The impact of conventional and organic agricultural approaches on blackcurrant polyphenol content and diversity. Sean Conner

Transcription

1 The impact of conventional and organic agricultural approaches on blackcurrant polyphenol content and diversity Sean Conner

2 Extraction Protocol 3 ml 60:40 water/acetonitrile 1% acetic acid 0.1 mg/ml morin (internal standard) 100 mg freeze dried (FD) powder Shaken for 60 minutes at 30 C Cyanidin glucoside Morin: Internal Standard y = 2E+09x R² =

3 Sample analysis LC-MS Targeted Anthocyanins (positive mode) & Flavanols (negative mode) Delphinidin glucoside Delphinidin rutinoside Cyanidin glucoside Cyanidin rutinoside Petunidin rutinoside Petunidin glucoside Pelargonidin glucoside Peonidin galactoside Pelargonidin rutinoside Peonidin rutinoside Cyanidin arabinoside Melvidin rutinoside Delphinidin coumaroylglucoside Myricetin-3-rutinoside Myricetin-6-glucoside Myricetin-3-(6"malonyl)glucoside Aureusidin glucoside Quercetin-3-rutinoside Quercetin-3-(6"-malonyl)glucoside Kaempferol-3-rutinoside Isorhamnetin-3-rutinoside Kampferol-3-glucoside Kaempferol-3-galactoside Myricetin-glucuronide Untargeted Totally unbiased approach

4 Experiment 1 - Organic vs Conventional Experimental Design Blackcurrant plants Two treatments sprayed and unsprayed Fruit harvest dates representative of ripe six harvesting events

5 t[2] PCA (1 vs 2) Targeted analysis Coloured by harvest date Organic vs Conventional 6 Positive & negative MS data combined Blackcurrant organic TARG results.m2 (PCA-X), Diluted samples removed t[comp. 1]/t[Comp. 2] Colored according to Obs ID (Company) 4 CV136PL56 CV140PL46 CV136PL CV177PL78 CV12PL45 CV176PL1 CV122PL68 CV12PL80 CV12PL16 CV177PL12 CV176PL94 CV122PL5 CV177PL72 CV177PL38 CV176PL67 CV122PL48 CV21PL6 CV21PL89 CV143PL15 CV143PL57 CV141PL50 CV143PL60 CV141PL69 CV1PL34 CV141PL90 CV56PL4 CV56PL71 CV57PL47 CV56PL35 CV56PL5 CV142PL49 CV142PL82 CV142PL3 CV142PL t[1] General Previously trend by harvest demonstrated date by harvest time experiment R2X[1] = R2X[2] = Ellipse: Hotelling T2 (0.95) SIMCA-P :06:56 (UTC+0)

6 p[2] Loadings scatter plot score 1v2 Organic vs Conventional Blackcurrant organic TARG results.m2 (PCA-X), Diluted samples removed p[comp. 1]/p[Comp. 2] Colored according to Var ID (mode2) n p 0.30 aureusidin_gluc isorham-3-rut kaemp-3-gala peonidin rut melv rut kamp-3-gluc kaemp3-rut querc-3-gluc querc-3-(6"-malonyl) Compounds driving trend peonidin gala petun rut petun gluc myric-gluc pelarg gluc delph coum myric-3-(6"mal)gluc delph rut cyan myric-3-rut gluc querc-3-rut myric-6-gluc cyan arab cyan rut delph g p[1] pelarg rut R2X[1] = R2X[2] = SIMCA-P :07:45 (UTC+0)

7 Targeted analysis Organic vs Conventional mg/100mg DFM

8 t[2] PCA (1 vs 2) Targeted analysis Coloured by cultivar Organic vs Conventional 6 Blackcurrant organic TARG results.m2 (PCA-X), Diluted samples removed t[comp. 1]/t[Comp. 2] Colored according to Obs ID (Study) Clustering by cultivar not treatment t[1] R2X[1] = R2X[2] = Ellipse: Hotelling T2 (0.95) SIMCA-P :36:09 (UTC+0)

9 Analysis of variance - Organic vs Conventional Of the 25 compounds analyses 16 are significantly different between cultivars compound Pval average SED Variate: aureusidin_gluc < Variate: isorham_3_rut < Variate: kaemp_3_gala < Variate: kamp_3_gluc < Variate: myric_3_6_mal_gluc < Variate: myric_6_gluc < Variate: myric_gluc < Variate: pelarg_gluc < Variate: pelarg_rut < Variate: melv_rut Variate: kaemp3_rut Variate: myric_3_rut Variate: delph_rut Variate: querc_3_6_malonyl_gluc Variate: petun_rut Variate: cyan_gluc Variate: cyan_arab Variate: delph_gluc Variate: cyan_rut Variate: querc_3_gluc Variate: querc_3_rut Variate: peonidin_rut Variate: petun_gluc Variate: delph_coum Variate: peonidin_gala

