Unlike animal GAPDH with only one cytosolic form, GAPDHs in higher plants are classified into three groups according to their subcellular locations: cytoplasmic GAPC for glycolysis, glycolytic GAPCp in non-green plastids, and chloroplastic GAPA/B for photosynthetic CO2 fixation1,36C38. of overexpression are abolished when Peptide 17 is usually deficient, the heat-induced nuclear accumulation of GAPC is usually suppressed, or the GAPC-NF-YC10 conversation is disrupted. overexpression also enhances the binding ability of NF-YC10 to its target promoter. The results reveal a cellular and molecular mechanism for the nuclear moonlighting of a glycolytic enzyme in herb response to environmental changes. and suffered a higher transpirational water loss than wild type plants2. GAPC affected multiple herb immune responses to bacterial pathogen, such as reactive oxygen species production, programmed cell death, and autophagy8,11. GAPC was also involved in viral contamination10,12. One mechanism for the GAPCs action in stress response is usually its stress-induced nuclear translocation. A small pool of GAPC accumulated in the nucleus in Arabidopsis response to treatments with cadmium, bacterial flagellin, PA, and hydrogen sulfide6C8,13,14. The nuclear accumulation of GAPC was also observed in tobacco BY-2 (bright-yellow 2) cells exposed to long-chain bases, regulators for programmed cell death in plants15. Since GAPC has no nuclear localization transmission, post-translational modifications of specific amino acid residues are believed to be important for the stress-induced intracellular translocation. Under certain stress conditions, the highly reactive catalytic cysteine of GAPC undergoes thiol modifications, such as itself, indicating that GAPC is usually a transcriptional activator of glycolytic function16. This study was undertaken to determine how GAPC Peptide 17 affected nuclear function in herb stress responses. Here we show that GAPC interacts with the transcription factor nuclear factor Y subunit C10 (NF-YC10) and regulates transcriptional and physiological responses to heat stress. Results NF-YC10 is usually identified as a GAPC-binding transcription factor The nuclear translocation of GAPC under stress raises a possibility that GAPC may play a role in stress-responsive gene expression by modulating transcriptional activity of transcription factor(s) through direct protein-protein conversation. To test this possibility, we screened an Arabidopsis transcription factor library for transcription factors potentially binding to and modulated by GAPC. We altered an Arabidopsis cDNA library composed of ~1500 transcription factors24 to produce recombinant proteins in clones was verified by separation of the proteins on a polyacrylamide gel (Fig.?1a). The protein mixture was then co-immunoprecipitated with GAPC2-Flag that was purified from Arabidopsis overexpressing the Peptide 17 recombinant protein or from control plants with vacant vector (EV) using an anti-Flag antibody. SDS-PAGE analysis revealed the successful immunoprecipitation of GAPC2 as determined by Tnfsf10 the obvious GAPC2 band (and immunoglobulin G heavy/light chain bands; Fig.?1b). To identify proteins co-immunoprecipitated with GAPC2, we sequenced the entire immunoprecipitants by mass spectrometry and compared the recognized proteins between the GAPC2 sample and EV control. The mass spectrometry-based protein sequencing recognized the nuclear factor Y subunit C10 (NF-YC10) co-precipitated specifically with GAPC2 (Fig.?1c). Open in a separate windows Fig. 1 Screening of Arabidopsis transcription factors to identify GAPC-binding proteins.a Mixture of the purified transcription factors. Proteins were purified by affinity chromatography and separated on a polyacrylamide gel. The gel was stained with Coomassie Amazing Blue. Protein marker size is usually on the left. b Gel image of co-immunoprecipitation. Transcription factors co-immunoprecipitated with GAPC2-Flag or vacant vector control (EV) were separated on a polyacrylamide gel and stained with Coomassie Amazing Blue. Protein marker size is usually on the left. Positions of GAPC2, immunoglobulin G (IgG) heavy and light chains are on the right. c Mass spectrometry-based identification of NF-YC10. Trypsin-digested peptides from your immunoprecipitated samples were sequenced by LC-MS/MS. Shown here is NF-YC10 sequence with the unique peptides in reddish that were recognized with probability 99%. GAPC conversation with NF-YC10 occurs in vitro and Peptide 17 in planta To verify the conversation between GAPC and NF-YC10, we cloned from Arabidopsis and expressed the recombinant protein as a fusion with 6xHis tag in cell lysate and purified to near homogeneity (Fig.?2a). The purified NF-YC10 was then co-immunoprecipitated using an anti-Flag antibody with GAPC1-Flag or GAPC2-Flag purified from Arabidopsis overexpressing the respective proteins or proteins purified from control plants with vacant vector (EV). Immunoblotting analysis using an anti-6xHis antibody exhibited that NF-YC10 was co-precipitated with both GAPC1 and GAPC2, but not with EV control (Fig.?2b). Next, we performed a bimolecular fluorescence complementation (BiFC) assay to verify the GAPC-NF-YC10 conversation Peptide 17 in planta. GAPC1 or GAPC2 fused with the N-terminal half of yellow fluorescence protein (GAPC-YFPN) and NF-YC10 with C-terminal half of YFP (NF-YC10-YFPC) were co-expressed transiently in tobacco leaves, and the fluorescent transmission was observed under a confocal microscope. For both GAPC1 and GAPC2, co-expression of GAPC.