Phytochemistry 72 (2011) 207–213

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Phytochemistry

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Temperature effect on leaf water deuterium enrichment and isotopic fractionationduring leaf lipid biosynthesis: Results from controlled growthof C3 and C4 land plants

Youping Zhou a,b,c,d,⇑, Kliti Grice a, Yoshito Chikaraishi e, Hilary Stuart-Williams b, Graham D. Farquhar b,Naohiko Ohkouchi e

a WA-Organic and Isotopic Geochemistry Centre, Department of Chemistry, Curtin University, Perth, Australiab RSB, Australian National University, Canberra, ACT 2601, Australiac State Key Laboratory of Loess and Quaternary Geology, IEE, CAS, Xi’an 710075, Chinad RSES, Australian National University, Canberra, ACT 0200, Australiae Institute of Biogeosciences, JAMESTEC, Natushima-cho 2-15, Yokosuka, Kanagawa 237-0061, Japan

a r t i c l e i n f o

Article history:Received 22 December 2009Received in revised form 1 October 2010Available online 15 December 2010

Keywords:Leaf waterLipidsHydrogen isotopeTemperature effectIsotope fractionation

0031-9422/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.phytochem.2010.10.022

⇑ Corresponding author at: WA-Organic and IsoDepartment of Chemistry, Curtin University, Perth,9792; fax: +61 8 9266 3547.

E-mail address: y.zhou@curtin.edu.au (Y. Zhou).

a b s t r a c t

The hydrogen isotopic ratios (2H/1H) of land plant leaf water and the carbon-bound hydrogen of leaf waxlipids are valuable indicators for climatic, physiological, metabolic and geochemical studies. Temperaturewill exert a profound effect on the stable isotopic composition of leaf water and leaf lipids as it directlyinfluences the isotopic equilibrium (IE) during leaf water evaporation and cellular water dissociation. It isalso expected to affect the kinetics of enzymes involved in lipid biosynthesis, and therefore the balance ofhydrogen inputs along different biochemical routes. We conducted a controlled growth experiment toexamine the effect of temperature on the stable hydrogen isotopic composition of leaf water and the bio-logical and biochemical isotopic fractionations during lipid biosynthesis. We find that leaf water 2Henrichment at 20 �C is lower than that at 30 �C. This is contrary to the expectation that at lower temper-atures leaf water should be more enriched in 2H due to a larger equilibrium isotope effect associated withevapotranspiration from the leaf if all other variables are held constant. A hypothesis is presented toexplain the apparent discrepancy whereby lower temperature-induced down-regulation of availableaquaporin water channels and/or partial closure of transmembrane water channel forces water flow to‘‘detour’’ to a more convoluted apoplastic pathway, effectively increasing the length over which diffusionacts against advection as described by the Péclet effect (Farquhar and Lloyd, 1993) and decreasing theaverage leaf water enrichment. The impact of temperature on leaf water enrichment is not reflected inthe biological isotopic fractionation or the biochemical isotopic fractionation during lipid biosynthesis.Neither the biological nor biochemical fractionations at 20 �C are significantly different from that at30 �C, implying that temperature has a negligible effect on the isotopic fractionation during lipidbiosynthesis.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

The hydrogen isotope ratio (2H/1H) of carbon-bound hydrogenin biosynthetic lipids of land plants is a valuable indicator for anumber of applications such as climate, hydrological and vegeta-tion reconstruction (Hou et al., 2008; Huang et al., 2004; Jiaet al., 2008; Krull et al., 2006; Li et al., 2009; Liu et al., 2005; Sachset al., 2009; Sachse et al., 2006; Sauer et al., 2001), organic mattersource provenancing (Englebrecht and Sachs, 2005; Chikaraishi

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topic Geochemistry Centre,Australia. Tel.: +61 8 9266

and Naraoka, 2005), assessment of thermal maturity (Dawsonet al., 2005, 2007; Schimmelmann et al., 2006), elucidation of met-abolic pathways and discrimination of bacterial life styles (Camp-bell et al., 2009; Hayes, 2001; Kreuzer-Martin et al., 2006;Schmidt et al., 2003; Sessions et al., 1999 ; Valentine et al., 2004;Zhang et al., 2009a), and physiological studies linking leaf evapora-tion to water utilisation efficiency (Farquhar and Gan, 2003; Yakiret al., 1990). Interpretation of the isotopic signal of lipid moleculesfrom land plants for such applications is, however, hindered by alack of a clear understanding of the biotic factors such as spe-cies-specific boundary layer and stomatal resistance, leaf morphol-ogy and venation pattern (Farquhar and Gan, 2003), biochemicalreactions leading to the incorporation of hydrogen into organicmolecules (Chikaraishi et al., 2009; Sessions et al., 1999), synchro-