10 t[3] Untargeted analysis PCA 2 vs 3 Organic vs Conventional SIEVE organic.m5 (OPLS/O2PLS-DA), OPLS all CVs t[comp. 2]/t[Comp. 3] Colored according to Obs ID (Cultivar) CV140PL81 CV140PL46 CV140PL61 As with targeted analysis we are seeing clustering Untargeted analysis PCA 2 vs 3 due to cultivar CV57PL47 CV1PL93 CV1PL59 CV1PL14 CV141PL13 CV1PL34 CV57PL83 CV57PL79 CV141PL50 CV21PL6 CV21PL70 CV21PL89CV141PL90 CV141PL69 CV142PL49 CV142PL82 CV142PL91 CV56PL71 CV122PL48 CV12PL45 CV142PL3 CV143PL36 CV56PL58 CV12PL80 CV12PL16 CV12PL39 CV143PL60 CV136PL56 CV122PL68 CV56PL4 CV136PL92 CV136PL17 CV122PL5 CV143PL57 CV143PL15 CV56PL35 CV176PL37 CV176PL94 CV176PL67 CV176PL1 CV177PL12 CV177PL38CV177P CV177PL7 11% variation on score 2 7% Variation on score t[2] R2X[2] = R2X[3] = Ellipse: Hotelling T2 (0.95) SIMCA-P :31:32 (UTC+0)

11 Intensity pq[3] Intensity Loadings scatter plot 2V3 Organic vs Conventional SIEVE organic.m5 (OPLS/O2PLS-DA), OPLS all CVs RT: SM: 5B pq[comp. 2]/pq[Comp. 3] Colored according to Var ID (Primary) pq[2] R2X[2] = R2X[3] SIMCA-P+ = :33:29 (UTC+0) Time (min) N P Cultivar number 57 m/z Intensity NL: 8.85E4 m/z= F: FTMS + p ESI Full ms [ ] MS CV57PL47_Climafruit_BCti metrialaapos_041 Compounds yet to be identified but are not of NL: 1.22E5 polyphenol m/z= F: FTMS + p ESI Full ms [ ] MS CV176PL67_Climafruit_BCt imetrialaapos_042 origin Cultivar number 176 m/z Intensity Var ID (MZ) Var ID (Time)

12 t[4] Untargeted analysis PCA 3 vs 4 Organic vs Conventional SIEVE organic.m5 (OPLS/O2PLS-DA), OPLS all CVs t[comp. 3]/t[Comp. 4] Colored according to Obs ID (Cultivar) CV56PL58 CV56PL35 CV56PL4 CV143PL57 CV56PL71 CV143PL60 CV143PL15 CV141PL50 CV21PL89 CV21PL6 CV141PL90 CV143PL36 CV21PL70 CV177PL78 CV177PL72 CV176PL1 CV141PL69 CV57PL79 CV57PL83 CV141PL13 CV176PL67 CV122PL5 CV176PL94 CV176PL37 CV136PL17 CV122PL48 CV122PL68 CV177PL38 CV177PL12 CV57PL47 CV1PL34 CV1PL93 CV1PL14 CV140PL46 CV140PL61 CV140PL As with PC 2 vs 3 clustering by cultivar is evident (7.5% variation on score 3) -5 CV136PL92 CV136PL56 CV12PL39 CV12PL80 CV12PL16 CV12PL45 CV1PL59-10 CV142PL91 CV142PL3 CV142PL82 CV142PL t[3] R2X[3] = R2X[4] = Ellipse: Hotelling T2 (0.95) SIMCA-P :32:09 (UTC+0)

13 pq[4] Loadings scatter plot score 3 vs 4 Organic vs Conventional 0.10 SIEVE organic.m5 (OPLS/O2PLS-DA), OPLS all CVs pq[comp. 3]/pq[Comp. 4] Colored according to Var ID (Primary) N P Var ID (MZ) Var ID (Time) m/z 304 Delphinidin Others not known phenolic In blackcurrant pq[3] R2X[3] = R2X[4] SIMCA-P+ = :36:11 (UTC+0) Var ID (MZ) Var ID (Time)

14 Experiment 1 Comments & Conclusions Harvest date has a major effect on polyphenol content Samples segregating by cultivar and NOT treatment Metabolites not from known polyphenol origin causing segregation: are these important?

