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In higher plants, the metabolism of carbon (C) and nitrogen nutrients (N) is mutually regulated and referred to as the C and N balance (C/N). Plants are thus able to optimize their growth depending on their cellular C/N status. Arabidopsis ATL31 and ATL6 encode a RING-type ubiquitin ligases which play a critical role in the C/N status response (Sato et al. in Plant J 60:852-864, 2009). Since many ATL members are involved in the plant defense response, the present study evaluated whether the C/N response regulators ATL31 and ATL6 are involved in defense responses. Our results confirmed that ATL31 and ATL6 expression is up-regulated with the microbe-associated molecular patterns elicitors flg22 and chitin as well as with infections with Pseudomonas syringae pv. tomato DC3000 (Pst. DC3000). Moreover, transgenic plants overexpressing ATL31 and ATL6 displayed increased resistance to Pst. DC3000. In accordance with these data, loss of ATL31 and ATL6 function in an atl31 atl6 double knockout mutant resulted in reduced resistance to Pst. DC3000. In addition, the molecular cross-talk between C/N and the defense response was investigated by mining public databases. The analysis identified the transcription factors MYB51 and WRKY33, which are involved in the defense response, and their transcripts levels correlate closely with ATL31 and ATL6. Further study demonstrated that the expression of ATL31, ATL6 and defense marker genes including MYB51 and WRKY33 were regulated by C/N conditions. Taken together, these results indicate that ATL31 and ATL6 function as key components of both C/N regulation and the defense response in Arabidopsis.

The ubiquitin-proteasome system (UPS) is a unique protein degradation mechanism conserved in the eukaryotic cell. In addition to the control of protein quality, UPS regulates diverse cellular signal transduction via the fine-tuning of target protein degradation. Protein ubiquitylation and subsequent degradation by the 26S proteasome are involved in almost all aspects of plant growth and development and response to biotic and abiotic stresses. Recent studies reveal that the UPS plays an essential role in adaptation to carbon and nitrogen availability in plants. Here we highlight ubiquitin ligase ATL31 and the homologue ATL6 target 14-3-3 proteins for ubiquitylation to be degraded, which control signaling for carbon and nitrogen metabolisms and C/N balance response. We also give an overview of the UPS function involved in carbon and nitrogen metabolisms.

The 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, 14-3-3χ, was selected for detailed characterization. The interaction between ATL31 and 14-3-3χ was confirmed by yeast two-hybrid and co-immunoprecipitation analyses. In vitro assays showed that ubiquitination of 14-3-3χ is catalyzed by ATL31. Degradation of 14-3-3χin vivo was shown to be correlated with ATL31 activity, and to occur in a proteasome-dependent 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 14-3-3χ 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., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Plants are able to sense and respond to changes in the balance between carbon (C) and nitrogen (N) metabolite availability, known as the C/N response. During the transition to photoautotrophic growth following germination, growth of seedlings is arrested if a high external C/N ratio is detected. To clarify the mechanisms for C/N sensing and signaling during this transition period, we screened a large collection of FOX transgenic plants, overexpressing full-length cDNAs, for individuals able to continue post-germinative growth under severe C/N stress. One line, cni1-D (carbon/nitrogen insensitive 1-dominant), was shown to have a suppressed sensitivity to C/N conditions at both the physiological and molecular level. The CNI1 cDNA encoded a predicted RING-type ubiquitin ligase previously annotated as ATL31. Overexpression of ATL31 was confirmed to be responsible for the cni1-D phenotype, and a knock-out of this gene resulted in hypersensitivity to C/N conditions during post-germinative growth. The ATL31 protein was confirmed to contain ubiquitin ligase activity using an in vitro assay system. Moreover, removal of this ubiquitin ligase activity from the overexpressed protein resulted in the loss of the mutant phenotype. Taken together, these data demonstrated that CNI1/ATL31 activity is required for the plant C/N response during seedling growth transition.

Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, its primary molecular mechanisms remain incompletely understood in plants and animals. Here we report unique Ca2+ signalling triggered by nitrate with live imaging of an ultrasensitive biosensor in Arabidopsis leaves and roots. A nitrate-sensitized and targeted functional genomic screen identifies subgroup III Ca2+-sensor protein kinases (CPKs) as master regulators that orchestrate primary nitrate responses. A chemical switch with the engineered mutant CPK10(M141G) circumvents embryo lethality and enables conditional analyses of cpk10 cpk30 cpk32 triple mutants to define comprehensive nitrate-associated regulatory and developmental programs. Nitrate-coupled CPK signalling phosphorylates conserved NIN-LIKE PROTEIN (NLP) transcription factors to specify the reprogramming of gene sets for downstream transcription factors, transporters, nitrogen assimilation, carbon/nitrogen metabolism, redox, signalling, hormones and proliferation. Conditional cpk10 cpk30 cpk32 and nlp7 mutants similarly impair nitrate-stimulated system-wide shoot growth and root establishment. The nutrient-coupled Ca2+ signalling network integrates transcriptome and cellular metabolism with shoot-root coordination and developmental plasticity in shaping organ biomass and architecture.