Low nitrogen conditions accelerate flowering by modulating the phosphorylation state of FLOWERING BHLH 4 in Arabidopsis.

Nitrogen (N) is an essential nutrient that affects multiple plant developmental processes, including flowering. As flowering requires resources to develop sink tissues for reproduction, nutrient availability is tightly linked to this process. Low N levels accelerate floral transition; however, the molecular mechanisms underlying this response are not well understood. Here, we identify the FLOWERING BHLH 4 (FBH4) transcription factor as a key regulator of N-responsive flowering in Arabidopsis Low N-induced early flowering is compromised in fbh quadruple mutants. We found that FBH4 is a highly phosphorylated protein and that FBH4 phosphorylation levels decrease under low N conditions. In addition, decreased phosphorylation promotes FBH4 nuclear localization and transcriptional activation of the direct target CONSTANS (CO) and downstream florigen FLOWERING LOCUS T (FT) genes. Moreover, we demonstrate that the evolutionarily conserved cellular fuel sensor SNF1-RELATED KINASE 1 (SnRK1), whose kinase activity is down-regulated under low N conditions, directly phosphorylates FBH4. SnRK1 negatively regulates CO and FT transcript levels under high N conditions. Together, these results reveal a mechanism by which N levels may fine-tune FBH4 nuclear localization by adjusting the phosphorylation state to modulate flowering time. In addition to its role in flowering regulation, we also showed that FBH4 was involved in low N-induced up-regulation of nutrient recycling and remobilization-related gene expression. Thus, our findings provide insight into N-responsive growth phase transitions and optimization of plant fitness under nutrient-limited conditions.

Involvement of the membrane-localized ubiquitin ligase ATL8 in sugar starvation response in Arabidopsis.

As major components of the ubiquitin system, ubiquitin ligases mediate the transfer of ubiquitin to specific target substrates, thereby playing important roles in regulating a wide range of cellular processes. The Arabidopsis Tóxicos en Levadura (ATL) family is a group of plant-specific RING-type ubiquitin ligases with N-terminal transmembrane-like domains. To date, 91 ATL isoforms have been identified in the Arabidopsis genome, with some reported to regulate plant responses to environmental stresses. However, the functions of most ATLs remain unclear. This study showed that ATL8 is a sugar starvation response gene and that ATL8 expression was significantly increased by sugar starvation conditions but repressed by exogenous sugar supply. The ATL8 protein was found to possess ubiquitin ligase activity in vitro and to localize to membrane-bound compartments in plant cells. In addition, Starch Synthase 4 was identified as a putative interactor with ATL8, suggesting that ATL8 may be involved in modulating starch accumulation in response to sugar availability. These findings suggest that ATL8 functions as a membrane-localized ubiquitin ligase likely to be involved in the adaptation of Arabidopsis plants to sugar starvation stress.

Methods for Elucidation of Plant Senescence in Response to C/N-Nutrient Balance.

Carbon (C) and nitrogen (N) are essential elements for metabolism, and the ratio of C to N availability is called the C/N balance. C/N balance is very important for plant growth, but little is known about the detailed mechanisms of plant C/N responses. Previously a method of treating Arabidopsis plants with sugar-supplemented medium for studying C/N responses at early post-germinative growth stages has been developed. This method, however, cannot be used to determine physiological C/N effects in plants of mature growth stages, including senescence. Here we present two methods of analyzing responses to C/N treatments in senescing plants: transient C/N treatment with liquid medium and long-term C/N treatment with elevated atmospheric CO2.

Sugar-responsive transcription factor bZIP3 affects leaf shape in Arabidopsis plants.