208 Y. Zhou et al. / Phytochemistry 72 (2011) 207–213

neity/asynchroneity of source water and biochemical synthesis oflipid molecules (Sessions, 2006), the photosynthetic mode of theplant (Chikaraishi et al., 2004; Smith et al., 2006; Sternberg et al.,1984), the biosynthetic pathways by which the lipid moleculesare made (Chikaraishi et al., 2004; Grice et al., 2008; Sessionset al., 1999; Zhou et al., 2010a,b) and abiotic factors such as the iso-topic composition of the source water, relative humidity andtemperature.

Of all these factors, temperature is expected to exert a profoundeffect on the stable isotopic composition of lipids synthesised byplants, with the individual fractionation factors generally decreas-ing with increasing temperature (i.e. the gas and liquid phases be-come more isotopically similar). Firstly, temperature controls theliquid–gas equilibrium isotope effect during leaf evapotranspira-tion. Secondly, it presumably reduces the isotopic depletion of H+

as one of the products of water dissociation (Luo et al., 1991),which is used for the generation of NADPH and for incorporationinto lipid molecules during complex biochemical reactions leadingto the synthesis of lipids. Thirdly, it affects directly, or indirectly,the isotopic fractionations of enzyme-catalysed biochemical reac-tions by changing the kinetics of the enzymes and altering the bal-ance of various pathways. Fourthly, even for those biochemicalsteps where enzymes are expected to play no role at all, the rateof isotopic exchange among metabolic intermediates and water,such as keto-enol tautomerism (Wang et al., 2009) is expected tobe temperature-dependent. Lastly, the water flow path (and there-fore, the average leaf water isotopic composition due to the Pécleteffect according to Farquhar and Lloyd, 1993) can be indirectly af-fected by temperature, as aquaporin (water channel protein) in thecellular membrane may be impacted by temperature as well. For arigorous use of hydrogen isotopic ratios, it is important to establishhow temperature affects leaf water enrichment and to establishwhich enzymes are involved in the synthesis of a particular lipid.Ideally isotope effects should be directly and quantitatively linkedto particular biophysical processes or biochemical reactions inplants. Although the sheer number of reactions and enzymes thatcatalyse the synthesis of lipid molecules and the size of the meta-bolic network in the lipid synthesis make it extremely difficult, ifnot impossible, to understand the effect of temperature on leafwater isotopic enrichment and overall biosynthetic isotopic frac-tionation, it is still valuable to make the attempt.

In this paper, we report the 2H/1H ratios of leaf water and lipidssynthesised by C3 and C4 plants grown under different temperatureregimes. Our results show that temperature does have a significantimpact on leaf water isotopic enrichment but that this is not evi-dent in the isotopic composition of leaf lipids.

2. Results and discussion

2.1. The effect of temperature on leaf water hydrogen isotopicenrichment

The isotopic compositions of the irrigation water, soil water, va-pour, leaf water are listed in Table 1. At a daytime temperature of20 �C, the d2H of observed leaf water is 3–12‰ (depending on spe-cies) lower than at 30 �C. This is contrary to the expectation thatlower temperature would translate to a higher enrichment of theleaf water due to increased fractionation between the liquid and va-pour phases. What causes the discrepancy? To answer this question,we looked at the isotopic spatial heterogeneity of the leaf water.

At the site of evaporation, leaf water isotopic enrichment oversource water De due to differential diffusion of 1H1HO and 2H1HOcan be modelled according to Craig and Gordon (1965):

De ¼ ee þ ek þ Dv � ek� �

� ea

ei

� �� �� ee þ 1½ � ð1Þ

where Dv is the isotopic enrichment of vapour over source water, ea

and ei are vapour pressures in the atmosphere immediately aroundthe leaf and intercellular spaces, respectively, ee is the equilibriumfractionation factor at the temperature of evaporation, ek is the ki-netic fractionation factor and depends on the boundary layer andstomatal resistances to gaseous molecular diffusion.

