-3s regulate global cleavage of their diverse binding partners in sugar-starved Arabidopsis cells. EMBO J. 19: 2869-2876.

Douglas P, Moorhead G, Hong Y, Morrice N, MacKintosh C 1998 Purification of a nitrate reductase kinase from Spinacea oleracea leaves, and its identification as a calmodulin-domain protein kinase. Planta 206: 435-442.

Foyer CH, Valadier MH, Migge A, Becker TW 1998 Drought-induced effects on nitrate reductase activity and mRNA and on the coordination of nitrogen and carbon metabolism in maize leaves. Plant Physiol. 117: 283-292.

Ferrario-Mery S, Valadier MH, Foyer CH 1998 Overexpression of nitrate reductase in tobacco delays drought-induced decreases in nitrate reductase activity and mRNA. Plant Physiol. 117: 293-302.

Huber JL, Huber SC, Campbell WH, Redinbaugh MG 1992 Reversible light/dark modulation of spinach leaf nitrate reductase activity involves protein phosphorylation. Arch. Biochem. Biophys. 296: 58-65.

Lillo C, Kazazaic S, Ruoff P, Meyer C 1997 Characterization of nitrate reductase from light- and dark-exposed leaves: comparison of different species and effects of 14-3-3 inhibitor proteins. Plant Physiol. 114: 1377-1383.

Lejay L, Quillere I, Roux Y, Tillard P, Cliquet J-B, Meyer C, Morot-Gaudry J-F, Gojon A 1997 Abolition of posttranscriptional regulation of nitrate reductase partially prevents the decrease in leaf NO₃^- reduction when photosynthesis is inhibited by CO₂ deprivation, but not in darkness. Plant Physiol. 115: 623-630.

MacKintosh C, Douglas P, Lillo C 1995 Identification of a protein that inhibits the phosphorylated form of nitrate reductase from spinach (Spinacia oleracea) leaves. Plant Physiol. 107: 451-457.

Moorhead G, Douglas P, Cotelle V, Harthill J, Morrice N, Meek S, Deiting U, Stitt M, Scarabel M, Aitken A, MacKintosh C 1999 Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins. Plant J. 18: 1-12.

Nussaume L, Vincentz M, Meyer C, Boutin JP, Caboche M 1995 Post-transcriptional regulation of nitrate reductase by light is abolished by an N-terminal deletion. Plant Cell 7: 611-621.

Olsson A, Svennelid F, Ek B, Sommarin M, Larsson C 1998 A phosphothreonine residue at the C-terminal end of the plasma membrane H⁺-ATPase is protected by fusicoccin-induced 14-3-3 binding. Plant Physiol. 118: 551-555.

Pigaglio E, Durand N, Meyer C 1999 A conserved acidic motif in the N-terminal domain of nitrate reductase is necessary for the inactivation of the enzyme in the dark by phosphorylation and 14-3-3 binding. Plant Physiol. 119: 219-229.

Provan F, Aksland LM, Meyer C, Lillo C 2000 Deletion of the nitrate reductase N-terminal domain still allows binding of 14-3-3 proteins but affects their inhibitory properties. Plant Physiol. 2000 123: 757-764.

Roberts MR, Bowles DJ 1999 Fusicoccin, 14-3-3 proteins, and defense responses in tomato plants. Plant Physiol. 119: 1243-1250.

Su W, Huber SC, Crawford NM 1996 Identification in vitro of a post-translational regulatory site in the hinge 1 region of Arabidopsis nitrate reductase. Plant Cell 8: 519-527.

Su W, Mertens JA, Kanamaru K, Campbell WH, Crawford NM 1997 Analysis of wild-type and mutant plant nitrate reductase expressed in the methylotrophic yeast Pichia pastoris. Plant Physiol. 115: 1135-1143.

Weiner H, Kaiser WM 1999 14-3-3 proteins control proteolysis of nitrate reductase in spinach leaves. FEBS Lett. 455: 75-78.

Wu K, Rooney MF, Ferl RJ 1997 The Arabidopsis 14-3-3 multigene family. Plant Physiol. 114: 1421-1431.