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Esters and other constituents of the foliar cuticular wax of a soybean variety
Biochemical Systematics and Ecology 63 (2015) 198e200
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Esters and other constituents of the foliar cuticular wax of a soybean variety C.E.J. Palacios a, G. Negri b, A. Salatino a, * a b
~o Paulo, Rua do Mata ~o 277, 05508-090, Sa ~o Paulo, Brazil Department of Botany, Institute of Biosciences, University of Sa ~o Paulo, CEBRID-UNIFESP, Sa ~o Paulo, Brazil Department of Psychobiology, Federal University of Sa
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 September 2015 Received in revised form 14 October 2015 Accepted 17 October 2015 Available online 2 November 2015
The constituents of the foliar cuticular wax of the variety ‘MG/BR 46 Conquista’ were isolated and chemically characterized. N-Alkanes, esters and triterpenoids are major constituents of the cuticular wax. Esters, taraxerol and steroids seem to be valuable cuticular wax constituents for characterization and distinction among soybean varieties. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Cuticular wax Fatty esters Glycine max Soybean Sterols Triterpenoids
1. Subject and source The cuticular wax is a crucial factor in the resistance against water loss by plant leaves. Evidence has been raised that cuticular wax from arid habitats may be more efficient as protection against water loss than their counterparts from other habitats (Oliveira et al., 2003). Drought affects severely soybean production (Seversike et al., 2014). The composition of the foliar cuticular wax is probably relevant to search varieties with higher drought resistance. Cuticular wax composition is also useful for characterization and distinction among crop species and varieties (Halinski et al., 2011; Kitagami et al., 2013). The present work deals with the composition of the foliar cuticular wax of ‘MG/BR 46 Conquista’, currently a soybean variety widely cultivated in Brazil. 2. Previous work To our knowledge, so far the paper by Kim et al. (2007) has been the only report addressing the composition of soybean foliar cuticular wax. 3. Present study Pesticide-free seeds of the variety ‘MG/BR Conquista’ of soybean (Glycine max (L.) Merril) were provided by EMBRAPA Soybean Division. Germinated seedlings were placed on trays with vermiculite and grown at 30 C during 4 days under * Corresponding author. E-mail address: [email protected] (A. Salatino). http://dx.doi.org/10.1016/j.bse.2015.10.012 0305-1978/© 2015 Elsevier Ltd. All rights reserved.
C.E.J. Palacios et al. / Biochemical Systematics and Ecology 63 (2015) 198e200
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photoperiod of 12 h. The plantlets were maintained in greenhouse for 10 days. The young plants were transferred to open air plots containing soil fertilized with vegetal ash in the experimental garden of the Department of Botany of the University of ~o Paulo (Brazil). The cultivation took place in JuneAug 2011 (winter season in southeast Brazil). Sa Total areas of five samples, each comprising 10 leaflets from 60 day old plants, were determined with the software ImageJ 1.44p. The cuticular wax was extracted by three consecutive immersions (30, 20 and 10 s) of the leaflets in dichloromethane. The solvent was evaporated and the wax weighed. Five fractions of wax constituents were isolated by preparative thin layer chromatography (TLC), using silicagel impregnated with 0.02% sodium fluoresceine (Oliveira et al., 2003), dichlorometane as mobile phase and visualization under long wave UV. The fractions of alkanes and fatty esters were analyzed directly by GC/ MS. Fractions containing fatty acids, triterpenes and sterols were derivatized with N,O-bis-(trimethylsilyl)-trifluoroacetamide (Jetter et al., 2000) prior to GC/MS analysis. Analysis of 1 mL of n-hexane solutions of n-alkane and derivatized fractions were performed with a GC/MS system Agilent GC/MS 6859/5975B, equipped with capillary column HP-5MS (crosslinked 5% MePh siloxane, 30 m 0.25 mm) according to Kitagami et al. (2013). The esters were analyzed starting at 250 C during 3 min, increasing 5 C min1 to 320 C and isotherm condition for 23 min. The n-alkane and fatty ester fractions were also analyzed in a HP 5890 series II gas chromatograph with FID detector. The relative percents of