Carbon and nitrogen metabolism regulated by the ubiquitin-proteasome system

Abstract

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 homolog 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 ubiquitin-proteasome system (UPS) plays a crucial role in the selective removal of short-lived target proteins, enabling finetuning of post-translation levels of the targets.¹ The UPS controls multiple phenomena in plant growth and development by regulating the stability of target proteins that govern specific cellular events including photomorphogenesis, cell cycle, senescence, defense response and phytohormone response.² To achieve precise degradation of target proteins, ubiquitin ligases have an essential role in this system. The ubiquitin ligase (E3) interacts with the target protein and adds ubiquitin molecules derived from the ubiquitin activating enzyme (E1) and the ubiquitin conjugating enzyme (E2). The poly-ubiquitinated substrate is then degraded by a multi-subunit protease complex, the proteasome. Ubiquitin ligase is the key enzyme to specify the target protein for degradation via UPS. The Arabidopsis genome contains more than 1,200 genes encoding ubiquitin ligases.³ Some of the essential signaling factors for phytohormone response were identified as the target of each ubiquitin ligase and showed an impressive mechanism of phytohormone signaling.⁴ However, the physiological function of most ubiquitin ligase is still unknown. Our recent studies have revealed that UPS modulates plant development in response to carbon/nitrogen (C/N) balance conditions.^5,6 Here we focus on the ubiquitin ligase and summarize the regulatory mechanism of carbon and nitrogen signaling via UPS.

Ubiquitin Ligase ATL31 and ATL6 Regulate Plant C/N Response

Plant growth and development is controlled by the concerted actions of signaling pathways that are triggered by various environmental conditions and developmental cues. Nutrient availability, in particularly that of carbon and nitrogen, is one of the most important factors for regulating plant metabolism and development. In addition to independent utilization, the ratio of carbohydrate to nitrogen metabolites in the cell is also important for regulation of plant growth and is referred to as the “C/N balance.”^7,8 However, little is known about the molecules involved in regulation of the C/N response and checkpoint for post-germinative growth arrest in plants. A PII-like protein (GLB1) and the glutamate receptor (AtGLR1.1) have been shown to have a role in the coordinated regulation of C- and N-metabolism, and are also thought to function in C/N signaling in plants.^9–11

To further clarify the mechanism of C/N response, we previously identified and characterized a novel C/N response mutant, cni1-D (carbon/nitrogen insensitive 1-D), which showed to survive in the medium containing excessive glucose under limited nitrogen conditions (300 mM glucose/0.1 mM nitrogen).⁵ The CNI1 gene encodes a RING-H2 type ubiquitin ligase and has been previously annotated as a member of the ATL family, ATL31 of which function was not understood.¹² ATL family proteins contain a transmembrane-like hydrophobic region at the N-terminus, a basic amino acid rich region, a region with highly conserved amino acid sequences (GLD), a RING-H2 type zinc finger domain and a non-conserved C-terminal region.¹³ There are 80 members of the ATL family in Arabidopsis, with ATL2 the best characterized and shown to be involved in defense responses.^13–15 Further experiments revealed that the cni1-D mutant, which is caused by overexpression of the ATL31 gene, leads to a less-sensitive phenotype while loss of the function mutant for the ATL31 gene shows a hypersensitive phenotype to C/N stress conditions. In addition, the ATL31 shares high sequence similarity with another ATL family member, ATL6, and the ATL6 loss-of function mutant also showed C/N hypersensitivity.⁵ 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 loss of the less-sensitive phenotype, suggesting that both ATL31 and ATL6 regulate plant C/N response via ubiquitination and following proteasome-dependent degradation of specific target proteins.⁵

