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Fractionation of hydrogen isotopes during phytol biosynthesis
Organic Geochemistry 40 (2009) 569–573
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Organic Geochemistry journal homepage: www.elsevier.com/locate/orggeochem
Fractionation of hydrogen isotopes during phytol biosynthesis Yoshito Chikaraishi a,*, Ryouichi Tanaka b, Ayumi Tanaka b, Naohiko Ohkouchi a a b
Institute for Research on Earth Evolution, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan Institute of Low Temperature Science, Hokkaido University, N19 W8, Kita-Ku, Sapporo 060-0819, Japan
a r t i c l e
i n f o
Article history: Received 24 September 2008 Received in revised form 9 February 2009 Accepted 23 February 2009 Available online 28 February 2009
a b s t r a c t Deuterium (D) depletion in phytol relative to ambient water as well as other lipids has been widely observed in various biological and geological samples; however, the mechanism for the depletion remains unknown. We have determined the hydrogen isotopic compositions of phytol and its precursors in cucumber cotyledons and have evaluated the fractionation of hydrogen isotopes during phytol biosynthesis. The hydrogen isotopic compositions of geranylgeraniol, dihydrogeranylgeraniol, tetrahydrogeranylgeraniol and phytol are 281‰, 302‰, 325‰ and 345‰, respectively. The results suggest that significantly D-depleted hydrogen is incorporated stepwise during hydrogenation of geranylgeraniol to phytol. We conclude that hydrogenation is important in controlling D depletion in phytol. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Because the isotopic effect for hydrogen is commonly large, marked variation in isotopic composition (400 to +50‰) is observed for organic compounds in biological and geological samples (e.g. Sessions et al., 1999; Sauer et al., 2001; Chikaraishi et al., 2004a,b,c; Huang et al., 2004; Radke et al., 2005; Chikaraishi and Naraoka, 2006; Dawson et al., 2007). In particular, phytol is significantly and consistently depleted in deuterium (D) relative to ambient water by ca. 300‰ in biological samples such as phytoplankton and terrestrial plants, whereas alkyl (e.g. fatty acids) and other isoprenoid (e.g. sterol) lipids are less depleted in D by ca. 100‰ and ca. 200‰, respectively (Sessions et al., 1999; Chikaraishi et al., 2004a,b). Similar isotopic distributions are also found in geological samples such as soils and sediments (Huang et al., 2004; Chikaraishi and Naraoka, 2005, 2006; Chikaraishi et al., 2007). One possible explanation for such strong D depletion in phytol is that it and other isoprenoid lipids are biosynthesized with isotopically distinct pools of NADPH (and/or organic substrates), as suggested by Sessions et al. (1999) and Sessions (2006). In fact, phytol is biosynthesized in the plastid via the 2-C-methyl-D-erythritol-4phosphate (MEP) pathway, whereas sterols are biosynthesized in the cytosol via the mevalonic-acid (MVA) pathway (Fig. 1; e.g. Lichtenthaler et al., 1997; Bohlmann et al., 1999; Lichtenthaler, 1999). The hydrogen isotopic composition of NADPH produced by photosynthetic water fission in the plastid would be different from that produced by the oxidative pentose phosphate cycle of glucose con* Corresponding author. Tel.: +81 46 867 9778; fax: +81 46 867 9775. E-mail address: [email protected] (Y. Chikaraishi). 0146-6380/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.orggeochem.2009.02.007
version in the cytosol (Sessions et al., 1999; Schmidt et al., 2003). This proposal is supported by an experimental finding that starch in the plastid is depleted in D by up to 190‰ relative to cytosolic sugar (Luo and Sternberg, 1991). Alternatively, Chikaraishi et al. (2004a) suggested pronounced isotopic fractionation associated with hydrogenation during phytol biosynthesis. In fact, the hydrogenation is specific to phytol biosynthesis downstream of geranylgeranyl pyrophosphate (GGPP, a branch point between phytol and other diterpenoids; Fig. 1) and it was also observed that phytol is depleted in D by up to 65‰ relative to other diterpenoids (Chikaraishi et al., 2004a). Thus, the reason for the strong D depletion in phytol remains uncertain; it is considered to reflect either the utilization of isotopically distinct pools of NADPH or isotopic fractionation during hydrogenation, or both. Phytol is primarily biosynthesized as the phytyl side chain of chlorophylls by two distinct pathways at different plastid stages in plants (Fig. 2; e.g. Thomson and Whatley, 1980; Keller et al., 1998; Schoefs and Bertrand, 2000; Sibata et al., 2004). At the etioplast stage, GGPP is first esterified to chlorophyllide and the product chorophyllide-geranylgeraniol is successively hydrogenated to ultimately form chorophyllide phytol (i.e. chlorophyll a), as a result of a scarcity of NADPH or low activity of GGPP reductase. The etioplast stage is found in cotyledons (seed leaves) at the beginning of greening, but is not usually found in green leaves themselves (e.g. Thomson and Whatley, 1980). In green leaves (i.e. at the chloroplast stage), GGPP is first converted to phytyl pyrophosphate and finally esterified with chlorophyllide to form chlorophyll a. However, the hydrogenations should be controlled by the same enzymes for both pathways (e.g. Tanaka et al., 1999).