15 Experiment 2 Organic vs Conventional Blackcurrant plants 2 field sites 2 Planting dates

16 t[2] Varietal trial Targeted Data PCA plot 1 vs 2; coloured by cultivar 6 Quant - SV collated - results from responce.m2 (PCA-X), Samples t[comp. 1]/t[Comp. 2] Colored according to Obs ID (Line) CV122PL129 CV159PL61 CV159PL47 CV57PL2 CV159PL34 CV149PL32 CV176PL4 CV176PL6 CV148PL38 CV155PL83 CV177PL10 CV176PL9 CV12PL11 CV146PL33 CV177PL19 Clustering by cultivar CV21PL97 CV148PL46 CV160PL71 CV160PL88 CV122PL111 CV57PL3 CV156PL104 CV155PL102 CV136PL126 CV147PL45 CV146PL48 CV91PL14 CV12PL20 CV91PL5 CV91PL8 CV136PL110 CV136PL114 CV170PL113 CV146PL62 CV168PL121 CV149PL66 CV57PL15 CV144PL57 CV147PL41 CV177PL22 CV143PL44 CV91PL25 CV1PL18 CV158PL81 CV156PL86 CV158PL89 CV161PL98 CV144PL40 CV170PL125 CV154PL91 CV21PL79 CV140PL68 CV145PL52 CV149PL49 CV145PL35 CV156PL90 CV169PL117 CV158PL103 CV155PL75 CV21PL101 CV142PL64 CV1PL2 CV158PL77 CV132PL112 CV141PL53 CV158PL72 CV142PL37 CV141PL36 CV154PL96 CV142PL50 CV12PL28 CV140PL39 CV147PL59 CV169PL122 CV148PL69 CV154PL78 CV169PL109 CV168PL108 CV56PL51 CV140PL55 CV157PL93 CV161PL76 CV161PL92 CV56PL58 CV145PL65 CV142PL70 CV168PL115 CV160PL95 CV143PL67 CV181PL26 CV154PL74 CV21PL84 CV132PL128 CV56PL43 CV181PL16 CV144PL54 CV157PL82 CV181PL13 CV181PL7 CV161PL105 CV170PL118 CV157PL99 CV132PL116 CV157PL t[1] CV160PL100 R2X[1] = R2X[2] = Ellipse: Hotelling T2 (0.95) SIMCA-P :52:01 (UTC+0)

17 t[2] Varietal trial Targeted Data PCA plot 1 vs 2; coloured by location Quant - SV collated - results from responce.m2 (PCA-X), Samples t[comp. 1]/t[Comp. 2] Colored according to Obs ID (Field/Year) M M CV122PL129 CV159PL61 CV159PL47 CV159PL34 CV149PL32 CV176PL4 CV176PL6 CV148PL38 CV155PL83 CV177PL10 CV176PL9 CV12PL11 CV146PL33 CV177PL19 CV21PL97 CV148PL46 CV160PL71 CV160PL88 CV155PL102 CV136PL126 CV122PL111 CV147PL45 CV57PL3 CV156PL104 CV146PL48 CV136PL110 CV91PL14 CV136PL114 CV170PL113 CV12PL20 CV91PL5 CV146PL62 CV168PL121 CV149PL66 CV91PL8 CV57PL15 CV144PL57 CV147PL41 CV177PL22 CV143PL44 CV91PL25 CV1PL18 CV158PL81 CV156PL86 CV158PL89 CV161PL98 CV144PL40 CV170PL125 CV154PL91 CV21PL79 CV140PL68 CV145PL52 CV149PL49 CV145PL35 CV156PL90 CV169PL117 CV158PL103 CV155PL75 CV21PL101 CV142PL64 CV1PL2 CV158PL77 CV132PL112 CV141PL53 CV158PL72 CV142PL37 CV141PL36 CV154PL96 CV142PL50 CV12PL28 CV140PL39 CV147PL59 CV169PL122 CV148PL69 CV154PL78 CV169PL109 CV168PL108 CV56PL51 CV140PL55 CV157PL93 CV161PL76 CV161PL92 CV56PL58 CV145PL65 CV142PL70 CV168PL115 CV160PL95 CV143PL67 CV181PL26 CV154PL74 CV21PL84 CV132PL128 CV56PL43 CV181PL16 CV144PL54 CV157PL82 CV181PL13 CV181PL7 CV161PL105 CV170PL118 CV157PL99 CV132PL116 Take a closer look at cultivar 157 CV157PL t[1] CV160PL100 CV57PL2 Clustering by cultivar Site independent R2X[1] = R2X[2] = Ellipse: Hotelling T2 (0.95) SIMCA-P :56:54 (UTC+0)