Since leaf water is spatially heterogeneous due to advection ofenriched water at the evaporative site toward the xylem wherewater is unenriched (the Péclet effect), the average leaf laminawater enrichment Dl is lower than De. According to Farquhar andLloyd (1993):

Dl ¼ Deð1� expð�}ÞÞ=} ð2Þ

where } ¼ EL=CD is the Péclet number, E is the evaporation rate, L isthe gradient length (the scaled effective path length of water flowbetween the xylem and sites of evaporation), C is the molar concen-tration of water (55.5 � 103 mol m�3), and D is the diffusivity of2H1HO in water (2.34 � 10�9 m2 s�1, a diffusivity change in re-sponse to temperature change from 20 to 30 �C (Mills, 1973) istranslated to a negligible 4% change in the estimates of effectivelength L).

According to Eq. (1), other things being equal, a higher temper-ature should result in a lower De due to a smaller ee. This results ina reduced depletion of the vapour relative to the liquid and a con-sequent reduction of isotopic enrichment of liquid remaining afterevaporation. As a higher temperature is also accompanied by ahigher E, this should result in reduced leaf water enrichment as aresult of the Péclet effect. The combined effect should result in alower Dl for higher temperatures. Thus the higher enrichment ofleaf water Dl at higher temperature may be a result of smaller gra-dient length L. This means that temperature could have an effect onthe gradient length, although the higher temperature plants alsohad higher E, and perhaps this reduces L.

We hypothesise here that the cause of the change in L is mostlikely due to changes in the transpiration and/or temperature-sen-sitive aquaporin-based water channels. Water flow in the leaf cantake either an apoplastic or a symplastic route. In an apoplasticroute, water flows along a continuum of cell walls of adjacentcells as well as the extracellular spaces. In a symplastic route,water diffusion is through either plasmodesmata or transmem-brane water channel protein, aquaporin (Barbour et al., 2000;Heinen et al., 2009; Steudle and Peterson, 1998). As the path-length of apoplastic water flow is set by the geometry of the cellwall, it is therefore not expected to change significantly due to therigidity of the cell wall. The pathlength of symplastic water flow,in particular the flow through aquaporins is, however, expected tochange significantly due to regulation of aquaporins in responseto environmental stimuli. As apoplastic flow is essentially a flowthat ‘‘flows around’’ the cell wall while symplastic flow is a flowthat ‘‘flows through’’ cytoplasm, it is expected to have a longerpathlength than the latter. At lower temperatures, the down-reg-ulation of genes responsible for aquaporin synthesis reduces thenumber of available water channels and/or partially closes thechannels (Luu et al., 2005; Wei et al., 2006; Maurel et al., 2008),forcing water to ‘‘detour’’ via the longer apoplastic route increas-ing the average pathlength of water flow. In fact, a recent obser-vation of an inverse relationship between hydrogen fractionation(between lipids and environmental water) and salinity underwhich the lipids are produced by cyanobacteria (Sachse et al.,2008) highlighted the role of aquaporin in regulating transmem-brane water flow and lends circumstantial support for ourhypothesis here. Similarly, perhaps greater E causes relatedeffects. This would explain the inverse relationship sometimesobserved between L and E (Zhou, 2005, PhD thesis).

Y. Zhou et al. / Phytochemistry 72 (2011) 207–213 209

2.2. The effect of temperature on lipid isotopic composition

Before we discuss the effect of temperature on lipid isotopiccomposition, we need to know the isotopic composition of thewater used for the biosynthesis of lipids. Since biosynthesis of lip-ids occurs in the cytoplasm, lipids are likely to record averagemesophyll lamina water dl. As physical isolation of leaf laminawater is impossible, bulk leaf water db (which includes contribu-tions of water from lamina, veins and xylem and ground tissues)rather than average lamina water is sampled. Therefore, the contri-bution of non-lamina water should be quantified in order to cor-rect the lamina water isotopic composition.

Neglecting the negligible contribution of veins (<1%, Gan et al.,2002), bulk leaf water (which includes water in veins, lamina andxylem and ground tissue) enrichment Db can be modelled accord-ing to Farquhar and Gan (2003):

