#METABOLOMICS WORKBENCH jemolina_20210626_043323 DATATRACK_ID:2719 STUDY_ID:ST001854 ANALYSIS_ID:AN003005 VERSION 1 CREATED_ON 03-30-2024 #PROJECT PR:PROJECT_TITLE Metabolic profiling of Rafflesia-infected Tetrastigma and applications for PR:PROJECT_TITLE propagation PR:PROJECT_SUMMARY Endemic to the forests of Southeast Asia, Rafflesia (Rafflesiaceae) is a genus PR:PROJECT_SUMMARY of holoparasitic plants producing the largest flowers in the world, yet PR:PROJECT_SUMMARY completely dependent on its host, the tropical grape vine, Tetrastigma. PR:PROJECT_SUMMARY Rafflesia species are threatened with extinction, making them an iconic symbol PR:PROJECT_SUMMARY of plant conservation. Thus far, propagation has proved challenging, greatly PR:PROJECT_SUMMARY decreasing efficacy of conservation efforts. This study compared the metabolites PR:PROJECT_SUMMARY in the shoots of Rafflesia-infected and non-infected Tetrastigma loheri to PR:PROJECT_SUMMARY examine how Rafflesia infection affects host metabolomics and elucidate the PR:PROJECT_SUMMARY Rafflesia infection process. Results from LC-MS-based untargeted metabolomics PR:PROJECT_SUMMARY analysis showed benzylisoquinoline alkaloids were significantly elevated in PR:PROJECT_SUMMARY non-infected shoots and are here reported for the first time in the genus PR:PROJECT_SUMMARY Tetrastigma, and in the grape family, Vitaceae. These metabolites have been PR:PROJECT_SUMMARY implicated in plant defense mechanisms and may prevent a Rafflesia infection. In PR:PROJECT_SUMMARY Rafflesia-infected shoots, oxygenated fatty acids, or oxylipins, and a PR:PROJECT_SUMMARY flavonoid, previously shown involved in plant immune response, were abundant. PR:PROJECT_SUMMARY This study provides a preliminary assessment of metabolites that differ between PR:PROJECT_SUMMARY Rafflesia-infected and non-infected Tetrastigma hosts and may have applications PR:PROJECT_SUMMARY in Rafflesia propagation to meet conservation goals. PR:INSTITUTE Long Island University PR:LAST_NAME Molina PR:FIRST_NAME Jeanmaire PR:ADDRESS 1 University Plaza PR:EMAIL jeanmaire.molina@liu.edu PR:PHONE 7182466410 PR:DOI http://dx.doi.org/10.21228/M8M40V #STUDY ST:STUDY_TITLE Metabolic profiling of Rafflesia-infected Tetrastigma and applications for ST:STUDY_TITLE propagation ST:STUDY_SUMMARY Endemic to the forests of Southeast Asia, Rafflesia (Rafflesiaceae) is a genus ST:STUDY_SUMMARY of holoparasitic plants producing the largest flowers in the world, yet ST:STUDY_SUMMARY completely dependent on its host, the tropical grape vine, Tetrastigma. ST:STUDY_SUMMARY Rafflesia species are threatened with extinction, making them an iconic symbol ST:STUDY_SUMMARY of plant conservation. Thus far, propagation has proved challenging, greatly ST:STUDY_SUMMARY decreasing efficacy of conservation efforts. This study compared the metabolites ST:STUDY_SUMMARY in the shoots of Rafflesia-infected and non-infected Tetrastigma loheri to ST:STUDY_SUMMARY examine how Rafflesia infection affects host metabolomics and elucidate the ST:STUDY_SUMMARY Rafflesia infection process. Results from LC-MS-based untargeted metabolomics ST:STUDY_SUMMARY analysis showed benzylisoquinoline alkaloids were significantly elevated in ST:STUDY_SUMMARY non-infected shoots and are here reported for the first time in the genus ST:STUDY_SUMMARY Tetrastigma, and in the grape family, Vitaceae. These metabolites have been ST:STUDY_SUMMARY implicated in plant defense mechanisms and may prevent a Rafflesia infection. In ST:STUDY_SUMMARY Rafflesia-infected shoots, oxygenated fatty acids, or oxylipins, and a ST:STUDY_SUMMARY flavonoid, previously shown involved in plant immune response, were abundant. ST:STUDY_SUMMARY This study provides a preliminary assessment of metabolites that differ between ST:STUDY_SUMMARY Rafflesia-infected and non-infected Tetrastigma hosts and may have applications ST:STUDY_SUMMARY in Rafflesia propagation to meet conservation goals. ST:INSTITUTE Long Island University ST:LAST_NAME Molina ST:FIRST_NAME Jeanmaire ST:ADDRESS 1 University Plaza ST:EMAIL jeanmaire.molina@liu.edu ST:PHONE - ST:SUBMIT_DATE 2021-06-26 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Tetrastigma loheri SU:TAXONOMY_ID 1006131 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Sample_14A_Jean_LIU_GB5_01_3547 factor:Infected RAW_FILE_NAME=Sample_14A_Jean_LIU_GB5_01_3547 SUBJECT_SAMPLE_FACTORS - Sample_14A_Jean_LIU_GB5_01_3548 factor:Infected RAW_FILE_NAME=Sample_14A_Jean_LIU_GB5_01_3548 SUBJECT_SAMPLE_FACTORS - Sample_16A_Jean_LIU_GB4_01_3543 factor:Infected RAW_FILE_NAME=Sample_16A_Jean_LIU_GB4_01_3543 SUBJECT_SAMPLE_FACTORS - Sample_16A_Jean_LIU_GB4_01_3545 factor:Infected RAW_FILE_NAME=Sample_16A_Jean_LIU_GB4_01_3545 SUBJECT_SAMPLE_FACTORS - Sample_16A_Jeanmaire_LIU_GA7_01_2912 factor:Infected RAW_FILE_NAME=Sample_16A_Jeanmaire_LIU_GA7_01_2912 SUBJECT_SAMPLE_FACTORS - Sample_12A_Jean_LIU_GB6_01_3536 factor:Non-infected RAW_FILE_NAME=Sample_12A_Jean_LIU_GB6_01_3536 SUBJECT_SAMPLE_FACTORS - Sample_12A_Jeanmaire_LIU_GA6_01_2911 factor:Non-infected RAW_FILE_NAME=Sample_12A_Jeanmaire_LIU_GA6_01_2911 SUBJECT_SAMPLE_FACTORS - Sample_18A_Jean_LIU_GB3_01_3534 factor:Non-infected RAW_FILE_NAME=Sample_18A_Jean_LIU_GB3_01_3534 SUBJECT_SAMPLE_FACTORS - Sample_18A_Jean_LIU_GB3_01_3540 factor:Non-infected RAW_FILE_NAME=Sample_18A_Jean_LIU_GB3_01_3540 #COLLECTION CO:COLLECTION_SUMMARY Cuttings of Rafflesia lagascae-infected Tetrastigma loheri Gagnep. and CO:COLLECTION_SUMMARY non-infected shoots were collected from San Lorenzo Ruiz Municipality, Mt. CO:COLLECTION_SUMMARY Guinatungan, Camarines Norte, Philippines. The non-infected cuttings were taken CO:COLLECTION_SUMMARY from sufficiently mature woody host vines that did not have any visible sign of CO:COLLECTION_SUMMARY Rafflesia infection (i.e. Rafflesia floral buds/scars absent), but mature enough CO:COLLECTION_SUMMARY that they could presumably support an infection since Rafflesia has never been CO:COLLECTION_SUMMARY observed to infect juvenile vines. Sections within ca. 5 cm of a Rafflesia bud, CO:COLLECTION_SUMMARY as well as comparable sections from non-infected cuttings, were subjected to CO:COLLECTION_SUMMARY LC-MS experiments. CO:SAMPLE_TYPE Plant #TREATMENT TR:TREATMENT_SUMMARY Sections within ca. 5 cm of a Rafflesia bud (i.e. infected), as well as TR:TREATMENT_SUMMARY comparable sections from non-infected cuttings, were subjected to LC-MS TR:TREATMENT_SUMMARY experiments. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were first extracted in methanol (25 mg ground in 700 µL methanol). The SP:SAMPLEPREP_SUMMARY extracts were evaporated to dryness under a gentle stream of nitrogen. Samples SP:SAMPLEPREP_SUMMARY were prepared for injection by reconstituting in 0.3 mL of 1:1 MeOH/water. #CHROMATOGRAPHY CH:INSTRUMENT_NAME Thermo Scientific Ultimate-3000 UHPLC system CH:COLUMN_NAME Agilent Acclaim 120 C18-column (2.1 mm x 100 mm, 5 µm) CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker Daltonics maXis-II UHR-ESI-QqTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:MS_COMMENTS Raw data were analyzed by using the online version of XCMS metabolomics software MS:MS_COMMENTS (version 1.10.9; Tautenhahn et al. 2012). To analyze the data in XCMS, we MS:MS_COMMENTS applied a pairwise comparison between infected and non-infected samples with MS:MS_COMMENTS default parameters for Bruker Q-TOF. After XCMS analysis, the difference reports MS:MS_COMMENTS were filtered. The features from XCMS with p-value < 0.05, intensities above MS:MS_COMMENTS 50000, and fold difference of at least 5, were analyzed further in Bruker MS:MS_COMMENTS Compass Data Analysis v4.3 and Metfrag Web (Ruttkies et al. 2016; MS:MS_COMMENTS https://msbi.ipb-halle.de/MetFragBeta/) to identify metabolites of interest. The MS:MS_COMMENTS neutral molecular formula of the precursor ions (desired features) and their MS:MS_COMMENTS MS/MS fragmentation spectra were then obtained in Bruker Compass Data Analysis MS:MS_COMMENTS and given as input in the MS/MS peak list in Metfrag. All other settings were MS:MS_COMMENTS kept at default values. Candidate metabolites were then retrieved with the MS:MS_COMMENTS highest scoring candidates subjected to additional analysis in CFM-ID (Allen et MS:MS_COMMENTS al. 2014; http://cfmid.wishartlab.com/) to confirm Metfrag candidates. Metfrag MS:MS_COMMENTS and CFM-ID are silico fragmentation tools that utilize known compounds from MS:MS_COMMENTS structure databases to calculate fragments that are matched to experimentally MS:MS_COMMENTS obtained spectra (Blaženović et al. 2018). In addition to these automated MS:MS_COMMENTS approaches, we have also performed a manual dereplication approach to verify the MS:MS_COMMENTS metabolites of interest, as described in previous publications (Gödecke et al. MS:MS_COMMENTS 2009; Nikolić et al. 2012; Nikolić et al. 2015; Nikolic et al. 2017). MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST001854_AN003005_Results.txt UNITS:ion intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END