n-alkanes and esters were estimated by the area under the corresponding peaks. Compounds identification followed mass spectroscopy, comparison with NIST 05 Library and authentic samples. The esters were identified by the base peak (corresponding to the protonated acid moiety) and the molecular ion (Moldovan et al., 2002). Identification of TMSi-derivatized triterpenoids was based on Ogunkoya (1981) and Carvalho et al. (2010), and the derivatized sterols on Dumazer et al. (1986), Buckley et al. (1999) and Moreau et al. (2002). The leaves analyzed contained 15.2 ± 1.1 mg cm2 of cuticular wax. Contents in the range 10e30 mg cm2 are common on leaves of cultivated plants (Baker, 1982). An average of 8.26 ± 1.67 mg cm2 of cuticular wax was reported for 18 soybean varieties (Kim et al., 2007). Five fractions were obtained from the wax: n-alkanes (10.8% of the products recovered from TLC), esters (25.7%), triterpenoids plus primary alcohols plus unknowns (16.2%), and sterols plus unknowns (37.5%). The n-alkane homologues ranged from C23 to C33, with predominance of C31 (27.5%). Acid moieties of the esters were C16, C18 and C20 and of alcohols were C18, C20, C26 and C28. The predominant ester was octacosanoyl-eicosanoate (C28eC20, 46.7%). The primary alcohols detected were C16, C18, C20, C22, C26 and C28, with high predominance (47%) of the latter homologue. Amyrins (a and b), lupeol and taraxerol were the triterpenoids detected. Taraxerol was characterized by the molecular ion (Mþ, m/z 498) and the base peak m/z 204, and eluted before a-amyrin (Bauer et al., 2004). The intensity of the taraxerol peak in the chromatogram, relative the total triterpenoids, was 9.6%. Sterols (sitosterol, stigmasterol and campesterol) were also detected as minor constituents. 4. Chemotaxonomic significance Cuticular wax constituents have been widely used as chemotaxonomic markers, particularly the distribution of n-alkanes ski et al., 2011; Jingjing et al., 2013). Little has been done regarding the chemotaxonomic potential of esters of cuticular (Halin wax. The distribution of wax esters can be analyzed by GC/MS without derivatization (Furuhasi et al., 2015; present work). Wax esters seem to be relevant chemotaxonomic markers for characterization of soybean varieties and distinction among them. In fact, they are major constituents of the foliar cuticular wax of the variety ‘MG/BR 46 Conquista’ and have not been detected in the cuticular wax of 18 other varieties of soybean (Kim et al., 2007). Not only presence/absence, but also the homologue distribution of cuticular esters may be chemotaxonomically relevant, as has been observed regarding n-alkanes; for example, the cuticular waxes of Coffea arabica and C. canephora are characterized by the predominance of the C29, and C. racemosa by the C31 n-alkane (Kitagami et al., 2013). Caution must be taken, however, regarding presence/absence of esters as soybean chemotaxonomic markers, since these constituents may have been overlooked or were not detected by Kim et al. (2007), due to the methodology they employed, which was quite distinct from the one used in the present work. Wax triterpenes also seem to be useful chemotaxonomic markers for distinction among soybean varieties: taraxerol was detected in the cuticular wax of ‘MG/BR 46 Conquista’ and not in the varieties analyzed by Kim et al. (2007), while the reverse holds for ski et al., 2012). They lupenone and 3-keto-olean-12-ene. Sterols are rare foliar wax constituents, except in Solanaceae (Halin were detected in the foliar wax of ‘MG/BR 46 Conquista’, but were not mentioned in the work by Kim et al. (2007). Acknowledgments ~o de Aperfeiçoamento do Pessoal de Ensino Superior) is Provision of MSc scholarship to CPJ by CAPES (Coordenaça acknowledged. AS is a research fellow of CNPq. References Baker, E.A., 1982. In: Cutler, A.D.K., Alvin, L., Price, C.E. (Eds.), The Plant Cuticle. Academic Press, London, pp. 139e165. Bauer, S., Schulte, E., Thier, H.-P., 2004. Composition of the surface waxes from tomatoes. Eur. Food Res. Technol. 219, 223. Buckley, S.A., Stott, A.W., Evershed, R.P., 1999. Analyst 124, 443. sio, S.R., Crotti, A.E.M., Figueiredo, U.S., Vieira, P.C., Furtado, N.A.J., 2010. Carvalho, T.C., Polizeli, A.M., Turatti, I.C., Severiano, M.E., Carvalho, C.E., Ambro Molecules 15, 6140. Dumazer, M., Farines, M., Soulier, J., 1986. Rev. Fr. Corps Gras 4, 151.