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Fig. 1. Compartmentation of isoprenoid biosynthesis in higher plants between plastid and cytosol (after Lichtenthaler et al., 1997; Bohlmann et al., 1999; Lichtenthaler, 1999). Abbreviations: acetyl-CoA, acetyl coenzyme-A; FPP, farnesyl pyrophosphate; GA-3-P, D-glyceraldehyde-3-phosphate; GGPP, geranylgeranyl pyrophospate; GPP, geranyl pyrophospate; IPP, isopentenyl pyrophosphate; MEP, 2-C-methyl-D-erythritol-4-phosphate; MVA, mevalonic acid.
Comparison of the hydrogen isotopic composition of phytol and its precursors is straightforward in terms of understanding the isotopic signature of phytol in biological and geological samples. The three precursors—geranylgeraniol (GG), dihydrogeranylgeraniol (DHGG), and tetrahydrogeranylgeraniol (THGG)—can be observed in cotyledons simultaneously with phytol as saponified products, but not in green leaves. Therefore, in this study we determine the hydrogen isotopic compositions of phytol and its precursors in plant cotyledons and evaluate the fractionation of hydrogen isotopes during phytol biosynthesis. 2. Materials and methods Cucumber (Cucumis sativus, cv. Aonagajibai) cotyledons were cultivated under the conditions reported by Aarti et al. (2006, 2007). Seeds were soaked in water for ca. 4–5 h prior to sowing in vermiculite and were allowed to germinate for 4 days in complete darkness in a growth chamber at 22 °C. Seedlings were subjected to continuous white fluorescent light (40–45 lE m2 s1) for 4 h. Cotyledons were harvested and used for analysis. The cotyledons were frozen by liquid N2 and crushed to a fine powder before analysis. The preparation method employed for phytol and its precursors followed the procedure described by Chikaraishi et al. (2004b) and Chikaraishi and Naraoka (2006). In brief, the powdered cotyledons were saponified with KOH/CH3OH/H2O and extracted with CH2Cl2/CH3OH. The compounds were separated by way of silica gel column chromatography and urea adduction followed by acetylation with acetic anhydride/pyridine. They were identified and quantified using gas chromatography/mass spec-
trometry (GC/MS) with an Agilent Technologies 6890N GC/5973A MSD system. Quantification was based on comparison of peak areas with an external phytol standard. The components were further separated using AgNO3 silica gel column chromatography (3.5 cm 6 mm) following the improved procedure reported by Chikaraishi et al. (2004c, 2005a). Phytol and THGG were eluted in the first 2 ml followed by a further 2 ml CH2Cl2/ethyl acetate (98:2, v/v), respectively. DHGG and GG were eluted in the first 2 ml followed by a further 4 ml n-hexane/ethyl acetate (4:1, v/v), respectively. The recovery of the compounds following AgNO3 silica gel column chromatography was >95%. Hydrogen isotopic compositions were determined using GC/ pyrolysis/isotope ratio MS (GC/pyrolysis/IRMS) with an Agilent Technologies 6890N GC instrument interfaced to a Thermo Finnigan Delta plus XP IRMS instrument via a pyrolysis furnace (Chikaraishi et al., 2005b, 2007). Pyrolysis was performed in a microvolume ceramic tube with graphite at 1440 °C (Burgøyne and Hayes, 1998; Hilkert et al., 1999). Isotopic compositions are expressed in the dD notation relative to Vienna Standard Mean Ocean Water (VSMOW). The H3 factor was 3.95, with variation before and after measurements within 0.05. For calibration, three pulses of reference H2 gas (250‰) were introduced at the beginning and end of each measurement (Fig. 3b). An n-alkane mixture (C15–C36: 37 to 280‰) and phytol (313‰) with known dD values were used as standards and analyzed every four GC/pyrolysis/IRMS runs to confirm the reproducibility of the measurements. The isotopic compositions of the standards gave an analytical uncertainty of 3.7‰ for an m/z 2 intensity of 0.5–5.0 V. The memory effect (see Wang and Sessions,
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Fig. 2. Two distinct pathways for phytol biosynthesis at different plastid stages in plants (after Keller et al., 1998; Schoefs and Bertrand, 2000; Sibata et al., 2004). Abbreviations: Chl a, chlorophyll a; Chlide, chlorophyllide; Chlide-DHGG, chlorophyllide-dihydrogeranylgeraniol; Chlide-GG, chlorophyllide-geranylgeraniol; ChlideTHGG,chlorophyllide-tetrahydrogeranylgeraniol; DHGGPP, dihydrogeranylgeranyl pyrophospate; PPP, phytyl pyrophospate; THGGPP, tetrahydrogeranylgeranyl pyrophospate.
