Sodium chloride (nacl)

Cadmium oxide (CdO, 99.99%), zinc oxide (ZnO, 99.99%, power), sulfur (S, 99.98%, power), selenium (Se, 99.99%, power), oleic acid (OA, 90%), 1-octadecene (ODE, 90%), and 2-(N-morpholino) ethanesulfonic acid (MES) were purchased from Aldrich. NaOH, HCl, NaCl, KCl, Na₂CO₃, NaHCO₃, KH₂PO₄, Na₂HPO₄, H₃BO₃, Na₂B₄O₇∙10H₂O, Tris, Hepes, and Tween-20 were purchased from Shanghai Sangon Ltd (China). Bovine serum albumin (BSA) and calf serum were purchased from Sigma. 1-ethyl-3-(3-(dimethylamino) propyl) carbodiimide (EDC), N-Hydroxysulfosuccinimide (sulfo-NHS) and the microplates were purchased from Thermo Fisher Scientific (USA). Mouse anti C-reaction protein monoclonal antibody and CRP antigen were obtained from Abcam (USA). The fluorescence spectra were detected using SpectraMaxi3 (Molecular Devices, Sunnyvale, USA). Images of electrophoresis gels were taken using a gel imaging system (GenoSens1860, Shanghai, China). The sizes of QDs and QD-antibody probe were recorded using dynamic light scattering (Nano-ZS 90, Malvern Instruments, UK). Purified water (18.2 mΩ, Millipore USA) was used in all experiments.

The seeds of hulless barley used for this study were purchased from the seed company of Xining City, Qinghai Province in China. After soaking in sterilized water for 16 h, the seeds were first treated with 70% ethanol for 3 min, and surface sterilized with 0.1% HgCl₂ for 10 min. After fine washing with sterile water five times, the pretreated seeds were placed onto plastic dishes, containing filter paper soaked with sterile water for germination, in an incubator under 100 μE•M⁻²•s⁻¹ constant illumination provided via cool white fluorescents at 25°C for 10 days. When the height of seedlings reached 10 cm, the illumination condition was adjusted from constant to a 12/12 h light/dark cycle for 2 days before simulating drought, cold, and salt stress treatments, as an entrainment of the transcriptome-wide gene expression.
For the cold treatment, seedlings were treated for 1 day in the 12/12 h light/dark cycle at 5°C, and then 0.1 g leaf tissues were then harvested every 4 h under constant light. For the drought and salt treatments, seedlings were first cultured in 15% (w/v) PEG6000 (Sangon, China) or 300 mM NaCl (Sangon, China) for 24 h under the 12/12 h light/dark cycle, and then 0.1 g leaf tissues were harvested every 4 h under constant light. All plant samples were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction.

Cells were lysed in NP-40 buffer containing 50 mM Tris-HCl (pH 7.5) (Sigma, St Louis, MO, USA), 150 mM NaCl (Sangon, Shanghai, China), 0.5% Nonidet P-40 (Sigma), 1 μg/ml aprotinin (Sigma), 1 μg/ml leupeptin (Sigma), 1 μg/ml pepstatin (Sigma), 1 mM Na3VO4 (Sigma) and 1 mM PMSF (Sigma). For immunoprecipitation, 500 μl of cell lysate was incubated with HA antibody (provided by the Zhao lab of Fudan University) for 3 h at 4 °C with rotation. Then, 30 μl of Protein A Agarose (Millipore, Billerica, MA, USA) was added for 12 h at 4 °C with rotation, and the beads were washed three times with lysis buffer before proteins were dissolved in loading buffer. Deacetylation assays were carried out in the presence of 5 μg enzyme and 0.3 μg peptide in 30 μl reaction buffer [30 mM HEPES (Sigma), 0.6 mM MgCl2 (Sangon), 1 mM DTT (Sigma), 1 mM NAD+ (Sigma), 10 mM PMSF (Sigma)]. The deacetylation reaction was incubated for 2 h at 37 °C before the mixture was desalted by passing it through a C18 ZipTip (Millipore). The desalted samples were analyzed using a MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Grand Island, NY, USA). The acetylated peptide used in the assay was TRKDYPAAK (Ac) RVKLDSVR (Glssale, Shanghai, China).

Dithiothreitol (DTT), Tris-base, phenyl methylsulfonyl fluoride (PMSF) and NaCl, were purchased from Sangon (Shanghai, China). KOD-plus mutagenesis kits were obtained from TOYOBO (Osaka, Japan). All restriction endonucleases, T4 DNA ligase, T4 Polynucleotide Kinase, Dynabeads protein G and Lipofectamine 2000 transfection reagent were obtained from Thermo Scientific (Waltham, MA, USA). All materials for the yeast two-hybrid screening and assay, including the Matchmaker Gold Yeast Two-Hybrid System, media, reagents, and a normalized Mate & Plate human cDNA library, were purchased from Clontech (Shiga, Japan). Escherichia coli Rosetta (DE3) cells were purchased from Stratagene (Santa Clara, CA, USA). Polyvinylidene fluoride (PVDF) membranes were obtained from Millipore (Darmstadt, Germany). DNA sequencing was performed by Biosune (Shanghai, China).