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C.E.J. Palacios et al. / Biochemical Systematics and Ecology 63 (2015) 198e200
Furuhasi, T., Obawa, T., Nakai, R., Nakazawa, M., Okazawa, A., Paderschoke, A., Nishio, K., Hirai, M.Y., Arita, M., Ohta, D., 2015. Metabolomics 11, 175. ski, Ł.P., Paszkiewicz, M., Gołe˛ biowski, M., Stepnowski, P., 2012. J. Food Comp. Anal. 25, 74. Halin ski, Ł.P., Szafranek, J., Stepnowski, P., 2011. Plant Biol. 13, 932. Halin €ffer, S., Riederer, M., 2000. Plant Cell Environ. 23, 619. Jetter, R., Scha Jingjing, L.J., Huang, J.H., Ge, J.W., Huang, X.Y., Xie, S.C., 2013. Biochem. Syst. Ecol. 49, 30. Kim, K.S., Park, S.H., Kim, D.K., Jenks, M.A., 2007. Int. J. Plant Sci. 168, 307. Kitagami, J.T., Salatino, A., Guerreiro-Filho, O., Salatino, M.L.F., 2013. Biochem. Syst. Ecol. 46, 119. Moldovan, Z., Jover, E., Bayona, J.M., 2002. J. Chrom. A 952, 193. Moreau, R.A., Whitaker, B.D., Hicks, K.B., 2002. Prog. Lipid Res. 41, 457. Ogunkoya, L., 1981. Phytochemistry 20, 121. Oliveira, A.F.M., Meirelles, S.T., Salatino, A., 2003. An. Acad. Bras. Cienc. 75, 431. Seversike, T.M., Sermons, S.M., Sinclair, T.R., Carter Jr., T.E., Rufty, T.W., 2014. Plant Soil 374, 359.
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Esters and other constituents of the foliar cuticular wax of a soybean variety C.E.J. Palacios a, G. Negri b, A. Salatino a, * a b
~o Paulo, Rua do Mata ~o 277, 05508-090, Sa ~o Paulo, Brazil Department of Botany, Institute of Biosciences, University of Sa ~o Paulo, CEBRID-UNIFESP, Sa ~o Paulo, Brazil Department of Psychobiology, Federal University of Sa
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 September 2015 Received in revised form 14 October 2015 Accepted 17 October 2015 Available online 2 November 2015
The constituents of the foliar cuticular wax of the variety ‘MG/BR 46 Conquista’ were isolated and chemically characterized. N-Alkanes, esters and triterpenoids are major constituents of the cuticular wax. Esters, taraxerol and steroids seem to be valuable cuticular wax constituents for characterization and distinction among soybean varieties. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Cuticular wax Fatty esters Glycine max Soybean Sterols Triterpenoids
1. Subject and source The cuticular wax is a crucial factor in the resistance against water loss by plant leaves. Evidence has been raised that cuticular wax from arid habitats may be more efficient as protection against water loss than their counterparts from other habitats (Oliveira et al., 2003). Drought affects severely soybean production (Seversike et al., 2014). The composition of the foliar cuticular wax is probably relevant to search varieties with higher drought resistance. Cuticular wax composition is also useful for characterization and distinction among crop species and varieties (Halinski et al., 2011; Kitagami et al., 2013). The present work deals with the composition of the foliar cuticular wax of ‘MG/BR 46 Conquista’, currently a soybean variety widely cultivated in Brazil. 2. Previous work To our knowledge, so far the paper by Kim et al. (2007) has been the only report addressing the composition of soybean foliar cuticular wax. 3. Present study Pesticide-free seeds of the variety ‘MG/BR Conquista’ of soybean (Glycine max (L.) Merril) were provided by EMBRAPA Soybean Division. Germinated seedlings were placed on trays with vermiculite and grown at 30 C during 4 days under * Corresponding author. E-mail address: [email protected] (A. Salatino). http://dx.doi.org/10.1016/j.bse.2015.10.012 0305-1978/© 2015 Elsevier Ltd. All rights reserved.