14-3-3 Protein as a Target of ATL31 and ATL6

We recently identified 14-3-3 protein as interactor of ATL31 with proteomics analysis and demonstrated the ATL31 has ubiquitination activity to the 14-3-3.⁶ ATL6 was also confirmed to interact and ubiquitinate the 14-3-3 protein. Further experiments showed the 14-3-3 protein is degraded by proteasome-dependent manner and the stability of the 14-3-3 is affected by C/N conditions under control by ATL31 and ATL6 activity. Finally, it was also demonstrated that overexpression of the 14-3-3 protein results in hypersensitivity to C/N stress conditions, which is enhanced by the absence of ATL31 and ATL6 activity. These data indicate that the ATL31 regulates the C/N response by degradation of the 14-3-3 protein via ubiquitination followed by proteasome-mediated degradation (Fig. 1A). The Arabidopsis 14-3-3 protein family contains at least 13 members that show high amino acid sequence similarity,^16,17 and 8 members were detected as interactors of ATL31 by our MS analysis while the functional specificity of each member has remained unclear. The 14-3-3 proteins bind phosphorylated motifs and function in multiple developmental processes by regulating the activity of a wide variety of target proteins.^17–20 In particular, 14-3-3 has been reported to regulate primary carbon and nitrogen metabolism by direct interaction with essential enzymes such as the plasma membrane H⁺-ATPase, nitrate reductase, sucrose phosphate synthase, ADP-glucose pyrophosphorylase and glutamine synthetase, for example.^21–26 In addition to direct regulation of enzyme activity, binding with the 14-3-3 protein changes the cleavage of some of these enzymes in response to nutrient conditions²⁵ and regulates the sub-cellular localization of target proteins.^27,28 On the other hand, the 14-3-3 protein may act as an adaptor as well as a scaffold protein, which recruits the other target protein X to be degraded, mediated by the ATL31/ATL6 ubiquitin ligase (Fig. 1B). Since the 14-3-3 protein forms a homo- or hetero-dimer and each 14-3-3 has a site to associate with a typical motif including phosphorylated Ser/Thr, the 14-3-3 protein complex binds with at least two different client proteins.^29,30

Figure 1.

Proposed model of ATL31 and ATL6 function to regulate plant C/N response during post-germinative growth (A) ATL31/ATL6 binds and ubiquitinates to the 14-3-3 protein to promote protein degradation via the 26S proteasome, which affects the activity of 14-3-3 client proteins and results in proper C/N response. Under high C/N stress conditions, over-accumulation of 14-3-3 proteins leads to growth arrest (red-colored arrow). Under low C/N ratios, the 14-3-3 proteins are degraded and post-germination growth continues through the phase transition checkpoint (green-colored arrow). On the other hand, we propose another hypothesis (B) that the 14-3-3 proteins function as adaptor proteins to connect the ATL31/ATL6 and target protein X for ubiquitination to be degraded. In this model, the target protein X for ubiquitination is the client of 14-3-3 proteins, indicating that the ATL31/ATL6 directly regulates degradation of multiple proteins via the proteasome.

Other Ubiquitin Ligase Involved in Carbon and Nitrogen Signaling

Carbon (sugar) signaling.

Ubiquitin ligases involved in carbon and nitrogen signaling are listed in Table 1. KEEP ON GOING (KEG) is a RING-HCa type ubiquitin ligase which contains a series of ankyrin repeats and Ser/Thr kinase domains.³¹ The loss-of function mutant of KEG (keg) showed hypersensitivity to sugars and post-germinative growth was arrested by exogenous glucose and sucrose in the medium. The keg mutant also showed a hypersensitive phenotype to abscisic acid (ABA). Furthermore, it was demonstrated that KEG directly interacted with the ABI5 protein, a bZIP transcription factor, which positively regulates the ABA signaling, and the ABI5 protein is accumulated in the mutant. Taken together, the authors concluded that KEG regulates the ABA signaling by the degradation of ABI5 via UPS. An ABI3-interacting protein 2 (AIP2) and a RING-H2 type ubiquitin ligase were isolated as interactors of abscisic acid insensitive 3 (ABI3) by yeast-two hybrid analysis.³² It was demonstrated that AIP2 ubiquitinates ABI3 and negatively regulates the ABA signaling.³³ Although the sugar sensitivity of the AIP2 mutant is not evaluated in the paper, the AIP2 is also expected to regulate sugar response since the abi3 mutant showed glucose-insensitivity at the seedling growth stage.³⁴

Table 1.