Fig. 3. (a) Total ion chromatogram (TIC) for phytol and its precursors obtained from GC/MS analysis and (b) m/z 2 chromatograms for each compound obtained from GC/C/ IRMS analysis.
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2008) on the values was not substantial. The sample phytol and THGG were analyzed in triplicate, with 1r of 3.2 and 2.4‰, respectively. Because of their low abundance in the extract, DHGG and GG were concentrated 20 times and the isotopic composition of each was determined in a single analysis (Fig. 3). The isotopic composition was corrected for the contribution of hydrogen incorporated during acetylation using an isotope mass balance calculation: the isotopic composition of the incorporated acetate hydrogen (204‰) was obtained during the acetylation of alcohol standards with known dD values, including n-alkanols, phytol and sterols (Chikaraishi et al., 2004b). We also determined the hydrogen isotopic compositions of leaf water and other typical lipids (i.e. fatty acids and sterol), according to the procedures described by Chikaraishi et al., 2004b,c, 2005b. 3. Results and discussion Phytol and the three precursors (THGG, DHGG, and GG) occur in a relative abundance of 87.7, 7.6, 2.3 and 2.4 mol%, respectively (Fig. 3a). This is consistent with the general distribution of the corresponding chlorophylls in plant cotyledons (e.g. Keller et al., 1998; Schoefs and Bertrand, 2000; Sibata et al., 2004). As summarized in Table 1, phytol (345‰) is significantly depleted in D relative to leaf water (52‰) in the cotyledons, whereas fatty acids (132‰ to 174‰) and b-sitosterol (278‰) are less depleted. This isotopic distribution and fractionation is almost consistent with those in plant leaves generally (e.g., Chikaraishi et al., 2004b), even though the cotyledons have little activity of photosynthesis. The hydrogen isotopic compositions of GG, DHGG, THGG and phytol are 281‰, 302‰, 325‰, and 345‰, respectively (Table 1), showing a gradual D depletion from GG to phytol. This strongly suggests pronounced isotopic variation even within the plastid terpenoids, including a lack of significant D depletion in the phytol precursors (especially GG, which has essentially the same dD value as b-sitosterol). Phytol and its precursors are clearly biosynthesized with the same pools of hydrogen source in the plastid (Fig. 1). The only difference between them is in hydrogen incorporation during hydrogenation (Fig. 2); therefore, this gradual D depletion can be ascribed to the incorporation of significantly Ddepleted hydrogen into GG to form DHGG, into DHGG to form Table 1 Hydrogen isotopic composition of phytol, its precursors, other lipids and leaf water from cucumber cotyledons. Compound
Abbreviation
Hydrogen isotopic composition (‰, vs. VSMOW) Whole molecules
Phytol and precursors Phytol Tetrahydrogeranylgeraniol Ditrahydrogeranylgeraniol Geranylgeraniol Other lipids Palmitic acid Stearic acid a-Linolenic acid b-Sitosterol Leaf water
THGG DHGG GG C16:0 C18:0 C18:3, n3 29D5
Incorporated hydrogen
dD
1r
dDinHa
rinHb
345 325 302 281
3.2 (n = 3) 2.4 (n = 3) n.d. (n = 1) n.d. (n = 1)
715 728 649
79 81 92
174 132 176 278 52
0.6 1.2 0.9 1.5 3.0
(n = 3) (n = 3) (n = 3) (n = 3) (n = 9)
THGG, and finally into THGG to form phytol. Assuming isotopic mass balance, the isotopic composition of the incorporated hydrogen is estimated by to be 715 to 649‰ for each hydrogenation step (Table 1). The incorporated hydrogen is probably derived from NADPH and H+ (cellular water) in the plastid. Although the isotopic compositions of NADPH and H+ were not determined, Schmidt et al. (2003) reported net values of ca. 250‰ for NADPH. Also, it is likely that the isotopic composition of leaf water is similar to that of H+. Taking into account the large error in the estimated isotopic composition of incorporated hydrogen (as large as 90‰, Table 1), we roughly estimate the isotopic fractionation factor (a) based on the isotopic compositions of incorporated hydrogen, NADPH and H+ of 700‰, 250‰ and 50‰, respectively. Since two hydrogens are incorporated during each reduction (probably one from NADPH and another one from H+), a is estimated to be ca. 0.35 from the following equation:
