Light-Dark Changes in Cytosolic Nitrate Pools Depend on Nitrate Reductase Activity in Arabidopsis Leaf Cells

Abstract

; and Crop Performance and Improvement Division, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom (S.J.C., A.J.M.)Several different cellular processes determine the size of the metabolically available nitrate pool in the cytoplasm. These processes include not only ion fluxes across the plasma membrane and tonoplast but also assimilation by the activity of nitrate reductase (NR). In roots, the maintenance of cytosolic nitrate activity during periods of nitrate starvation and resupply (M. van der Leij, S.J. Smith, A.J. Miller [1998] [356][357][358][359][360][361] suggests that this pool is regulated. Under nitrate-replete conditions vacuolar nitrate is a membranebound store that can release nitrate to the cytoplasm; after depletion of cytosolic nitrate, tonoplast transporters would serve to restore this pool. To study the role of assimilation, specifically the activity of NR in regulating the size of the cytosolic nitrate pool, we have compared wild-type and mutant plants. In leaf mesophyll cells, light-to-dark transitions increase cytosolic nitrate activity (1.5-2.8 mM), and these changes were reversed by dark-to-light transitions. Such changes were not observed in nia1nia2 NR-deficient plants indicating that this change in cytosolic nitrate activity was dependent on the presence of functional NR. Furthermore, in the dark, the steady-state cytosolic nitrate activities were not statistically different between the two types of plant, indicating that NR has little role in determining resting levels of nitrate. Epidermal cells of both wild type and NR mutants had cytosolic nitrate activities that were not significantly different from mesophyll cells in the dark and were unaltered by dark-to-light transitions. We propose that the NR-dependent changes in cytosolic nitrate provide a cellular mechanism for the diurnal changes in vacuolar nitrate storage, and the results are discussed in terms of the possible signaling role of cytosolic nitrate.Nitrogen is the mineral nutrient required in the highest amounts by plants and is most frequently limiting growth and yield. Nitrate is often the most abundant form of nitrogen available to roots especially in temperate agricultural soils. Nitrate incorporation into biological molecules involves the reduction of nitrate to nitrite via the cytosolic enzyme nitrate reductase (NR). The key position of this enzyme early in the pathway and the cellular toxicity of the product, nitrite, has stimulated research to identify if NR is a critical regulatory step in nitrate assimilation (Stitt et al., 2002). Measurements of both gene expression and protein levels of NR have shown that the amounts of the enzyme are not what limit the NR activity in cells (for review, see Kaiser and Huber, 2001;MacKintosh and Meek, 2001). NR is known to be under complex regulation that occurs at both transcription and posttranscriptional levels. The expression of NR is induced by the application of nitrate, and the activity of the protein is altered in response to changes in environmental ...

CLC-mediated anion transport in plant cells

“…Considering AtClCa within the plant cell context, with a gradient of 2 pH units (pHvac 5.5, pHcyt 7.5) and a transmembrane potential of K30 mV between the cytosol and the vacuole (figure 1), the 2NO K 3 =1H C exchange mechanism allows an accumulation of nitrate in the vacuole by a factor of 50, whereas a channel could only drive a threefold accumulation. Interestingly, the measured NO K 3 concentration gradient between vacuole and cytosol in A. thaliana mesophyll cells (Coockson et al 2005) falls within the same range as that potentially built up by the AtClCa NO K 3 /H C exchanger. A transient transformation system of mesophyll protoplasts extracted from mutant plants was set up with a dual objective: (i) to demonstrate that the knockout mutants were functionally complemented by AtClCa cDNA, and (ii) to set up an expression system suitable for electrophysiological experiments to investigate AtClCa structure-function relationships.…”

ATP Binding to the C Terminus of the Arabidopsis thaliana Nitrate/Proton Antiporter, AtCLCa, Regulates Nitrate Transport into Plant Vacuoles

“…Nitrate storage in the vacuole takes place during the night, when nitrate reductase is inactive. NO 3 Ϫ is then released from the vacuole during the day, when the requirement of nitrate by nitrate reductase is high (34). The cytosolic ATP and AMP pools have been shown to regulate nitrate reductase activity and to vary together with the photosynthetic status of the cell (35).…”

Comparing nitrate storage and remobilization in two rice cultivars that differ in their nitrogen use efficiency

“…In the middle of the wall there was a 2.53130.5 cm hole and a tie to hold the tissue in place during the recording. This design was modified from a chamber described previously (Miller et al, 2001;Cookson et al, 2005). The electrophysiology solution covered half of the leaf area, and the site for impaling was just above the liquid surface.…”

“…Some recent papers have suggested that there is a close link between cytosolic nitrate activity and nitrate reductase activity (NRA). For example, in leaf cells of Arabidopsis (Cookson et al, 2005) and barley root cells (Fan et al, 2006), changes in cytosolic nitrate activity could be measured under conditions when cellular NRA was altered. As cytosolic nitrate activity is important for determining the thermodynamic gradients for transport to and from the vacuole (Miller and Smith, 1992;De Angeli et al, 2006) how NRA and mRNA expression changed during the remobilization of stored nitrate has also been examined.…”