Developmental variation in floral volatiles composition of a fragrant orchid (Kunth) Garay.

Paramita Beraa, Syamali Chakrabartib, Nitin K Gaikwada, Nithya N Kuttya, Monica Barmana, Adinpunya Mitraa*

aNatural Product Biotechnology Group, Agricultural and Food Engineering Department, Indian Institute of Technology Kharagpur, Kharagpur - 721 302, India b ICAR-National Research Centre for Orchids, Indian Council of Agricultural Research, Pakyong - 723 106, Sikkim, India

*Corresponding author. Fax: +91 3222 282244/255303 Email addresses: [email protected] / [email protected]

Experimental Study species and material

Z. maculatum were grown in the normal light/dark cycle in the green house of Genetics Section, ICAR-National Research Centre for Orchids, Pakyong, Sikkim. Volatiles were collected from the during the month of November in two consecutive years (2015 and 2016). During the experimental processes, the plants were watered once in a day. The plant was identified by one of the authors (S Chakrabarti), who has long-standing experience in orchid research. Herbariums prepared with the voucher specimens (NPBTH-2/15, for Z. maculatum) are safely stored in the Agricultural Biotechnology Laboratory at Kharagpur.

Headspace floral scent collection Floral scent was collected using dynamic headspace extraction as described by Bera et al. (2015). Adsorbent matrices (30 mg each) were packed between two layers of non-adsorbent cotton in a purposely-built glass column. A single full attached was inserted into the transparent polypropylene bag and the scent was allowed to saturate in the headspace for 30 mins. One end of the column was inserted into the bag and the other end was attached to a vacuum pump. After saturation, volatiles were sampled for 30 minutes in all the stages. The volatiles were then eluted from the column with 200 μL of hexane and samples were stored in a -20°C freezer until further analysis. Because floral scent emission rates can vary over time (for example, Hansted et al., 1994 reported increased day time emission in Ribe snigrum), all scent collection was completed before noon on each sampling dayin order to maximize the consistency of the emission rate. Four different adsorbent materials, namely, Porapak Type Q polymer (Waters, Milford, MA, USA, mesh size: 80/100), Tenax (Sigma-Aldrich, mesh size: 60/80), activated charcoal (HIMEDIA) and graphite were used to get a comprehensive volatile profile of Z. maculatum.To determine the volatile profiles, floral mass (in grams) and number of flowers sampled for each plant were recorded. For each type of adsorbent, a flower from three different plants was taken as the study material. Gas chromatography-Mass Spectrometry for emitted volatile profiling The volatile samples of Z. maculatum were analyzed on a Shimadzu Gas Chromatography- Mass Spectrometry system (GCMS-QP2010SE). A ZB-5 column (30 m × 0.25 mm id, film thickness 0.25 μm) was used for the separation of volatile compounds with helium as a carrier gas. Identification of the volatile compounds was done according to the previously described method (Bera et al., 2015). Geraniol (9 ng/μl) was added as an internal standard to each sample before injection and quantification of volatile emission rate (ng/ g fresh weight/h) was carried out according to Bera et al., (2015). Sampling of volatiles from flower extracts Endogenous glycosyl-bound volatiles The glycosidically bound endogenous volatiles was extracted by the method described in the literature with minor modifications (Oka et al., 1999; Picone et al., 2004). The fresh flower was first macerated by using liquid nitrogen and the powdered sample was treated with 1 mL of hexane to remove the volatiles (emitted and free endogenous). The sample was then vortexed and incubated in hot water bath for 60 s. The sample was centrifuged at 10,000 rpm for 10 min and the pellet was collected. This washing process was done thrice and the final pellet was kept at room temperature for drying. Then, 500 µL of phosphate-citrate buffer (pH 5.4) was added to the pellet and vortexed for 30 s. The sample was subjected to sonication in a water bath for 15 min and centrifuged at 10,000 rpm for 10 min. The supernatant was then transferred to a new 1.5 mL microcentrifuge tube and 200 µL of Viscozyme® L (cellulolytic enzyme mixture, Sigma-Aldrich, India) was added for deglycosylation. The sample was vortexed for 30 s followed by addition of 500 µL of DCM and incubated at 33ºC for 16 h. After incubation, the sample was vortexed for 1 min followed by 5 min centrifugation at 5,000 rpm at room temperature. The DCM layer was extracted quickly and treated with anhydrous Na2SO4 to remove water from the sample and again centrifuged at 10,000 rpm for 10 min. The supernatant was collected and concentrated by passing a gentle stream of nitrogen gas before GC-MS analysis. To set a control reaction, the assay was performed without Viscozyme® L. Geraniol (9 ng/μl) was added as an internal standard to each sample before injection and quantification of amount volatile present was carried out in terms of ng/ g fresh weight of flower.