C.E.J. Palacios et al. / Biochemical Systematics and Ecology 63 (2015) 198e200
199
photoperiod of 12 h. The plantlets were maintained in greenhouse for 10 days. The young plants were transferred to open air plots containing soil fertilized with vegetal ash in the experimental garden of the Department of Botany of the University of ~o Paulo (Brazil). The cultivation took place in JuneAug 2011 (winter season in southeast Brazil). Sa Total areas of five samples, each comprising 10 leaflets from 60 day old plants, were determined with the software ImageJ 1.44p. The cuticular wax was extracted by three consecutive immersions (30, 20 and 10 s) of the leaflets in dichloromethane. The solvent was evaporated and the wax weighed. Five fractions of wax constituents were isolated by preparative thin layer chromatography (TLC), using silicagel impregnated with 0.02% sodium fluoresceine (Oliveira et al., 2003), dichlorometane as mobile phase and visualization under long wave UV. The fractions of alkanes and fatty esters were analyzed directly by GC/ MS. Fractions containing fatty acids, triterpenes and sterols were derivatized with N,O-bis-(trimethylsilyl)-trifluoroacetamide (Jetter et al., 2000) prior to GC/MS analysis. Analysis of 1 mL of n-hexane solutions of n-alkane and derivatized fractions were performed with a GC/MS system Agilent GC/MS 6859/5975B, equipped with capillary column HP-5MS (crosslinked 5% MePh siloxane, 30 m 0.25 mm) according to Kitagami et al. (2013). The esters were analyzed starting at 250 C during 3 min, increasing 5 C min1 to 320 C and isotherm condition for 23 min. The n-alkane and fatty ester fractions were also analyzed in a HP 5890 series II gas chromatograph with FID detector. The relative percents of n-alkanes and esters were estimated by the area under the corresponding peaks. Compounds identification followed mass spectroscopy, comparison with NIST 05 Library and authentic samples. The esters were identified by the base peak (corresponding to the protonated acid moiety) and the molecular ion (Moldovan et al., 2002). Identification of TMSi-derivatized triterpenoids was based on Ogunkoya (1981) and Carvalho et al. (2010), and the derivatized sterols on Dumazer et al. (1986), Buckley et al. (1999) and Moreau et al. (2002). The leaves analyzed contained 15.2 ± 1.1 mg cm2 of cuticular wax. Contents in the range 10e30 mg cm2 are common on leaves of cultivated plants (Baker, 1982). An average of 8.26 ± 1.67 mg cm2 of cuticular wax was reported for 18 soybean varieties (Kim et al., 2007). Five fractions were obtained from the wax: n-alkanes (10.8% of the products recovered from TLC), esters (25.7%), triterpenoids plus primary alcohols plus unknowns (16.2%), and sterols plus unknowns (37.5%). The n-alkane homologues ranged from C23 to C33, with predominance of C31 (27.5%). Acid moieties of the esters were C16, C18 and C20 and of alcohols were C18, C20, C26 and C28. The predominant ester was octacosanoyl-eicosanoate (C28eC20, 46.7%). The primary alcohols detected were C16, C18, C20, C22, C26 and C28, with high predominance (47%) of the latter homologue. Amyrins (a and b), lupeol and taraxerol were the triterpenoids detected. Taraxerol was characterized by the molecular ion (Mþ, m/z 498) and the base peak m/z 204, and eluted before a-amyrin (Bauer et al., 2004). The intensity of the taraxerol peak in the chromatogram, relative the total triterpenoids, was 9.6%. Sterols (sitosterol, stigmasterol and campesterol) were also detected as minor constituents. 