a ¼ ðdDinH þ 1000Þ=fðdDNADPH þ dDHþ Þ=2 þ 1000g Thus, significant isotopic fractionation represents a plausible explanation for the gradual D depletion observed from GG (281‰) to phytol (345‰). In the chloroplast, the NADPH is initially strongly depleted in D (up to 600‰) by way of a large isotope effect during the reduction of NADP+ to NADPH in photosynthesis but finally enriched in D to around 250‰ (as a net value) as a result of other isotope effects and exchange during various substrate reactions (e.g. Luo et al., 1991; Hayes, 2001); however, it is relevant for green leaves that are actively photosynthesizing and the isotopic composition of NADPH in the cotyledons is unknown. It is also likely that NADPH is mainly processed via the oxidative cycle of starches stored in the seed. If the initially produced NADPH with a value as low as 600‰ is quantitatively utilized for the hydrogenation of phytol precursors in the cotyledons, a large isotopic fractionation (a ca. 0.35) is not necessary for explaining the gradual D depletion from GG to phytol. Thus, although we cannot rule out the potential importance of a utilization of isotopically distinct pools of NADPH, the gradual D depletion from GG (281‰) to phytol (345‰) clearly indicates that significantly D-depleted hydrogen is incorporated stepwise during hydrogenation from GG to phytol. Since the hydrogenations should be controlled by the same enzymes for both cotyledons and green leaves (e.g. Tanaka et al., 1999), the phenomenon probably provides a general explanation for the significant D depletion in plant phytol. Various compounds undergo hydrogenation (or dehydrogenation) along their biosynthetic and metabolic pathways. If the above process is applicable, hydrogenation products could be depleted in D relative to their producers (as with phytol), whereas dehydrogenation agents may be enriched relative to their products. Indeed, a large difference in isotopic composition is observed among the different degrees of unsaturation of fatty acids in marine macroalgae (Chikaraishi et al., 2004c) and alkenones in cultured Emiliania huxleyi, suspended particles and sediments (D’Andrea et al., 2007; Schwab and Sachs, 2009). Thus, isotopic fractionation associated with hydrogenation and dehydrogenation is likely to be an important factor in explaining the wide variation in hydrogen isotopic composition observed among compounds in biological and geological samples. Acknowledgments
a
Hydrogen isotopic composition of hydrogens incorporated during hydrogenation (dDinH) calculated with an isotope mass balance dDinH = (dDproduct nproduct dDprecursor nprecursor)/2; n is the number of hydrogen atoms. b Error in estimated dDinH value: r2inH ¼ 1r2product ðnproduct =2Þ2 þ 1r2precursor ðnprecursor =2Þ2 ; 1r for DHGG and GG is used as 1r of standard analysis (3.7‰, see text).
We are grateful to N.O. Ogawa, Y. Kashiyama, Y. Takano and H. Kitazato for expert advice and constructive discussion. Hydrogen isotope measurement of leaf water using TCEA/IRMS was kindly supported by K. Tamura and N. Kurita (JAMSTEC, IORGC). We thank
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A.L. Sessions and R.D. Pancost for valuable suggestions and comments. The work was partly supported by a Grant-in-Aid for Creative Scientific Research (19GS0211) to N.O. Associate Editor — R.D. Pancost References Aarti, P.D., Tanaka, R., Tanaka, A., 2006. Effects of oxidative stress on chlorophyll biosynthesis in cucumber (Cucumis sativus) cotyledons. Physiologia Plantarum 128, 186–197. Aarti, P.D., Tanaka, R., Ito, H., Tanaka, A., 2007. High light inhibits chlorophyll biosynthesis at the level of 5-aminolevulinate synthesis during de-etiolation in cucumber (Cucumis sativus) cotyledons. Photochemistry and Photobiology 83, 171–176. Bohlmann, J., Phillips, M., Ramachandiran, V., Katoch, A., Croteau, R., 1999. cDNA cloning, characterization, and functional expression of four new monoterpene synthase members of the Tpsd gene family from grand fir (Abies grandis). Archives of Biochemistry and Biophysics 368, 232–243. Burgøyne, T.W., Hayes, J.M., 1998. Quantitative production of H2 by pyrolysis of gas chromatographic effluents. Analytical Chemistry 70, 5136–5141. Chikaraishi, Y., Naraoka, Hiroshi, 2005. d13C and dD identification of sources of lipid biomarkers in sediments of Lake Haruna (Japan). Geochimica et Cosmochimica Acta 69, 3285–3297. Chikaraishi, Y., Naraoka, H., 2006. Carbon and hydrogen isotope variation of plant biomarkers in a plant–soil system. Chemical Geology 231, 190–202. Chikaraishi, Y., Naraoka, H., Poulson, S.R., 2004a. Carbon and hydrogen isotopic fractionation during lipid biosynthesis in a higher plant (Cryptomeria japonica). Phytochemistry 65, 323–330. Chikaraishi, Y., Naraoka, H., Poulson, S.R., 2004b. Hydrogen and carbon isotopic fractionations of lipid biosyntheses among terrestrial (C3, C4 and CAM) and aquatic plants. Phytochemistry 65, 1369–1381. Chikaraishi, Y., Suzuki, Y., Naraoka, H., 2004c. Hydrogen isotopic fractionations during desaturation