#METABOLOMICS WORKBENCH xmyu0616_20240128_193417 DATATRACK_ID:4618 STUDY_ID:ST003061 ANALYSIS_ID:AN005016 PROJECT_ID:PR001907 VERSION 1 CREATED_ON 05-29-2024 #PROJECT PR:PROJECT_TITLE Characterization of CsUGT73AC15 as a Multifunctional Glycosyltransferase PR:PROJECT_TITLE Impacting Flavonol Triglycoside Biosynthesis in Tea Plants PR:PROJECT_SUMMARY Flavonol glycosides, essential for the health benefits and astringent flavors of PR:PROJECT_SUMMARY tea (Camellia sinensis), are abundant in tea leaves as diglycosides and PR:PROJECT_SUMMARY triglycosides. However, the UDP-glycosyltransferases (UGTs) responsible for PR:PROJECT_SUMMARY flavonol multi-glycosylation have remained elusive. In this study, integrated PR:PROJECT_SUMMARY proteomic and metabolomic analyses were performed to identify potential UGTs PR:PROJECT_SUMMARY involved in flavonol triglycoside biosynthesis. Among the tested recombinant PR:PROJECT_SUMMARY enzymes, rCsUGT75AJ1 exhibited flavonoid 4ʹ-O-glucosyltransferase activity, PR:PROJECT_SUMMARY while rCsUGT75L72 catalyzed glucosylation of flavonoids preferentially at the PR:PROJECT_SUMMARY 3-OH position. Remarkably, rCsUGT73AC15 demonstrated substrate promiscuity and PR:PROJECT_SUMMARY regioselectivity, enabling glucosylation of rutin at multiple positions as well PR:PROJECT_SUMMARY as kaempferol 3-O-rutinoside at the 7-OH position. Expression analysis revealed PR:PROJECT_SUMMARY an inverse correlation between the level of CsUGT73AC15 and rutin abundance PR:PROJECT_SUMMARY across different cultivars, suggesting its involvement in flavonol triglycoside PR:PROJECT_SUMMARY biosynthesis in planta. These findings advance the understanding of flavonol PR:PROJECT_SUMMARY triglycoside biosynthesis in tea plants and provide a foundation for further PR:PROJECT_SUMMARY in-vivo characterization of gene functions. PR:INSTITUTE Fujian Agriculture and Forestry University PR:LAST_NAME Yu PR:FIRST_NAME Xiaomin PR:ADDRESS Shang Xia Dian Road No. 15, Fuzhou, Fujian, 350002, China PR:EMAIL xmyu0616@sina.com PR:PHONE (86)18649802306 PR:PUBLICATIONS https://doi.org/10.1021/acs.jafc.4c03824 PR:DOI http://dx.doi.org/10.21228/M8714X #STUDY ST:STUDY_TITLE Untargeted metabolomics analysis of three tea cultivars. ST:STUDY_TYPE leaf tissue ST:STUDY_SUMMARY To compare flavonoid metabolism in three tea cultivars, namely Camellia sinensis ST:STUDY_SUMMARY cv. "Jinfenghuang" (JFH), "Fuyun 6" (FY6) and "Bantianyao" (BTY), fresh tea ST:STUDY_SUMMARY leaves were plucked, rapidly ground to fine powders in liquid nitrogen and ST:STUDY_SUMMARY subjected to extraction by 70% methanol. The extracts were analyzed by ST:STUDY_SUMMARY UPLC-Quadrupole Time-of-Flight Mass Spectrometry (UPLC-QTOFMS) using an ACQUITY ST:STUDY_SUMMARY UPLC system in tandem to a SYNAPT G2-Si QTOF mass spectrometer (Waters, Milford, ST:STUDY_SUMMARY MA, USA), following the previously described method. Raw data were processed in ST:STUDY_SUMMARY Progenesis QI (v2.4, Nonlinear Dynamics) with default settings for peak picking ST:STUDY_SUMMARY and alignment. Metabolites were annotated by searching against our in-house ST:STUDY_SUMMARY MS/MS spectral library and public databases. ST:INSTITUTE Fujian Agriculture and Forestry University ST:LAST_NAME Yu ST:FIRST_NAME Xiaomin ST:ADDRESS Shangxia Dian Road No. 15, Fuzhou 350002, Fujian, China ST:EMAIL xmyu0616@sina.com ST:PHONE (86)18649802306 ST:SUBMIT_DATE 2024-01-28 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Camellia sinensis SU:TAXONOMY_ID 4442 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - BTY-1 Genotype:Bantianyao RAW_FILE_NAME=BTY-1.mzML SUBJECT_SAMPLE_FACTORS - BTY-2 Genotype:Bantianyao RAW_FILE_NAME=BTY-2.mzML SUBJECT_SAMPLE_FACTORS - BTY-3 Genotype:Bantianyao RAW_FILE_NAME=BTY-3.mzML SUBJECT_SAMPLE_FACTORS - FY6-1 Genotype:Fuyun 6 RAW_FILE_NAME=FY6-1.mzML SUBJECT_SAMPLE_FACTORS - FY6-2 Genotype:Fuyun 6 RAW_FILE_NAME=FY6-2.mzML SUBJECT_SAMPLE_FACTORS - FY6-3 Genotype:Fuyun 6 RAW_FILE_NAME=FY6-3.mzML SUBJECT_SAMPLE_FACTORS - JFH-1 Genotype:Jinfenghuang RAW_FILE_NAME=JFH-1.mzML SUBJECT_SAMPLE_FACTORS - JFH-2 Genotype:Jinfenghuang RAW_FILE_NAME=JFH-2.mzML SUBJECT_SAMPLE_FACTORS - JFH-3 Genotype:Jinfenghuang RAW_FILE_NAME=JFH-3.mzML #COLLECTION CO:COLLECTION_SUMMARY Mature leaves from the tea cultivars were plucked and flash-frozen in liquid CO:COLLECTION_SUMMARY nitrogen. CO:SAMPLE_TYPE Leaves #TREATMENT TR:TREATMENT_SUMMARY No treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Leaves were ground into a fine powder using a pre-cooled mortar and pestle. Tea SP:SAMPLEPREP_SUMMARY leaf powder (30 mg) was extracted with 1.0 mL of 70% methanol using SP:SAMPLEPREP_SUMMARY ultrasonication for 20 min. After centrifugation, the supernatant was filtered SP:SAMPLEPREP_SUMMARY through a PVDF filter (0.22 µm, Millipore). #CHROMATOGRAPHY CH:INSTRUMENT_NAME Waters Acquity I-Class CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:COLUMN_TEMPERATURE 40 CH:FLOW_GRADIENT 1–7% B (0–2 min), 7–40% B (2–13 min), 40–60% B (13–17 min), CH:FLOW_GRADIENT immediately elevated to 99% B (17 min), held at 99% B (17–22 min) CH:FLOW_RATE 0.3 mL/min CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:MS_COMMENTS MassLynx software (ver 4.1) was used to operate the instrument. Progenesis QI MS:MS_COMMENTS software (ver 2.4) was used for peak picking, alignment and normalization. MS:ION_MODE NEGATIVE MS:MS_RESULTS_FILE ST003061_AN005016_Results.txt UNITS:Da Has m/z:Yes Has RT:Yes RT units:Minutes #END