Metabolomics Reveals How Cucumber (Cucumis. sativus) Reprograms Metabolites to Cope with. Silver Nanoparticle-Induced Oxidative Stress
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1 1 Supporting Informtion for Metbolomics Revels How Cucumber (Cucumis stivus) Reprogrms Metbolites to Cope with Silver Nnoprticle-Induced xidtive Stress Huiling Zhng, Wencho Du, Jose R. Perlt-Vide ζ, Jorge L. Grde-Torresdey ζ, Json C. White, Arturo Keller, Hongyn Guo, Rong Ji *, Lijun Zho * Stte Key Lbortory of Pollution Control nd Resource Reuse, School of Environment, Nnjing University, Nnjing , Chin ζ Chemistry Deprtment, The University of Texs t El Pso, 500 West University Avenue, El Pso, Texs 79968, United Sttes Deprtment of Anlyticl Chemistry, The Connecticut Agriculturl Experiment Sttion (CAES), New Hven, Connecticut 06504, United Sttes Bren School of Environmentl Science & Mngement, University of Cliforni, Snt Brbr, Cliforni , United Sttes *Corresponding uthor. Tel: ; fx: Emil ddress: ji@nju.edu.cn; ljzho@nju.edu.cn S1
2 21 22 Pge S3 nd S4. Discussion regrding other significntly chnged secondry metbolites in cucumber leves exposed to different doses of Ag ion or AgNPs. 23 Pge S5. Figure S1 24 Pge S6. Figure S2 25 Pge S7. Figure S3 26 Pge S8. Figure S4 27 Pge S9. Figure S5 28 Pge S10. Figure S6 29 Pge S11. Figure S7 30 Pge S12. Figure S8 31 Pge S13. Figure S9 32 Pge S14. Figure S10 33 Pge S15. Figure S S2
3 ther Secondry Metbolites induced by AgNPs. Primry metbolites such s sugrs, mino cids, nd TCA cycle intermedites re known to hve significnt role in plnt response to biotic stresses. Chnges in the levels of these biomolecules re considered to be n indiction of photosynthetic dysfunction nd/or osmotic redjustment. 41 Conversely, secondry metbolites re known to be ltered in response to prticulr stress conditions, such s pthogens, ntioxidnts, RS scvengers, coenzymes, nd regultory molecules. 42 In ddition to phenolic compounds, there re number of secondry metbolites tht re significntly chnged by Ag tretment, indicting either dmge results or the development of tolernce/resistnce. A significnt (p 0.05) increse in the ccumultion of phytosphingosine ws identified upon 0.4 mg Ag + nd 4 nd 40 mg AgNPs tretment (Figure S9). Peer et l. suggested tht phytosphingosine (t18:0) is positive regultor of the hypersensitive rection during the plnts pthogen defense response. 43 Nicotinmide (NIC) ws not detected in the unexposed control plnts; however, significnt increse ws observed under Ag + or AgNPs tretment (Figure S9). NIC is stress-ssocited signling compound tht cn induce defensive metbolism t the genetic level nd cn regulte secondry metbolic ccumultion. 44 Itconic cid, which is known to hve ntimicrobil properties, ws observed to significntly (p<0.01) increse up to 2-fold upon exposure to 4 nd 40 mg AgNPs. Michemucci et l. demonstrted tht reduction of intrcellulr itconic cid levels resulted in significntly reduced ntimicrobil ctivity during bcteril infections furoic cid (2FA) ws observed to significntly (p<0.05) increse in dose dependent mnner with both Ag + nd Ag NPs. This compound hs been reported to induce resistnce ginst the tomto wilt disese cused by Fusrium oxysporumf. sp. lycopersici Deoxyerythritol ws S3
4 significntly (p<0.05) decresed in dose-dependent wy with Ag ions nd Ag NPs (Figure S9); the physiologicl role of this compound in Ag exposure is unknown. In ddition, we found tht 5,6-dimethylbenzimidzole (DMB), precursor of Vitmin B12, ws not detected in the unexposed control plnts nd the 0.04 mg Ag + tretment, but the biomolecule ws significntly incresed by the other Ag tretments. 3-cynolnine ws found to significntly (p<0.05) increse in dose dependent mnner; 40 mg AgNPs induced 6-fold increse of 3-cynolnine in exposed leves. The bet-cynolnine pthwy is involved in plnt response to vrious biotic or biotic stress; 46 it is possible tht 3-cynolnine is involved in tolernce or dpttion mechnisms ssocited with Ag induced stress S4
5 80 Silver ion concentrtion in plnt tissues (mg kg-1 dry weight) A-Root B-Stem C-Lef b Control 0.1 ppm Ag ions 1 ppm Ag ions 10 ppm AgNPs 100 ppm AgNPs b c c b d d c Figure S1. Ag content in cucumber roots (A), stems (B), nd leves (C) fter 7 d folir ppliction of 0, 0.04 nd 0.4 mg Ag+; 4 nd 40 mg AgNPs. Dt re the men ± SD of five replictes. The dt re nlyzed using one-wy ANVA, followed by Tukey- Honestly Significnt Difference multiple comprisons test. The letters represent sttisticl differences t p 0.05 compred with other groups. 86 S5
