Ice plant (Mesembryanthemum crystallinum L.) is a model plant for studying salt-tolerance mechanism in higher plants. The unique responses for salinity tolerance of ice plant can be induced at specific developmental stages. McCPN1, a plant-specific RING-type copine, is identified from halophyte Mesembryanthemum crystallinum L. and has a copine vWA domain at the N-terminus for protein-protein interaction and a C-terminal really interesting new gene (RING)-finger domain which contains ubiquitin E3 ligase activity. McCPN1 interacts and ubiquitinates a salt-induced protein McSKD1, a protein involving in protein trafficking. McCPN1 has high similarity to AtRGLG2, an E3 ligase mediates auxin transport and salt and drought stress response in Arabidopsis. To explore the role of McCPN1 in the salt stress adaptation process of ice plant, full-length McCPN1 was used as a bait to perform yeast two hybrid (Y2H) screen in the cDNA library constructed from roots of salt-treated ice plant. There were 120 candidate proteins identified from Y2H screening and eight candidates, E3, E9, E35, E36, E45, E46, E65 and E67 were chosen to determine the DNA sequences, after nutrition screening and β-galactosidase activity assays. Candidate E3 and E9 were Argonaute 4 (AGO4) which participates in small RNAs interference-mediated regulation of genes expression. E35 was a galactose-binding protein with galactose-binding domain-like and Sad 1/ UNC-like (SUN) C-terminal domain and, based on domain analysis, involved in cell adhesion, proliferation, apoptosis, nuclear anchoring and migration, and pre-mRNA splicing. E36 was xyloglucan endotransglucosylase/hydrolases and participates in cell wall extensibility, abscission, fruit growth, ripening and softening. E45 was DCD (Development and Cell Death) domain protein which interacts with cytoskeleton proteins for its movement and participates in programmed cell death under endoplasmic reticulum stress and osmotic stress. E46 was 26S ribosomal RNA in DNA blasting and unknown function protein of maize in protein sequence searching. E65 was heat shock protein 70 and responsible for proteins folding, refolding, transport and degradation in lysosomes or proteasomes and preventing protein aggregation. E67 was SPIKE1 protein which mediates tissue organization, cell morphology by controlling cytoskeleton microtubule, vesicle trafficking from the ER to Golgi in early secretory pathway homeostasis. According to Y2H result, McCPN1 can interact with candidate proteins to regulate cell morphology, development, death processes, and help protein trafficking, refolding and degradation after salt stress through ubiquitination. Cellular localization of McCPN1-YFP fusion protein was found in the plasma membrane, ER and Golgi, and distributed over the cytoplasm. The result suggests that McCPN1 widely distributes in several compartments and participates the ubiquitination process in these campartments. Candidates E3 and E9 blasted as AGO4 were chosen for further analyses. Full-length McAGO4 was identified by rapid amplification of cDNA ends (RACE). The domain analysis showed it has an amino-terminal domain DUF1785, a central PAZ domain and PIWI domain at C-terminus. Full-length McCPN1 and full-length McAGO4 have weak interaction as shown by pair-wise Y2H, pull-down assay, protoplast two-hybrid and bimolecular fluorescence complementation (BiFC). The weak protein-protein interaction might be the result of steric hindrance between two proteins. It is known that AGO4 locates at the nucleus and is involved in RNA-directed DNA methylation (RdDM). The BiFC results showed McCPN1 and McAGO4 colocalized at the nucleus suggesting that cytoplasmic localized McCPN1 moves to the nucleus to participate McAGO4-mediated gene silencing. In conclusion, McCPN1 is involved in many salt stress responses and one of them is to modulate McAGO4 activity by ubiquitination and changes gene expression profiles to increase salt tolerance of ice plant.
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