Sugars are essential for plant metabolism, growth and development. Plants must therefore manage their growth and developmental processes in response to sugar availability. Sugar signaling pathways constitute a complicated molecular network and are associated with global transcriptional regulation. However, the molecular mechanisms underlying sugar signaling remain largely unclear. This study reports that the protein basic-region leucine zipper 3 (bZIP3) is a novel sugar-responsive transcription factor in Arabidopsis plants. The expression of bZIP3 was rapidly repressed by sugar. Genetic analysis indicated that bZIP3 expression was modulated by the SNF1-RELATED KINASE 1 (SnRK1) pathway. Moreover, transgenic plants overexpressing bZIP3 and dominant repressor form bZIP3-SRDX showed aberrant shaped cotyledons with hyponastic bending. These findings suggest that bZIP3 plays a role in plant responses to sugars and is also associated with leaf development.

Membrane-localized ubiquitin ligase ATL15 functions in sugar-responsive growth regulation in Arabidopsis.

Ubiquitin ligases play important roles in regulating various cellular processes by modulating the protein function of specific ubiquitination targets. The Arabidopsis Tóxicos en Levadura (ATL) family is a group of plant-specific RING-type ubiquitin ligases that localize to membranes via their N-terminal transmembrane-like domains. To date, 91 ATL isoforms have been identified in the Arabidopsis genome, with several ATLs reported to be involved in regulating plant responses to environmental stresses. However, the functions of most ATLs remain unknown. This study, involving transcriptome database analysis, identifies ATL15 as a sugar responsive ATL gene in Arabidopsis. ATL15 expression was rapidly down-regulated in the presence of sugar. The ATL15 protein showed ubiquitin ligase activity in vitro and localized to plasma membrane and endomembrane compartments. Further genetic analyses demonstrated that the atl15 knockout mutants are insensitive to high glucose concentrations, whereas ATL15 overexpression depresses plant growth. In addition, endogenous glucose and starch amounts were reciprocally affected in the atl15 knockout mutants and the ATL15 overexpressors. These results suggest that ATL15 protein plays a significant role as a membrane-localized ubiquitin ligase that regulates sugar-responsive plant growth in Arabidopsis.

Arabidopsis CBL-Interacting Protein Kinases Regulate Carbon/Nitrogen-Nutrient Response by Phosphorylating Ubiquitin Ligase ATL31.

In response to the ratio of available carbon (C) and nitrogen (N) nutrients, plants regulate their metabolism, growth, and development, a process called the C/N-nutrient response. However, the molecular basis of C/N-nutrient signaling remains largely unclear. In this study, we identified three CALCINEURIN B-LIKE (CBL)-INTERACTING PROTEIN KINASES (CIPKs), CIPK7, CIPK12, and CIPK14, as key regulators of the C/N-nutrient response during the post-germination growth in Arabidopsis. Single-knockout mutants of CIPK7, CIPK12, and CIPK14 showed hypersensitivity to high C/low N conditions, which was enhanced in their triple-knockout mutant, indicating that they play a negative role and at least partly function redundantly in the C/N-nutrient response. Moreover, these CIPKs were found to regulate the function of ATL31, a ubiquitin ligase involved in the C/N-nutrient response via the phosphorylation-dependent ubiquitination and proteasomal degradation of 14-3-3 proteins. CIPK7, CIPK12, and CIPK14 physically interacted with ATL31, and CIPK14, acting with CBL8, directly phosphorylated ATL31 in a Ca2+-dependent manner. Further analyses showed that these CIPKs are required for ATL31 phosphorylation and stabilization, which mediates the degradation of 14-3-3 proteins in response to C/N-nutrient conditions. These findings provide new insights into C/N-nutrient signaling mediated by protein phosphorylation.

Regulation of senescence under elevated atmospheric CO2 via ubiquitin modification.

Elevated atmospheric CO2 concentration is a serious global environmental problem. Elevated CO2 affects plant growth by changing primary metabolism, closely related to carbon (C) and nitrogen (N) availability. Under sufficient N conditions, plant growth is dramatically promoted by elevated CO2. When N availability is limited, however, elevated CO2 disrupts the balance between cellular C and N (C/N). Disruption of the C/N balance is regarded as an important factor in plant growth defects. Here we highlight the regulation of senescence in higher plants by atmospheric CO2 and N, and the physiological function of C/N-related ubiquitin ligase ATL31 under condition of elevated CO2. We also provide an overview of the ubiquitin ligases and related enzymes involved in regulating senescence in plants.