Db � De /xe�}r þ ð1� /xÞ1� e�}

}

� �ð3Þ

where /x is the proportion of leaf water associated with the xylemand ground tissue, }r is the Péclet effect associated with radial flow(i.e. from the veins to the sites of evaporation) gradients in enrich-ment in the veinlets (}vr) and the lamina mesophyll (}), }r = } + }rv.As Db, De, and E are either directly measured or calculated from airtemperature (Tair), leaf temperature (Tl) and relative humidity(RHair), estimation of L is therefore dependent on /x, }r. When /x,}r are varied within their respective likely ranges (/x < 0.3,e�}r < 0:2De according to Cernusak et al. (2003), Gan et al. (2002,2003), Barbour and Farquhar (2005, unpublished results), E and Lpresent a negative correlation R(E, L), which is a function of /x, }r.R(E, L) and decreases with increasing /x while remaining much lesssensitive to }r. As L is a species-specific property and largely deter-mined by leaf anatomy, the actual /x should be represented by thepoint where R(E, L) becomes insignificant. Using the critical R(E, L)at confidence level P (P < 0.05), the estimated /x for each specieswithin the range of }r used here are 0.22–0.25 for S. bicolor, 0.15–0.17 for Z. mays, 0.02–0.02 for P. coloratum, 0.04–0.05 for G. hirsu-tum, 0.23–0.26 for N. tabacum and 0.13–0.15 for R. communis. Usingthe estimate /x and L, the corrected lamina leaf water enrichmentover source water (here refers to the source water, i.e. irrigationwater) Dcor according to Eq. (3) is presented in Table 1 togetherwith observed leaf water enrichment DL and modelled enrichmentDe.

The biological ebiol and biochemical ebiochem isotopic fractiona-tions are calculated using:

ebiochem ¼d2Hlipid þ 1000d2Hcor þ 1000

� 1

!� 1000‰

ebiol ¼d2Hlipid þ 1000d2Hirg þ 1000

� 1

!� 1000‰

The calculated ebiol and ebiochem are presented in Table 1. For allthe species investigated here, there is no difference in ebiol and ebio-

chem among the three sterols synthesised via the mevalonic (MVA)pathway if the overall analytical error is taken into account. Thedifference in ebiol and ebiochem between 20 and 30 �C is also statisti-cally insignificant. The same is true for phytol, the diterpene syn-thesised via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway.For the n-alkanes synthesised via the acetogenic (ACT) pathway,ebiochem for individual n-C27 and n-C29 or their weighted averagedof N. tabacum is higher at 30 �C, but ebiol shows no difference withinanalytical error while for n-C29 to n-C33 or their weighted averageof Z. mays, ebiochem and ebiol are lower at 30 �C by 29‰ and 25‰,respectively. Since there is only one C3 and one C4 species for

which we have d2Y data, we are uncertain if the observed apparentdifference between N. tabacum and Z. mays represents the differ-ence between C3 and C4 plants. If it does, the absence of tempera-ture effect on sterols and phytol and the presence of a temperatureeffect (although small) on n-alkanes suggest that temperature mayhave a different effect on the enzymes involved in the DXP, MVAand ACT pathways.

The 3–12‰ d2H difference in observed leaf water dL betweenthe 30 and 20 �C treatment is not reflected in ebiol. This means thereis a similar, albeit small, biochemical fractionation ebiochem differ-ence between the 30 and 20 �C treatments which offsets the dL dif-ference. However as the overall error for ebiol is 4–7‰, it could alsobe that that real ebiol difference is masked by the analytical error.

There is a lack of data on the effect of temperature on isotopicfractionation in higher plant leaf lipid biosynthesis, although thereare four papers (Stiller and Nissenbam, 1980; Estep and Hoering,1981; Schouten et al., 2006; Zhang et al., 2009b) to our knowledgethat directly address the effect of temperature on hydrogen isoto-pic fractionation during biosynthesis of organic matter in freshwater or marine algae and phytoplankton. The isotopic analysesin the first two were conducted on bulk organic matter and makedirect comparison with our results impossible. Are algae and phy-toplankton good higher plant analogues in terms of photosynthesisand lipid biosynthesis? Can we assume that there is a constant d2Hoffset between bulk organic matter and lipids in these plants, andthat the isotopic composition of water used for biosynthesis isequal to that of the plants’ environmental water? If so, then ourfinding of a statistically negligible effect of temperature on landplant leaf sterols is at odds with the suggestion in these papers thata higher temperature is associated with a greater net isotopic frac-tionation and therefore a lower lipid d2H.

3. Concluding remarks

Despite the fact that the isotopic ratios (2H/1H) of carbon-boundhydrogen in the lipids of plants have been used widely in climatic,vegetational and hydrological studies, the effect of environmentalfactors such as temperature, humidity, light level, CO2/O2 ratioand water supply on this parameter is largely unknown. For a ro-bust and unambiguous interpretation of the isotopic signaturesof lipids retrieved from ancient samples these effects need to bemeasured.

