I-qMS/MS analysis was carried out by the Metabolomic Core Facility at the Agricultural Biotechnology Research Center, Academia Sinica, Taiwan. A HALO C18 (Advanced Materials Technologies, Inc., Wilmington, DE, USA) column (inner diameter, two.1 mm; column length, 75 mm; particle size, 2.7 ) was used, and gradient elution was performed with water and 0.05 glacial acetic acid (solvent A) and acetonitrile with 0.05 glacial acetic acid (solvent B) at a constant flow price of 0.6 mL min-1 . The following gradient profile was applied: t (min), A): (0, 99), (two.20, 0), (2.40, 0), (two.60, 99), (three, 99). The MS and MS/MS experiments had been performed with an API 3000 triple quadrupole mass spectrometer (PE Sciex, Concord, Ont., Canada) with all the following parameters: temperature of 400 C, nebulizer gas (N2 ) 10 (arbitrary units), curtain gas (N2 ) 12 (arbitrary units), collision gas (N2 ) four (arbitrary units), plus the Dopamine Receptor Modulator list capillary voltage of -3.five kV. The mass spectrometer was operated in many reaction mode (MRM). To germinate seeds for the ABA sensitivity assay, Arabidopsis seeds have been sterilized and spread around the MS medium with or without having 0.11 ABA, along with the growth situations had been observed at 7 days immediately after germination.Viruses 2021, 13,four of2.five. Real-Time Quantitative PCR qRT-PCR was performed to validate the expression patterns of chosen DEGs in the HTP network. Total RNA was extracted from every single biological replicate in the Col-0, P1/HC-ProTu , and P1/HC-ProZ plants (each replicate consisted of 250 seedlings) applying a plant total RNA extraction miniprep program (Viogene-Biotek Corporation, New Taipei City, Taiwan). The obtained RNA was treated with a TURBO DNA-free Kit (Ambion Thermo Fisher Scientific, Waltham, MA, USA) then subjected to phenol/chloroform extraction and alcohol precipitation to take away contaminating genomic DNA. First-strand cDNA was synthesized using MMLV reverse transcriptase (Invitrogen, iNOS Inhibitor custom synthesis Carlsbad, CA, USA). Gene-specific primers for the DEGs were made applying Primer3Plus [9]. The primer sets of OZF1_qPCR_F1 (five -CGGATTCGTAAACCGGAGTGTCTG-3 ) and OZF1_qPCR_R1 (5 GAGGAATCTCCCTCGAATCATCGATTATG-3 ) for OZF1, MYB44_qPCR_F1 (five -GGAGTT GGGAGAATCGAGTAGACAAAGTG-3 ) and MYB44_qPCR_R1 (5 -CGTCACTACGTCCC CAGCTCTC-3 ) for MYB44, MYB96_qPCR_F1 (five -GCTCTACAACACTCTTTTCCCCTTTTG G-3 ) and MYB96_qPCR_R1 (five -GCATAACCATATGAGCCACAAAGTGAAAC-3 ) for MYB96, ABF4_qPCR_F1 (five -TGGTGCAAATGAGGCCATGATTGG-3 ) and ABF4_qPCR_R1 (five -GGCAAAACAAATCATGCAGTGTACCTG-3 ) for ABF4, IQM4_qPCR_F1 (five -GCCTTG TCAACTTAACTCACCAAGAAGTG-3 ) and IQM4_qPCR_R1 (five -CCTTGGGCATTTCACC TAAACCAGAAG-3 ) for IQM4, CaLB_qPCR_F1 (5 -TCCTTGGTTTTGTGTGTTCATCATC CTC-3 ) and CaLB_qPCR_R1 (five -GCGATGATTATACGCCGATAAGTTCCG-3 ) for CaLB, CPK28_qPCR_F1 (five -CGCAGCAAAACAAAGAGAGAAAGTGG-3 ) and CPK28_qPCR_R1 (five -ATTCAGGGAATGCCACGTGTCCTC-3 ) for CPK28 and P_Actin2 (5 -CCTCAATCTCA TCTTCTTCCGCTC-3 ) and M_Actin2 (5 -AGCATCATCTCCTGCAAATCCAGC-3 ) for ACT2 had been made use of for expressional detection. The qRT-PCR assays had been performed making use of the Light Cycler 480 Method (Roche) with all the KAPA SYBR Rapid qPCR Master Mix (two Kit (Sigma-Aldrich, St. Louis, MO, USA). Three biological replicates and three technical replicates had been incorporated inside the assays. The expression levels of ACT2 were utilised because the internal control, and normalized mRNA expression levels have been calculated employing the formula 2-Ct . 2.six. Expression-Based Heatmaps and Principal Component Analysis (PCA) The identified DEGs were functionally annotated based on their sequence