NO3- Enhances the Kinase Activity for Phosphorylation of Phosphoenolpyruvate Carboxylase and Sucrose Phosphate Synthase Proteins in Wheat Leaves: Evidence from the Effects of Mannose and Okadaic Acid

Abstract

The aim of this work was to determine which of the two reactions (i.e. phosphorylation or dephosphorylation) involved in the establishment of the phosphorylated status of the wheat leaf phosphoenolpyruvate carboxylase and sucrose phosphate synthase protein responds in vivo to NO(3) (-) uptake and assimilation. Detached mature leaves of wheat (Triticum aestivum L. cv Fidel) were fed with N-free (low-NO(3) (-) leaves) or 40 mm NO(3) (-) solution (high-NO(3) (-) leaves). The specific inhibition of the enzyme-protein kinase or phosphatase activities was obtained in vivo by addition of mannose or okadaic acid, respectively, in the uptake solution. Mannose at 50 mm, by blocking the kinase reaction, inhibited the processes of NO(3) (-)-dependent phosphoenolpyruvate carboxylase activation and sucrose phosphate synthase deactivation. Following the addition of mannose, the deactivation of phosphoenolpyruvate carboxylase and the activation of sucrose phosphate synthase, both due to the enzyme-protein dephosphorylation, were at the same rate in low-NO(3) (-) and high-NO(3) (-) leaves, indicating that NO(3) (-) had no effect per se on the enzyme-protein phosphatase activity. Upon treatment with okadaic acid, the higher increase of phosphoenolpyruvate carboxylase and decrease of sucrose phosphate synthase activities observed in high NO(3) (-) compared with low NO(3) (-) leaves showed evidence that NO(3) (-) enhanced the protein kinase activity. These results support the concept that NO(3) (-), or a product of its metabolism, favors the activation of phosphoenolpyruvate carboxylase and deactivation of sucrose phosphate synthase in wheat leaves by promoting the light activation of the enzyme-protein kinase(s) without affecting the phosphatase(s).

Mannose Inhibits Arabidopsis Germination via a Hexokinase-Mediated Step1

“…Man-6-P is then only slowly processed by the plant, since the enzymes required for this are either absent or exist in very low concentrations (Sheu-Hwa et al, 1975;Walker and Sivak, 1986). Due to this property, Man has been used in the past to provoke ATP and phosphate depletion in adult leaves of some plant species (Siegl and Stitt, 1990;Van Quy and Champigny, 1992). The Man concentrations used in these studies (50-200 mm) were much higher than the ones we used (7.5 mm).…”

“…The lowest concentration of Man (0.5 mm) still allowed germination, but 7.5 and 50 mm repressed it entirely. A concentration of 50 mm Man has previously been used to provoke ATP depletion in detached adult wheat leaves (Van Quy and Champigny, 1992). The ATP levels were then measured at 24-h intervals for several days (Fig.…”

In Vivo and in Vitro Phosphorylation of the Phosphoenolpyruvate Carboxylase from Wheat Seeds during Germination

“…Nonphotosynthetic PEPC isoforms have been shown to be radiolabeled in vivo in detached wheat leaves and soybean nodules (Van Quy et al, 1991;Zhang et al, 1995). Likewise, changes in the malate sensitivity related to the phosphorylation status of PEPC have been reported in guard cells during stomatal opening (Schnabl et al, 1992;Zhang et al, 1994) and in C, leaves during illumination of N-recovering seedlings (Van Quy and Champigny, 1992;. In seeds, a putative phosphorylation domain was shown to be present in the deduced N terminus of PEPC from soybean (Sugimoto et al, 1992).…”

“…It has been shown that they are subject to allosteric control (Andreo et al, 1987), but there is little experimental evidence to illustrate the in vivo posttranslational regulation, the signal transduction pathway, and the nature of the stimulus operating on the covalent modification of these nonphotosynthetic isoforms. The nonphotosynthetic PEPCs were shown to be in vivo phosphorylated in illuminated, N-recovering wheat (Trificum aestivum) leaves (Van Quy et al, 1991;Van Quy and Champigny, 1992;, in soybean nodules (Zhang et al, 1995), and in stomatal guard cells (Schnabl et al, 1992;Zhang et al, 1994). Changes in the properties of an in vitro phosphorylated enzyme were established only for the recombinant C,-type Sorghum PEPC (Pacquit et al, 1993) and the soybean nodule enzyme (Schuller and Dietrich, 1993).…”

Potato plants contain multiple forms of sucrose phosphate synthase, which differ in their tissue distributions, their levels during development, and their responses to low temperature

“…Phosphorylation leads to a lowering of the substrate affinity of the enzyme, and makes it more susceptible to inhibition by Pj and more dependent on Glc6P Siegl, Mackintosh & Stitt 1990). Reversible phosphorylation is involved in the regulation of SPS in response to rising rates of photosynthesis (Stitt et al 1988), accumulation of sucrose (Stitt et al 1988), the assimilation of nitrate (Van Quy & Champigny 1992) and water stress (Quick et al 1989;Zrenner & Stitt 1991;, although all the details of the regulation circuits have not yet been elucidated (see Huber & Huber 1996).…”