and elongation associated with polyunsaturated fatty acid biosynthesis in marine macroalgae. Phytochemistry 65, 2293–2300. Chikaraishi, Y., Yamada, Y., Naraoka, H., 2005a. Carbon and hydrogen isotopic compositions of sterols from riverine and marine sediments. Limnology and Oceanography 50, 1763–1770. Chikaraishi, Y., Matsumoto, K., Ogawa, N.O., Suga, H., Kitazato, H., Ohkouchi, N., 2005b. Hydrogen, carbon and nitrogen isotopic fractionations during chlorophyll biosynthesis in C3 higher plants. Phytochemistry 66, 911–920. Chikaraishi, Y., Matsumoto, H., Kitazato, H., Ohkouchi, N., 2007. Sources and transformation processes of pheopigments: stable carbon and hydrogen isotopic evidence from Lake Haruna, Japan. Organic Geochemistry 38, 985–1001. D’Andrea, W.J., Liu, Z., Alexander, M.D.R., Wattey, S., Herbert, T.D., Huang, Y., 2007. An efficient method for isolating individual long-chain alkenones for compoundspecific hydrogen isotope analysis. Analytical Chemistry 79, 3430–3435. Dawson, D., Grice, K., Alexander, R., Edwards, D., 2007. The effect of source and maturity on the stable isotopic compositions of individual hydrocarbons in sediments and crude oils from the Vulcan Sub-basin, Timor Sea, Northern Australia. Organic Geochemistry 38, 1015–1038. Hayes, J.M., 2001. Fractionation of carbon and hydrogen isotopes in biosynthetic processes. In: Valley, J.W., Cole, D.R. (Eds.), Reviews in Mineralogy and Geochemistry, vol. 43. The Mineralogical Society of America, pp. 225–277.
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Contents lists available at ScienceDirect
Organic Geochemistry journal homepage: www.elsevier.com/locate/orggeochem
Fractionation of hydrogen isotopes during phytol biosynthesis Yoshito Chikaraishi a,*, Ryouichi Tanaka b, Ayumi Tanaka b, Naohiko Ohkouchi a a b
Institute for Research on Earth Evolution, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan Institute of Low Temperature Science, Hokkaido University, N19 W8, Kita-Ku, Sapporo 060-0819, Japan
a r t i c l e
i n f o
Article history: Received 24 September 2008 Received in revised form 9 February 2009 Accepted 23 February 2009 Available online 28 February 2009
a b s t r a c t Deuterium (D) depletion in phytol relative to ambient water as well as other lipids has been widely observed in various biological and geological samples; however, the mechanism for the depletion remains unknown. We have determined the hydrogen isotopic compositions of phytol and its precursors in cucumber cotyledons and have evaluated the fractionation of hydrogen isotopes during phytol biosynthesis. The hydrogen isotopic compositions of geranylgeraniol, dihydrogeranylgeraniol, tetrahydrogeranylgeraniol and phytol are 281‰, 302‰, 325‰ and 345‰, respectively. The results suggest that significantly D-depleted hydrogen is incorporated stepwise during hydrogenation of geranylgeraniol to phytol. We conclude that hydrogenation is important in controlling D depletion in phytol. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Because the isotopic effect for hydrogen is commonly large, marked variation in isotopic composition (400 to +50‰) is observed for organic compounds in biological and geological samples (e.g. Sessions et al., 1999; Sauer et al., 2001; Chikaraishi et al., 2004a,b,c; Huang et al., 2004; Radke et al., 2005; Chikaraishi and Naraoka, 2006; Dawson et al., 2007). In particular, phytol is significantly and consistently depleted in deuterium (D) relative to ambient water by ca. 300‰ in biological samples such as phytoplankton and terrestrial plants, whereas alkyl (e.g. fatty acids) and other isoprenoid (e.g. sterol) lipids are less depleted in D by ca. 100‰ and ca. 200‰, respectively (Sessions et al., 1999; Chikaraishi et al., 2004a,b). Similar isotopic distributions are also found in geological samples such as soils and sediments (Huang et al., 2004; Chikaraishi and Naraoka, 2005, 2006; Chikaraishi et al., 2007). One possible explanation for such strong D depletion in phytol is that it and other isoprenoid lipids are biosynthesized with isotopically distinct pools of NADPH (and/or organic substrates), as suggested by Sessions et al. (1999) and Sessions (2006). In fact, phytol is biosynthesized in the plastid via the 2-C-methyl-D-erythritol-4phosphate (MEP) pathway, whereas sterols are biosynthesized in the cytosol via the mevalonic-acid (MVA) pathway (Fig. 1; e.g. Lichtenthaler et al., 1997; Bohlmann et al., 1999; Lichtenthaler, 1999). The hydrogen isotopic composition of NADPH produced by photosynthetic water fission in the plastid would be different from that produced by the oxidative pentose phosphate cycle of glucose con* Corresponding author. Tel.: +81 46 867 9778; fax: +81 46 867 9775. E-mail address: [email protected] (Y. Chikaraishi). 0146-6380/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.orggeochem.2009.02.007