GC-MS analysis of endogenous glycosyl-bound floral volatiles The samples were analyzed on a Trace 1300 Gas Chromatograph and ISQ-QD Mass Spectrometer (Thermo Scientific) using helium as the carrier gas (1.5 mL/min). Separation of the sample was carried out using TG-5 MS column (length: 30 m, ID: 0.32 μm). Sample (1 µL) was injected and the split ratio was kept at 10:1. The injector temperature of the GC was set at 260ºC and the initial column temperature was set at 50ºC which was held for 2 min. The temperature was further raised to 60ºC at the rate of 2ºC per min and held for 2 min. Following which the temperature of the oven was raised to 210ºC at the rate of 3ºC/min and held for 2 min. Finally, the oven temperature was raised to 270°C at the rate of 10ºC/min and held for 7 min. The MS transfer line and ion source temperature were set at 280ºC and 200ºC and the scan range was set at 40-600 amu. The resulting files were analyzed using Thermo XcaliburTM software and the mass spectra of compounds were confirmed with NIST 14 library (National Institute of Standards and Technology, Gaithersburg, USA) and compared with the retention indices of those published in the literature (Fernando &Grün 2001; Högnadóttir & Rouseff 2003; Takaku et al. 2007; Leela et al. 2009).

Figure S1 Z. maculatum at different developmental stages. These stages are indicated here as S1: bud stage; S2: early blooming stage, S3: mid blooming stage, S4: full blooming stage

Table S1 Comparison of emitted scent volatile compounds of full bloomed flower of Z. maculatum by using different adsorbents

Sr. No. Emission rate (ng scent/g fresh flower/h) Compound aID cRI dRT (min) P T C G 1 Benzaldehyde 0.1862±0.240 0.069±0.002 - - bMS, cRI 964 10.54 2 β-Myrcene 0.8128±0.195 1.791±0.120 - - MS, RI 989 12.37 0.1336±0.0 3 Decane - - - MS, RT 1000 12.89 21 4 Limonene - - - - MS, RI 1047 14.40 5 trans-α-Ocimene 0.1425±0.012 0.3388±0.055 - - MS, RI 1050 15.04 6 2-Phenyl ethanal 0.1796±0.006 0.0980±0.098 - - MS, RI, RT 1052 15.22 7 cis-β-Ocimene - 4.4402±0.034 - - MS, RI 1055 15.62 8 o-Diethylbenzene 11.026±0.124 - - - MS, RI, RT 1056 15.63 9 p-Diethylbenzene 12.844±0.1 - - - MS, RI 1066 15.99 10 Cymene 0.1792±.096 - - - MS, RI 1073 17.54 0.1188 11 Linalool 0.0742±0.157 - - MS, RI 1102 18.49 ±0.023 0.1881±0.0 12 Benzyl acetate 2.5112±0.109 1.9892±0.129 - MS, RI, RT 1167 21.92 03 13 Naphthalene - - - - MS, RI, RT 1179 22.76 0.0222±0.0 14 Methyl salicylate 0.3969±0.251 0.5088±0.1 - MS, RI, RT 1193 23.42 01 0.323±0.06 15 Dodecane - - - MS, RI, RT 1200 23.88 1 0.3444±0.0 16 2-Phenethyl acetate 2.8155±0.220 2.3013±0.129 - MS, RI, RT 1264 26.69 45 0.2089±0.0 17 Tridecane - - - MS, RI, RT 1300 28.87 01 0.6146±0.111 18 Eugenol 0.2759±0.019 - - MS, RI 1348 31.58 5 0.2451±0.0 19 Tetradecane - - - MS, RI, RT 1400 33.53 09 20 Cinnamyl acetate 0.2847±0.109 0.2961±0.016 - - MS, RI, RT 1454 35.53 0.2266±0.1 21 (E,E)-α-Farnesene 0.8383±0.098 0.3204±0.016 - MS, RI,RT 1494 38.23 30 aID, Identification: Identification was carried out by comparing mass spectrum (bMS) of the component to that of mass spectral library from NIST 14 and Wiley 8.0 (Wiley, New York, USA); cRI, Retention indices, identification by comparison of Retention indices with the published literatures and dRT, Retention time: identification by comparison of retention time to that of authentic standards. P: Porapak Q (mess size 80-100); T: Tenax; C: activated charcoal; G: Graphite. Each value is the mean ± SE (standard error) of triplicate analysis from at least three independent volatile extractions. Table S2. Comparison of scent volatile compounds emitted from Z. maculatum flowers of four different developmental stages