18 p[2] Varietal trial Loadings scatter plot score1 vs 2 Coloured pos/neg Quant - SV collated - results from responce.m2 (PCA-X), Samples p[comp. 1]/p[Comp. 2] Colored according to Var ID (Mode) n p 0.15 Cyanidin_c 0.10 kampferolkaempferol quercetin- quercetin querce myricetin- myricetinmyricetin- aureusidin myricitinisorhamnet kaempferol Melvidin_r Peonidin_g Peonidin_r Petunidin_ Petunidin_ Delphinidi Cyanidin_a Delphinidi Cyanidin_g Delphinidi Pelargonid cyanidin_r Pelargonid p[1] R2X[1] = R2X[2] SIMCA-P+ = :54:09 (UTC+0)

19 t[4] Varietal Trial - Untargeted Analysis PCA 3 vs 4 Coloured by cultivar SIEVE results.m2 (PCA-X), refs and bad removed t[comp. 3]/t[Comp. 4] Colored according to Obs ID (pos cv) CV56 CV56 CV176 CV176 CV177 CV177 CV181 CV169 CV146 CV145 CV176 CV143 CV136 CV142 CV154 CV160CV157 CV154 CV136 CV21 CV168 CV158 CV147 CV21 CV57CV12 CV56 CV12CV91 CV122 CV156 CV144 CV168 CV169 CV142CV155 CV159 CV170 CV159 CV161 CV144 CV157 CV1 CV91 CV158 CV160 CV161 CV181 CV169 CV147 CV122 CV21 CV143 CV149 CV140 CV145 CV132 CV156 CV176 CV149 CV141 CV91CV91 CV168 CV181 CV154 CV136CV141 CV157 CV1 CV157 CV145 CV160 CV21 CV170 CV146 CV161 CV132 CV181CV143 CV12 CV147CV158 CV161 CV149 CV132 CV140 CV142 CV57 CV146 CV160 CV57CV155 CV154CV159 CV142 CV170 CV155 CV158 CV140 CV158 CV12 CV148 CV148 CV177 CV144 Indication of clustering by cultivar e.g. 176 & 177 CV122 CV t[3] R2X[3] = R2X[4] = Ellipse: Hotelling T2 (0.95) SIMCA-P :06:11 (UTC+0)

20 p[4] Varietal trial Loadings scatter plot Var ID (MZ) score 3v4 Coloured to pos neg SIEVE results.m2 (PCA-X), refs and bad removed p[comp. 3]/p[Comp. 4] Colored according to Var ID (Primary) N P Var ID (RT) Metabolites not polyphenols p[3] R2X[3] = R2X[4] = SIMCA-P :15:16 (UTC+0)

21 Organic and Traditional confirmation cross comparison cultivar 176 Traditional Organic MZ RT MZ RT

22 data_set_yaxis_ptr Delphinidin_glucoside_TRAD Delphinidin_rutinoside_TRAD Delphinidin_coumaroylglucoside_T cyanidin_rutinoside_trad Cyanidin_glucoside_TRAD Cyanidin_arabinoside_TRAD Melvidin_rutinoside_TRAD myricetin_3_6_malonyl_glugoside_ pelarg_gluc_org myricetin_3_rutinoside_trad myricetin_6_glucoside_trad myricitin_glucuronide_trad Petunidin_rutinoside_TRAD Petunidin_glucoside_TRAD Pelargonidin_glucoside_TRAD Pelargonidin_rutinoside_TRAD Peonidin_galactoside_TRAD Peonidin_rutinoside_TRAD aureusidin_glucoside_trad isorhamnetin_3_rutinoside_trad kaempferol_3_galactoside_trad kaempferol_3_rutinoside_trad kampferol_3_glucoside_trad quercetin_3_6_malonyl_glucoside_ quercetin_3_glucoside_trad quercetin_3_rutinoside_trad delph_gluc_org delph_rut_org delph_coum_org cyan_rut_org cyan_gluc_org cyan_arab_org melv_rut_org myric_3_6_mal_gluc_org myric_3_rut_org myric_6_gluc_org myric_gluc_org petun_rut_org petun_gluc_org pelarg_gluc_org pelarg_rut_org peonidin_gala_org peonidin_rut_org aureusidin_gluc_org isorham_3_rut_org kaemp_3_gala_org kaemp3_rut_org kamp_3_gluc_org querc_3_6_malonyl_gluc_org querc_3_gluc_org Cyanidin_arabinoside_TRAD querc_3_rut_org Pelargonidin_glucoside_TRAD 0.040

23 Experiment 2 Comments & Conclusions Samples clustered by cultivar and not origin Good correlation between cultivars grown for the organic and traditional experiments