version in the cytosol (Sessions et al., 1999; Schmidt et al., 2003). This proposal is supported by an experimental finding that starch in the plastid is depleted in D by up to 190‰ relative to cytosolic sugar (Luo and Sternberg, 1991). Alternatively, Chikaraishi et al. (2004a) suggested pronounced isotopic fractionation associated with hydrogenation during phytol biosynthesis. In fact, the hydrogenation is specific to phytol biosynthesis downstream of geranylgeranyl pyrophosphate (GGPP, a branch point between phytol and other diterpenoids; Fig. 1) and it was also observed that phytol is depleted in D by up to 65‰ relative to other diterpenoids (Chikaraishi et al., 2004a). Thus, the reason for the strong D depletion in phytol remains uncertain; it is considered to reflect either the utilization of isotopically distinct pools of NADPH or isotopic fractionation during hydrogenation, or both. Phytol is primarily biosynthesized as the phytyl side chain of chlorophylls by two distinct pathways at different plastid stages in plants (Fig. 2; e.g. Thomson and Whatley, 1980; Keller et al., 1998; Schoefs and Bertrand, 2000; Sibata et al., 2004). At the etioplast stage, GGPP is first esterified to chlorophyllide and the product chorophyllide-geranylgeraniol is successively hydrogenated to ultimately form chorophyllide phytol (i.e. chlorophyll a), as a result of a scarcity of NADPH or low activity of GGPP reductase. The etioplast stage is found in cotyledons (seed leaves) at the beginning of greening, but is not usually found in green leaves themselves (e.g. Thomson and Whatley, 1980). In green leaves (i.e. at the chloroplast stage), GGPP is first converted to phytyl pyrophosphate and finally esterified with chlorophyllide to form chlorophyll a. However, the hydrogenations should be controlled by the same enzymes for both pathways (e.g. Tanaka et al., 1999).
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Fig. 1. Compartmentation of isoprenoid biosynthesis in higher plants between plastid and cytosol (after Lichtenthaler et al., 1997; Bohlmann et al., 1999; Lichtenthaler, 1999). Abbreviations: acetyl-CoA, acetyl coenzyme-A; FPP, farnesyl pyrophosphate; GA-3-P, D-glyceraldehyde-3-phosphate; GGPP, geranylgeranyl pyrophospate; GPP, geranyl pyrophospate; IPP, isopentenyl pyrophosphate; MEP, 2-C-methyl-D-erythritol-4-phosphate; MVA, mevalonic acid.
Comparison of the hydrogen isotopic composition of phytol and its precursors is straightforward in terms of understanding the isotopic signature of phytol in biological and geological samples. The three precursors—geranylgeraniol (GG), dihydrogeranylgeraniol (DHGG), and tetrahydrogeranylgeraniol (THGG)—can be observed in cotyledons simultaneously with phytol as saponified products, but not in green leaves. Therefore, in this study we determine the hydrogen isotopic compositions of phytol and its precursors in plant cotyledons and evaluate the fractionation of hydrogen isotopes during phytol biosynthesis. 2. Materials and methods Cucumber (Cucumis sativus, cv. Aonagajibai) cotyledons were cultivated under the conditions reported by Aarti et al. (2006, 2007). Seeds were soaked in water for ca. 4–5 h prior to sowing in vermiculite and were allowed to germinate for 4 days in complete darkness in a growth chamber at 22 °C. Seedlings were subjected to continuous white fluorescent light (40–45 lE m2 s1) for 4 h. Cotyledons were harvested and used for analysis. The cotyledons were frozen by liquid N2 and crushed to a fine powder before analysis. The preparation method employed for phytol and its precursors followed the procedure described by Chikaraishi et al. (2004b) and Chikaraishi and Naraoka (2006). In brief, the powdered cotyledons were saponified with KOH/CH3OH/H2O and extracted with CH2Cl2/CH3OH. The compounds were separated by way of silica gel column chromatography and urea adduction followed by acetylation with acetic anhydride/pyridine. They were identified and quantified using gas chromatography/mass spec-