Emission rate (ng scent/g fresh flower/h) Early Sr. Mid blooming Full blooming Compound Name aID cRI dRT Bud stage blooming No. stage flower (Stage 1) stage (Stage 3) (Stage 4) (Stage 2) 1 Benzaldehyde bMS, RI 964 10.54 - - 0.1533±0.012 0.1899±0.014 2 β-Myrcene RI, MS, RT 989 12.37 *Tr - 0.4015±0.003 0.8461±0.066 3 (-) Limonene RI, MS 1047 14.40 Tr 0.0981±0.002 0.2819±0.008 - 4 trans-α-Ocimene RI, MS 1050 15.04 Tr - 0.043577±0.019 0.1425±0.017 5 2-Phenylethanal RI, MS 1052 15.22 Tr - 0.1493±0.045 0.1696±0.195 1.1360 ±0.011 6 o-Diethylbenzene RI, MS 1056 15.63 - 6.1994±0.029 11.0969±0.060

7 p-Diethylbenzene RI, MS 1066 15.99 - 1.8178±0.008 7.3732±0.141 12.8346±0.095 8 Cymene RI 1073 17.54 - 0.0882±0.021 0.1692±0.026 9 Linalool RI, MS, RT 1102 18.49 - - 0.0358±0.011 0.0709±0.025 10 Benzylacetate RI, MS, RT 1167 21.92 Tr 0.0583±0.002 1.3910±0.005 2.6112±0.264 11 Naphthalene RI, MS, RT 1179 22.76 - - 0.2267±0.006 - 12 Methylsalicylate RI, MS 1193 23.42 - - 0.2293±0.109 0.4302±0.066 13 2-Phenylethylacetate RI, MS, RT 1264 26.69 Tr - 1.6051±0.120 2.8155±0.1 14 Eugenol RI, MS, RT 1348 31.58 - - 0.2274±0.014 0.6813±0.115 15 Teradecane MS, RT 1400 33.53 - 0.1255±0.201 - - 16 Cinnamylacetate RI, MS 1454 35.53 - - - 0.2647±0.026 17 (E,E)-α-Farnesene RI, MS, RT 1494 38.23 - - 0.3120±0.011 0.8383±0.152 aID, Identification: Identification was carried out by comparing mass spectrum (bMS) of the component to that of mass spectral library from NIST 14; cRI, Retention indices, identification by comparison of Retention indices with the published literature and dRT, Retention time: identification by comparison of retention time to that of authentic standards. *Tr, trace amount. Each value is the mean ± SE (standard error) of triplicate analysis from at least three independent volatile extractions.

Table S3. Endogenous-glycosyl bound volatile components present in fully opened flower of Z. maculatum aRT Amount (ng/ g fresh Sr. No. Compound Name bRI (min) flower weight) 1 Benzyl alcohol 11.78 1023 3.1979±0.012 2 Phenylethyl alcohol 15.87 1114 10.92±0.007 0.0926±0.0008 3 n-dodecane 20.48 1200 2.4198±0.0006 4 p-Hydroxyallyl benzene 23.20 1249 0.8072±0.0078 5 (E)-Cinnamyl alcohol 25.28 1302 6 Eugenol 27.57 1348 0.0413±0.0006 7 2-Nitrocumene 23.74 1324 0.3658±0.0056 8 3-Isopropoxybenzaldehyde 29.55 1234 0.0491±0.0045 9 2,5-Di-tert-butylphenol 33.47 1514 0.0096±0.0003 10 2-Butenedioic acid, dibutyl ester 35.14 1505 0.0692±0.0007 11 Limonen-6-ol, pivalate 35.63 1538 0.18±0.001 12 Methoxyeugenol 37.57 1615 0.1194±0.0008 13 Benzene, (1-butylheptyl)- 38.76 1628 0.0276±0.0009 14 Benzaldehyde, 4-hydroxy-3,5-dimethoxy- 39.62 1664 0.6248±0.008 0.2547±0.0006 15 trans-Sinapyl alcohol 44.61 2034 16 n-Hexadecanoic acid 52.82 1964 0.0788±0.0007 aRT, Retention time: identification by comparison of retention time to that of authentic standards. bRI, Retention indices, identification by comparison of Retention indices with the published literature. Each value is the mean ± SE (standard error) of triplicate analysis from at least three independent volatile extractions.

Supplementary References

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