6 Figure S2. Differentil toxicity on cucumber leves fter 7 d folir ppliction of 0, 0.04, 0.4 mg Ag+; 4 nd 40 mg AgNPs, which corresponded to the control, 0.1 ppm nd 1 ppm Ag ions, 10 ppm nd 100 ppm AgNPs for individul tretments S6
7 Biomss (g per plnt) Biomss (g per plnt) Control 0.1 ppm Ag ions 1 ppm Ag ions 10 ppm AgNPs 100 ppm AgNPs Control 0.1 ppm Ag ions 1 ppm Ag ions 10 ppm AgNPs 100 ppm AgNPs Figure S3. Biomss of root, stem nd lef (A) nd totl biomss (B) of cucumber fter 7 d folir exposure to 0, 0.04, 0.4 mg Ag+; 4 nd 40 mg AgNPs. Dt re the men of five replictes. Error brs stnd for stndrd devition. The dt re nlyzed using one-wy ANVA, followed by Tukey-Honestly Significnt Difference multiple comprisons test. The letters represent sttisticl differences t p 0.05 compred with other groups. b Lef Stem Root S7
8 rbutin slicin drenline cis-2-hydroxycinnmic cid Methyl Phosphte Citrconic cid ferulic cid Ethyl cinnmte Benzyl thiocynte methyl octnote 3-hydroxy-L-proline cnvnine degr prod Lctmide N-Methyl-DL-lnine N-Methyl-L-glutmic cid Crnitine 3-hydroxybutyric cid sprgine trns-4-hydroxy-l-proline Mono2-ethylhexylphthlte p-benzoquinone cetnilide 2-Ketocproic cid nicotinmide 2,2-Dimethylsuccinic Acid Nonnoic cid methyl ester lph-ecdysone pentdecnoic cid succinte semildehyde dibenzofurn denosine 2-Indnone Leucrose Gluconic lctone slicylic cid 2-Furoic Acid rchidonic cid Cysteinylglycine 2-minoethnethiol Figure S4. VIP scores from PLS-DA nlysis of cucumber leves showing the discriminting metbolites between unexposed control, 0.04 nd 0.4 mg Ag + ; 4 nd 40 mg AgNPs groups S8
9 rbutin Ethyl cinnmte slicin tetrcosne Methyl Phosphte nphthlene cffeic cid Cysteinylglycine cis-2-hydroxycinnmic cid 2-Indnone 5-hydroxytryptophn drenline 3-hydroxybenzyl lcohol 4-Hydroxymndelonitrile pyrogllol Isomltose ferulic cid 2-Furoic Acid rchidonic cid bet-hydroxypyruvte Acetol trns-4-hydroxy-l-proline butyrldehyde succinte semildehyde 2-mercptoethnesulfonic cid pentdecnoic cid cycloleucine nicotinmide 2-Deoxyerythritol Allntoic cid 2,2-Dimethylsuccinic Acid 2-Ketocproic cid epsilon-cprolctm pltinitol lph-ecdysone 3-3-hydroxyphenylpropionic cid 2-Deoxyuridine kyotorphin scchrin Meltonin indole-3-cetmide 2-hydroxybutnoic cid ornithine octnl phenylcetldehyde Figure S5. VIP scores from PLS-DA nlysis of cucumber leves showing how the discriminting metbolites induce metbolite seprtion between unexposed control, 0.04 nd 0.4 mg Ag S9
10 Citrconic cid 3-hydroxy-L-proline slicin N-Methyl-DL-lnine Crbzole methyl octnote rffinose Benzyl thiocynte ferulic cid rbutin drenline sprgine cycloleucine lctulose cis-2-hydroxycinnmic cid Crnitine cnvnine degr prod N-Methyl-L-glutmic cid Mono2-ethylhexylphthlte Methyl Phosphte Lctmide p-benzoquinone 3-hydroxybutyric cid Ethyl cinnmte cetnilide trns-4-hydroxy-l-proline 2-Ketocproic cid 2-Deoxyerythritol 3-3-hydroxyphenylpropionic cid pyrogllol dibenzofurn 2,2-Dimethylsuccinic Acid pentdecnoic cid Leucrose 2-Indnone denosine tetrcosne D-erythronolctone 4-Hydroxymndelonitrile phenylcetldehyde Figure S6. VIP scores from PLS-DA nlysis of cucumber leves showing how the discriminting metbolites induce metbolite seprtion between unexposed control, 4 nd 40 AgNPs groups S10
11 Figure S7. Up- nd Down- regulted metbolites of Ag ions. AgNPs nd the overlp between the two. Red nd green represent up- nd down-regultion of metbolites, respectively. The results re the combintion of one-wy ANVA ( p 0.05 ) nd prtil lest-squres discriminnt nlysis (VIP > 1). S11
12 Figure S8. Pthwy nlysis using MetboAnlyst 2.0 for exposed cucumber leves. 211 S12
13 Figure S9. Box plot of reltive bundnce of dditionl secondry metbolites in 4- week-old cucumber leves exposed to different doses of AgNPs (4 nd 40 mg per plnt) nd Ag + (0.04 nd 0.4 mg per plnt) S13
14 H N H H H H H H H H H H CH NH 2 H HS H H SH H H HN CH 3 H Figure S10. Box plot of reltive bundnce of metbolites only induced by AgNPs S14
15 Figure S11. Box plot of reltive bundnce of significntly decresed metbolites only induced by AgNPs S15
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