trometry (GC/MS) with an Agilent Technologies 6890N GC/5973A MSD system. Quantification was based on comparison of peak areas with an external phytol standard. The components were further separated using AgNO3 silica gel column chromatography (3.5 cm 6 mm) following the improved procedure reported by Chikaraishi et al. (2004c, 2005a). Phytol and THGG were eluted in the first 2 ml followed by a further 2 ml CH2Cl2/ethyl acetate (98:2, v/v), respectively. DHGG and GG were eluted in the first 2 ml followed by a further 4 ml n-hexane/ethyl acetate (4:1, v/v), respectively. The recovery of the compounds following AgNO3 silica gel column chromatography was >95%. Hydrogen isotopic compositions were determined using GC/ pyrolysis/isotope ratio MS (GC/pyrolysis/IRMS) with an Agilent Technologies 6890N GC instrument interfaced to a Thermo Finnigan Delta plus XP IRMS instrument via a pyrolysis furnace (Chikaraishi et al., 2005b, 2007). Pyrolysis was performed in a microvolume ceramic tube with graphite at 1440 °C (Burgøyne and Hayes, 1998; Hilkert et al., 1999). Isotopic compositions are expressed in the dD notation relative to Vienna Standard Mean Ocean Water (VSMOW). The H3 factor was 3.95, with variation before and after measurements within 0.05. For calibration, three pulses of reference H2 gas (250‰) were introduced at the beginning and end of each measurement (Fig. 3b). An n-alkane mixture (C15–C36: 37 to 280‰) and phytol (313‰) with known dD values were used as standards and analyzed every four GC/pyrolysis/IRMS runs to confirm the reproducibility of the measurements. The isotopic compositions of the standards gave an analytical uncertainty of 3.7‰ for an m/z 2 intensity of 0.5–5.0 V. The memory effect (see Wang and Sessions,
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Fig. 2. Two distinct pathways for phytol biosynthesis at different plastid stages in plants (after Keller et al., 1998; Schoefs and Bertrand, 2000; Sibata et al., 2004). Abbreviations: Chl a, chlorophyll a; Chlide, chlorophyllide; Chlide-DHGG, chlorophyllide-dihydrogeranylgeraniol; Chlide-GG, chlorophyllide-geranylgeraniol; ChlideTHGG,chlorophyllide-tetrahydrogeranylgeraniol; DHGGPP, dihydrogeranylgeranyl pyrophospate; PPP, phytyl pyrophospate; THGGPP, tetrahydrogeranylgeranyl pyrophospate.
Fig. 3. (a) Total ion chromatogram (TIC) for phytol and its precursors obtained from GC/MS analysis and (b) m/z 2 chromatograms for each compound obtained from GC/C/ IRMS analysis.
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2008) on the values was not substantial. The sample phytol and THGG were analyzed in triplicate, with 1r of 3.2 and 2.4‰, respectively. Because of their low abundance in the extract, DHGG and GG were concentrated 20 times and the isotopic composition of each was determined in a single analysis (Fig. 3). The isotopic composition was corrected for the contribution of hydrogen incorporated during acetylation using an isotope mass balance calculation: the isotopic composition of the incorporated acetate hydrogen (204‰) was obtained during the acetylation of alcohol standards with known dD values, including n-alkanols, phytol and sterols (Chikaraishi et al., 2004b). We also determined the hydrogen isotopic compositions of leaf water and other typical lipids (i.e. fatty acids and sterol), according to the procedures described by Chikaraishi et al., 2004b,c, 2005b. 3. Results and discussion Phytol and the three precursors (THGG, DHGG, and GG) occur in a relative abundance of 87.7, 7.6, 2.3 and 2.4 mol%, respectively (Fig. 3a). This is consistent with the general distribution of the corresponding chlorophylls in plant cotyledons (e.g. Keller et al., 1998; Schoefs and Bertrand, 2000; Sibata et al., 2004). As summarized in Table 1, phytol (345‰) is significantly depleted in D relative to leaf water (52‰) in the cotyledons, whereas fatty acids (132‰ to 174‰) and b-sitosterol (278‰) are less depleted. This isotopic distribution and fractionation is almost consistent with those in plant leaves generally (e.g., Chikaraishi et al., 2004b), even though the cotyledons have little activity of photosynthesis. The hydrogen isotopic compositions of GG, DHGG, THGG and phytol are 281‰, 302‰, 325‰, and 345‰, respectively (Table 1), showing a gradual D depletion from GG to phytol. This strongly suggests pronounced isotopic variation even within the plastid terpenoids, including a lack of significant D depletion in the phytol precursors (especially GG, which has essentially the same dD value as b-sitosterol). Phytol and its precursors are clearly biosynthesized with the same pools of hydrogen source in the plastid (Fig. 1). The only difference between them is in hydrogen incorporation during hydrogenation (Fig. 2); therefore, this gradual D depletion can be ascribed to the incorporation of significantly Ddepleted hydrogen into GG to form DHGG, into DHGG to form Table 1 Hydrogen isotopic composition of phytol, its precursors, other lipids and leaf water from cucumber cotyledons. Compound