Phosphorylation of Arabidopsis ubiquitin ligase ATL31 is critical for plant carbon/nitrogen nutrient balance response and controls the stability of 14-3-3 proteins.

Ubiquitin ligase plays a fundamental role in regulating multiple cellular events in eukaryotes by fine-tuning the stability and activity of specific target proteins. We have previously shown that ubiquitin ligase ATL31 regulates plant growth in response to nutrient balance between carbon and nitrogen (C/N) in Arabidopsis. Subsequent study demonstrated that ATL31 targets 14-3-3 proteins for ubiquitination and modulates the protein abundance in response to C/N-nutrient status. However, the underlying mechanism for the targeting of ATL31 to 14-3-3 proteins remains unclear. Here, we show that ATL31 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. We identified Thr(209), Ser(247), Ser(270), and Ser(303) as putative 14-3-3 binding sites on ATL31 by motif analysis. Mutation of these Ser/Thr residues to Ala in ATL31 inhibited the interaction with 14-3-3 proteins, as demonstrated by yeast two-hybrid and co-immunoprecipitation analyses. Additionally, we identified in vivo phosphorylation of Thr(209) and Ser(247) on ATL31 by MS analysis. A peptide competition assay showed that the application of synthetic phospho-Thr(209) peptide, but not the corresponding unphosphorylated peptide, suppresses the interaction between ATL31 and 14-3-3 proteins. Moreover, Arabidopsis plants overexpressing mutated ATL31, which could not bind to 14-3-3 proteins, showed accumulation of 14-3-3 proteins and growth arrest in disrupted C/N-nutrient conditions similar to wild-type plants, although overexpression of intact ATL31 resulted in repression of 14-3-3 accumulation and tolerance to the conditions. Together, these results demonstrate that the physiological role of phosphorylation at 14-3-3 binding sites on ATL31 is to modulate the binding ability and stability of 14-3-3 proteins to control plant C/N-nutrient response.

Ubiquitin ligase ATL31 functions in leaf senescence in response to the balance between atmospheric CO2 and nitrogen availability in Arabidopsis.

Carbon (C) and nitrogen (N) are essential elements for metabolism, and their availability, called the C/N balance, must be tightly coordinated for optimal growth in plants. Previously, we have identified the ubiquitin ligase CNI1/ATL31 as a novel C/N regulator by screening plants grown on C/N stress medium containing excess sugar and limited N. To elucidate further the effect of C/N balance on plant growth and to determine the physiological function of ATL31, we performed C/N response analysis using an atmospheric CO2 manipulation system. Under conditions of elevated CO2 and sufficient N, plant biomass and total sugar and starch dramatically increased. In contrast, elevated CO2 with limited N did not increase plant biomass but promoted leaf chlorosis, with anthocyanin accumulation and increased senescence-associated gene expression. Similar results were obtained with plants grown in medium containing excess sugar and limited N, suggesting that disruption of the C/N balance affects senescence progression. In ATL31-overexpressing plants, promotion of senescence under disrupted CO2/N conditions was repressed, whereas in the loss-of-function mutant it was enhanced. The ATL31 gene was transcriptionally up-regulated under N deficiency and in senescent leaves, and ATL31 expression was highly correlated with WRKY53 expression, a key regulator of senescence. Furthermore, transient protoplast analysis implicated the direct activation of ATL31 expression by WRKY53, which was in accordance with the results of WRKY53 overexpression experiments. Together, these results demonstrate the importance of C/N balance in leaf senescence and the involvement of ubiquitin ligase ATL31 in the process of senescence in Arabidopsis.