4. Chemotaxonomic significance Cuticular wax constituents have been widely used as chemotaxonomic markers, particularly the distribution of n-alkanes ski et al., 2011; Jingjing et al., 2013). Little has been done regarding the chemotaxonomic potential of esters of cuticular (Halin wax. The distribution of wax esters can be analyzed by GC/MS without derivatization (Furuhasi et al., 2015; present work). Wax esters seem to be relevant chemotaxonomic markers for characterization of soybean varieties and distinction among them. In fact, they are major constituents of the foliar cuticular wax of the variety ‘MG/BR 46 Conquista’ and have not been detected in the cuticular wax of 18 other varieties of soybean (Kim et al., 2007). Not only presence/absence, but also the homologue distribution of cuticular esters may be chemotaxonomically relevant, as has been observed regarding n-alkanes; for example, the cuticular waxes of Coffea arabica and C. canephora are characterized by the predominance of the C29, and C. racemosa by the C31 n-alkane (Kitagami et al., 2013). Caution must be taken, however, regarding presence/absence of esters as soybean chemotaxonomic markers, since these constituents may have been overlooked or were not detected by Kim et al. (2007), due to the methodology they employed, which was quite distinct from the one used in the present work. Wax triterpenes also seem to be useful chemotaxonomic markers for distinction among soybean varieties: taraxerol was detected in the cuticular wax of ‘MG/BR 46 Conquista’ and not in the varieties analyzed by Kim et al. (2007), while the reverse holds for ski et al., 2012). They lupenone and 3-keto-olean-12-ene. Sterols are rare foliar wax constituents, except in Solanaceae (Halin were detected in the foliar wax of ‘MG/BR 46 Conquista’, but were not mentioned in the work by Kim et al. (2007). Acknowledgments ~o de Aperfeiçoamento do Pessoal de Ensino Superior) is Provision of MSc scholarship to CPJ by CAPES (Coordenaça acknowledged. AS is a research fellow of CNPq. References Baker, E.A., 1982. In: Cutler, A.D.K., Alvin, L., Price, C.E. (Eds.), The Plant Cuticle. Academic Press, London, pp. 139e165. Bauer, S., Schulte, E., Thier, H.-P., 2004. Composition of the surface waxes from tomatoes. Eur. Food Res. Technol. 219, 223. Buckley, S.A., Stott, A.W., Evershed, R.P., 1999. Analyst 124, 443. sio, S.R., Crotti, A.E.M., Figueiredo, U.S., Vieira, P.C., Furtado, N.A.J., 2010. Carvalho, T.C., Polizeli, A.M., Turatti, I.C., Severiano, M.E., Carvalho, C.E., Ambro Molecules 15, 6140. Dumazer, M., Farines, M., Soulier, J., 1986. Rev. Fr. Corps Gras 4, 151.
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C.E.J. Palacios et al. / Biochemical Systematics and Ecology 63 (2015) 198e200
Furuhasi, T., Obawa, T., Nakai, R., Nakazawa, M., Okazawa, A., Paderschoke, A., Nishio, K., Hirai, M.Y., Arita, M., Ohta, D., 2015. Metabolomics 11, 175. ski, Ł.P., Paszkiewicz, M., Gołe˛ biowski, M., Stepnowski, P., 2012. J. Food Comp. Anal. 25, 74. Halin ski, Ł.P., Szafranek, J., Stepnowski, P., 2011. Plant Biol. 13, 932. Halin €ffer, S., Riederer, M., 2000. Plant Cell Environ. 23, 619. Jetter, R., Scha Jingjing, L.J., Huang, J.H., Ge, J.W., Huang, X.Y., Xie, S.C., 2013. Biochem. Syst. Ecol. 49, 30. Kim, K.S., Park, S.H., Kim, D.K., Jenks, M.A., 2007. Int. J. Plant Sci. 168, 307. Kitagami, J.T., Salatino, A., Guerreiro-Filho, O., Salatino, M.L.F., 2013. Biochem. Syst. Ecol. 46, 119. Moldovan, Z., Jover, E., Bayona, J.M., 2002. J. Chrom. A 952, 193. Moreau, R.A., Whitaker, B.D., Hicks, K.B., 2002. Prog. Lipid Res. 41, 457. Ogunkoya, L., 1981. Phytochemistry 20, 121. Oliveira, A.F.M., Meirelles, S.T., Salatino, A., 2003. An. Acad. Bras. Cienc. 75, 431. Seversike, T.M., Sermons, S.M., Sinclair, T.R., Carter Jr., T.E., Rufty, T.W., 2014. Plant Soil 374, 359.