Abbreviation
Hydrogen isotopic composition (‰, vs. VSMOW) Whole molecules
Phytol and precursors Phytol Tetrahydrogeranylgeraniol Ditrahydrogeranylgeraniol Geranylgeraniol Other lipids Palmitic acid Stearic acid a-Linolenic acid b-Sitosterol Leaf water
THGG DHGG GG C16:0 C18:0 C18:3, n3 29D5
Incorporated hydrogen
dD
1r
dDinHa
rinHb
345 325 302 281
3.2 (n = 3) 2.4 (n = 3) n.d. (n = 1) n.d. (n = 1)
715 728 649
79 81 92
174 132 176 278 52
0.6 1.2 0.9 1.5 3.0
(n = 3) (n = 3) (n = 3) (n = 3) (n = 9)
THGG, and finally into THGG to form phytol. Assuming isotopic mass balance, the isotopic composition of the incorporated hydrogen is estimated by to be 715 to 649‰ for each hydrogenation step (Table 1). The incorporated hydrogen is probably derived from NADPH and H+ (cellular water) in the plastid. Although the isotopic compositions of NADPH and H+ were not determined, Schmidt et al. (2003) reported net values of ca. 250‰ for NADPH. Also, it is likely that the isotopic composition of leaf water is similar to that of H+. Taking into account the large error in the estimated isotopic composition of incorporated hydrogen (as large as 90‰, Table 1), we roughly estimate the isotopic fractionation factor (a) based on the isotopic compositions of incorporated hydrogen, NADPH and H+ of 700‰, 250‰ and 50‰, respectively. Since two hydrogens are incorporated during each reduction (probably one from NADPH and another one from H+), a is estimated to be ca. 0.35 from the following equation:
a ¼ ðdDinH þ 1000Þ=fðdDNADPH þ dDHþ Þ=2 þ 1000g Thus, significant isotopic fractionation represents a plausible explanation for the gradual D depletion observed from GG (281‰) to phytol (345‰). In the chloroplast, the NADPH is initially strongly depleted in D (up to 600‰) by way of a large isotope effect during the reduction of NADP+ to NADPH in photosynthesis but finally enriched in D to around 250‰ (as a net value) as a result of other isotope effects and exchange during various substrate reactions (e.g. Luo et al., 1991; Hayes, 2001); however, it is relevant for green leaves that are actively photosynthesizing and the isotopic composition of NADPH in the cotyledons is unknown. It is also likely that NADPH is mainly processed via the oxidative cycle of starches stored in the seed. If the initially produced NADPH with a value as low as 600‰ is quantitatively utilized for the hydrogenation of phytol precursors in the cotyledons, a large isotopic fractionation (a ca. 0.35) is not necessary for explaining the gradual D depletion from GG to phytol. Thus, although we cannot rule out the potential importance of a utilization of isotopically distinct pools of NADPH, the gradual D depletion from GG (281‰) to phytol (345‰) clearly indicates that significantly D-depleted hydrogen is incorporated stepwise during hydrogenation from GG to phytol. Since the hydrogenations should be controlled by the same enzymes for both cotyledons and green leaves (e.g. Tanaka et al., 1999), the phenomenon probably provides a general explanation for the significant D depletion in plant phytol. Various compounds undergo hydrogenation (or dehydrogenation) along their biosynthetic and metabolic pathways. If the above process is applicable, hydrogenation products could be depleted in D relative to their producers (as with phytol), whereas dehydrogenation agents may be enriched relative to their products. Indeed, a large difference in isotopic composition is observed among the different degrees of unsaturation of fatty acids in marine macroalgae (Chikaraishi et al., 2004c) and alkenones in cultured Emiliania huxleyi, suspended particles and sediments (D’Andrea et al., 2007; Schwab and Sachs, 2009). Thus, isotopic fractionation associated with hydrogenation and dehydrogenation is likely to be an important factor in explaining the wide variation in hydrogen isotopic composition observed among compounds in biological and geological samples. Acknowledgments
a
Hydrogen isotopic composition of hydrogens incorporated during hydrogenation (dDinH) calculated with an isotope mass balance dDinH = (dDproduct nproduct dDprecursor nprecursor)/2; n is the number of hydrogen atoms. b Error in estimated dDinH value: r2inH ¼ 1r2product ðnproduct =2Þ2 þ 1r2precursor ðnprecursor =2Þ2 ; 1r for DHGG and GG is used as 1r of standard analysis (3.7‰, see text).
We are grateful to N.O. Ogawa, Y. Kashiyama, Y. Takano and H. Kitazato for expert advice and constructive discussion. Hydrogen isotope measurement of leaf water using TCEA/IRMS was kindly supported by K. Tamura and N. Kurita (JAMSTEC, IORGC). We thank
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