SUMMARYThe balance between carbon (C) and nitrogen (N) availability is an important determinant for various phases of plant growth; however, the detailed mechanisms regulating the C/N response are not well understood. We previously described two related ubiquitin ligases, ATL31 and ATL6, that function in the C/N response in Arabidopsis thaliana. Here, we used FLAG tag affinity purification and MS analysis to identify proteins targeted by ATL31, and thus likely to be involved in regulating the phase transition checkpoint based on C/N status. This analysis revealed that 14-3-3 proteins were associated with ATL31, and one of these, 14-3-3v, was selected for detailed characterization. The interaction between ATL31 and 14-3-3v was confirmed by yeast two-hybrid and co-immunoprecipitation analyses. In vitro assays showed that ubiquitination of 14-3-3v is catalyzed by ATL31. Degradation of 14-3-3v in vivo was shown to be correlated with ATL31 activity, and to occur in a proteasomedependent manner. Furthermore, 14-3-3 protein accumulation was induced by a shift to high-C/N stress conditions in Arabidopsis seedlings, and this regulated response required both ATL31 and ATL6. It was also shown that over-expression of 14-3-3v leads to hypersensitivity of Arabidopsis seedlings to C/N stress conditions. These results indicate that ATL31 targets and ubiquitinates 14-3-3 proteins for degradation via the ubiquitin-proteasome system during the response to cellular C/N status.

The upper part of a nodulated soybean root hydroponically cultured in a glass bottle was monitored using a computer microscope under controlled environmental conditions, and the diameter of individual nodules was measured from 10-24 d after planting. The diameter of a root nodule attached to the primary root increased from 1 mm to 6 mm for 2 weeks under nitrogen-free conditions. The increase in diameter of the nodules was almost completely stopped after 1 d of supplying 5 mM nitrate, and was due to the cessation of nodule cell expansion. However, nodule growth quickly returned to the normal growth rate following withdrawal of nitrate from the solution. The reversible depression of nodule growth by nitrate was similar to the restriction of photoassimilate supply by continuous dark treatment for 2 d followed by normal light/dark conditions. In addition, the inhibitory effect of nitrate was partially alleviated by the addition of 3% (w/v) sucrose to the medium. Plant leaves were exposed to (11)C or (14)C-labelled carbon dioxide to investigate the effects of 5 mM nitrate on the translocation and distribution of photosynthates to nodules and roots. Supplying 5 mM nitrate stimulated the translocation rate and the distribution of labelled C in nitrate-fed parts of the roots. However, the (14)C partitioning to nodules decreased from 9% to 4% of total (14)C under conditions of 5 mM nitrate supply. These results indicate that the decrease in photoassimilate supply to nodules may be involved in the quick and reversible nitrate inhibition of soybean nodule growth.

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The application of combined nitrogen, especially nitrate, to soybean plants is known to strongly inhibit nodule formation, growth and nitrogen fixation. In the present study, we measured the effects of supplying 5 mM nitrate on the growth of nodules, primary root, and lateral roots under light at 28 °C or dark at 18 °C conditions. Photographs of the nodulated roots were periodically taken by a digital camera at 1-h intervals, and the size of the nodules was measured with newly developed computer software. Nodule growth was depressed approximately 7 h after the addition of nitrate under light conditions. The nodule growth rate under dark conditions was almost half that under light conditions, and nodule growth was further suppressed by the addition of 5 mM nitrate. Similar results were observed for the extending growth rate of the primary root as those for nodule growth supplied with 5 mM nitrate under light/dark conditions. In contrast, the growth of lateral roots was promoted by the addition of 5 mM nitrate. The 2D-PAGE profiles of nodule protein showed similar patterns between the 0 and 5 mM nitrate treatments, which suggested that metabolic integrity may be maintained with the 5 mM nitrate treatment. Further studies are required to confirm whether light or temperature condition may give the primary effect on the growth of nodules and roots.

C and N are the most important essential elements constituting organic compounds in plants. The shoots and roots depend on each other by exchanging C and N through the xylem and phloem transport systems. Complex mechanisms regulate C and N metabolism to optimize plant growth, agricultural crop production, and maintenance of the agroecosystem. In this paper, we cover the recent advances in understanding C and N metabolism, regulation, and transport in plants, as well as their underlying molecular mechanisms. Special emphasis is given to the mechanisms of starch metabolism in plastids and the changes in responses to environmental stress that were previously overlooked, since these changes provide an essential store of C that fuels plant metabolism and growth. We present general insights into the system biology approaches that have expanded our understanding of core biological questions related to C and N metabolism. Finally, this review synthesizes recent advances in our understanding of the trade-off concept that links C and N status to the plant’s response to microorganisms.

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Nitrate is one of the major sources of nitrogen for higher plants and is taken up from the soil by active transporters coupled with H ϩ across the plasma membrane (PM) of root cells. Nitrate uptake systems have been classified into two groups: low-affinity transport systems (LATS) and high-affinity transport systems (HATS). The LATS contribute to nitrate uptake at high nitrate concentrations above 1 mM whereas the HATS operate at micromolar concentrations of external nitrate and display MichaelisMenten kinetics saturating at 0.2-0.5 mM nitrate. The HATS are further divided into two categories: constitutive HATS (cHATS) and inducible HATS (iHATS), which are significantly affected by the supply of external nitrate. Many studies of the molecular basis of nitrate uptake reveal the existence of two gene families, namely the NRT1 and NRT2 families, which potentially encode for LATS and HATS respectively. NRT2 genes are identified in a variety of organisms including fungi, certain yeasts, green algae, and higher plants ( Unkles et al. 1991;Quesada et al. 1994;Trueman et al. 1996;Pérez et al. 1997;Quesada et al. 1997;Amarasinghe et al. 1998;Zhuo et al., 1999;Araki and Hasegawa 2006;Tsujimoto et al. 2007). In most species, NRT2 genes are members of a multigene family: for example, seven Arabidopsis genes (AtNRT2.1-AtNRT2.7) and four rice genes (OsNRT2.1-OsNRT2.4) have been found in their genomes (Orsel et al. 2002; Araki and Hasegawa 2007), and at least four NRT2 genes (HvNRT2.1-HvNRT2.4) exist in barley (Vidmar 2000a). Amino acid sequences deduced from these genes indicate that the NRT2 proteins are typically 480-510 amino acids in length and predicted to be integral to membranes with 12 transmembrane helices (Forde 2000).It has been well documented that iHATS activity is strongly induced by nitrate supply, and is down-regulated by the accumulation of nitrate assimilation products, especially ammonium and glutamine (Crawford and Glass 1998). In several plant species, it has been shown that a particular member of the NRT2 gene family (e.g., NpNRT2.1 for Nicotiana plumbaginifolia, AtNRT2.1 for Arabidopsis, HvNRT2.1 for barley) contribute to iHATS, because those transcript levels are highly correlated with changes in iHATS activity in such species (Krapp et al. 1998;Lejay et al. 1999;Zhuo et al. 1999;Vidmar et al. 2000a Abstract A high affinity transport system (HATS) for nitrate in plants is operated by a two-component NRT2/NAR2 transport system. However, the regulation and localization of NRT2 and NAR2 at protein level are largely unknown and especially so in crop plant species. In this study with barley (Hordeum vulgare), membrane localization, protein expression in the roots, and a direct protein-protein interaction of HvNRT2 and HvNAR2 proteins were investigated. Immunochemical analysis showed that both HvNRT2 and HvNAR2 proteins were co-localized in the plasma membrane of the roots. Expression of HvNRT2 and HvNAR2 proteins was more strongly induced by treatment with higher concentrations of external nitrate, while H...