Ubiquitin ligase involved in carbon and nitrogen signaling

Ubiquitin ligase	Type	Function	Substrate	Ref
Carbon
KEG	RING-HCa	Inhibition of ABA signaling	ABI5	31
AIP2	RING-H2	Inhibition of ABA signaling	ABI3	33
SIS3	RING-H2	Sugar signaling independent on ABA	?	35
ATL43	RING-H2	ABA signaling	?	12
Nitrogen
NLA	RING-HCa	Low nitrogen adaptation/Phenylpropanoid metabolism	?	38, 39
C/N balance
ATL31/ATL6	RING-H2	C/N signaling	14-3-3	5, 6

SUGAR-INSENSITIVE3 (SIS3) is also a ubiquitin ligase containing a RING domain (RING-H2 type) and putative transmembrane domains.³⁵ SIS3 loss-of function leads to insensitive phenotype to excessive glucose and sucrose in the medium. On the other hand, the ABA response of sis3 mutant was similar to the wild-type plant, suggesting that the SIS3 regulates sugar response via an ABA-independent pathway. The ubiquitination target of SIS3 has not been reported.

Some members of the ATL family are also expected to be involved in sugar response. The T-DNA insertion line for ATL43 showed an increased germination ratio in the medium containing a high amount of glucose and also ABA,¹² suggesting that ATL43 is involved in sugar signaling via ABA pathway. Osuna et al. analyzed the temporal change of transcriptome in response to sugar status with microarray analysis and demonstrated that the ATL8 and ATL11 genes are transcriptionally repressed 30 min after application of sucrose to carbon-deprived Arabidopsis seedlings.³⁶ Usadel et al. also showed the repression of ATL8 expression dependent on the diurnal cycle.³⁷ Interestingly, a database search shows that the ATL8 mRNA is co-expressed with a group of enzymes involved in branched-chain amino acid degradation and galactose metabolism (ATTED II; http://atted.jp), which suggests that ATL8 functions in response to carbon source starvation. In contrast, the expression of the ATL31 gene increased in 3 h by sucrose treatment (Osuna et al. 2008, Sup. information).

Nitrogen Signaling.

A nitrogen limitation adaptation (nla) mutant was identified by the screening with T-DNA insertion mutants grown in low nitrogen conditions³⁸ (Table 1). The nla displayed an early senescence phenotype in limited nitrogen conditions, which did not occur when the mutant was grown in sufficient nitrogen conditions. NLA encodes RING-HCa type ubiquitin ligase with an SPX domain. While the ubiquitin ligase activity of the NLA was not confirmed with in vitro assay, it was demonstrated that the NLA is associated with UBC8, a member of the Arabidopsis E2 protein, in plant cells with a bimolecular fluorescence complementation assay using split YFP. Interestingly, the NLA and NLA-UBC8 complex were localized to nuclear speckles although the ubiquitination target and biochemical function of NLA have not been reported. Subsequent study in the nla mutant indicated that the phenylpropanoid metabolic flux is affected and switched to a lignin biosynthesis pathway from anthocyanin biosynthesis in limited nitrogen conditions.³⁹

Conclusion and Perspective

Our recent studies have revealed that ubiquitin ligases ATL31 and ATL6 target 14-3-3 proteins and regulate the C/N response via UPS-mediated degradation, which suggests a novel regulatory mechanism for primary carbon and nitrogen metabolism.^5,6 In the future studies, we will clarify the detailed mechanism of interaction between ATL31 and the 14-3-3 protein including up-stream signals such as the phosphorylation cascade. We should also reveal the dynamics of 14-3-3 proteins associated with target enzymes and how C/N metabolism is regulated.

In addition, studies on other ubiquitin ligases indicated that plant UPS contributes extensively toward regulation of carbon and nitrogen response. Identification of the targets for these ubiquitin ligases will provide us with further information about the regulation of the signaling and metabolism. Furthermore, integrated studies on information between signaling mechanisms including UPS and metabolite dynamics will be needed to elucidate the complicated mechanisms of how plants coordinate development in response to nutrient environments.