An abiotic factor, temperature, is expected to have an impact onthe isotopic composition of leaf water as EIE is temperature-depen-dent during leaf water evaporation. It is also expected to have animpact on the isotopic fractionation during organic molecule syn-thesis as the isotopic effect of water dissociation to produce H+

used for NADPH generation and other biochemical reactions is alsotemperature-dependent. We therefore conducted controlledgrowth experiment to examine the temperature effect on leafwater enrichment and isotopic fractionation during leaf lipid bio-synthesis. Our results show that the Péclet-effect-corrected leaflamina water enrichment at 20 �C is less than at 30 �C, contraryto the expectation that a lower temperature should result in great-er enrichment. We propose that the discrepancy is a result of tran-spiration or temperature-induced changes in transmembraneaquaporin transport, as aquaporins are the water channel proteinresponsible for direct cell-to-cell water movement. Lower temper-ature or lower evaporation may reduce the number and permeabil-ity of aquaporin channels, forcing water to ‘‘detour’’ to a moreconvoluted pathway. As the Péclet effect which dampens the aver-age lamina water isotopic enrichment is positively correlated tothe effective gradient length between the site of evaporation andxylem, a longer water flow path means less enrichment. The im-pact of temperature on leaf water enrichment was, however, not

Table 1Growth conditions and d2Y of soil water, leaf water, vapour and lipids of the temperature experiment. EIE/KIE: equilibrium/kinetic isotope effect; BLR/SR: boundary layer/stomatal resistance; Rel. Humid.: relative humidity; Transp. Rate:transpiration rate; the analytical error for water d2Y and lipids is 3‰. The measurement errors of temperature, humidity, boundary and stomatal resistances, water and lipids are propagated into leaf water enrichment and lipid isotopicfractionation calculation. The overall error (DY) of leaf water enrichment and lipid isotopic fractionation is calculated using DY ¼

Pn1j

@f@pnjDpn , where Y = f(p1, p2, . . ., pn) is the leaf water enrichment or isotopic fractionations, P1, P2, . . ., Pn

are the parameters used for the calculation, DP1, DP2, . . ., DPn are the measurement errors for parameters P1, P2, . . ., Pn, respectively. The calculated overall error varies from 4‰ to 7‰ from species to species and from lipid to lipid. KIE iscalculated as ek = (25rs + 17rb(‰))/(rb + rs)(‰), according to Merlivat (1978). EIE is calculated as �e according to Araguás-Araguás et al. (1998); Dif: is the difference between 30 and 20 �C. Dob–mo: is the difference between observed andmodelled leaf water d2Y. C28D

5: campesterol (24-methylcholest-5-en-3b-ol); C29D5,22: stigmasterol (24-ethylcholesta-5,22-dien-3b-ol); C29D

5: b-sitosterol (24-ethylcholest-5-en-3b-ol); Dob–mo: difference between observed andmodelled d2Y.

Treatment Latin names Temp. Rel. Humid. EIE BLR SR Transp. Rate KIE Vapour Leaf water d2H (‰) Leaf water 2H enrichment (‰)

�C % ‰ m2 s1 mol�1 mmol m�2 s�1 ‰ ‰ Observed Modelled Dob–mo Modelled Observed Corrected

Tair Tleaf RHair RHleaf ee Dif rb rs E ek Dif d2HV d2HL Dif d2He Dif D2He Dif D2HL Dif D2Hcor Dif d2Hcor

High temp. S. bicolor 30 32 70 58 69 2.7 15.5 1.3 24 �84 6.2 16 9.8 52 40 50 13Z. mays 30 32 70 60 69 5.4 8.5 2.3 22 �84 13.3 14 0.7 50 49 56 19P. coloratum 30 32 70 62 70 3.3 9.7 2 23 �84 8 13 6 49 42 43 6G. hirsutum 30 31 70 61 71 2.4 3.1 4.7 22 �84 14.4 15 0.6 50 50 51 14N. tabacum 30 31 70 60 71 0.9 3.7 3.9 23 �84 10.3 14 3.7 55 52 67 22R. communis 30 30 70 65 71 2.5 1.7 6.8 20 �84 8.1 10 1.9 51 50 57 13

Moderate temp. S. bicolor 20 21 68 62 80 �11 1.2 14.6 0.7 24 �1 �81 2.9 3 26 �10 23.1 61 �10 38 2 47 3 10Z. mays 20 22 68 62 80 �10 1.2 13.4 0.8 24 �2 �81 1.1 12 23 �9 21.9 64 �15 42 6 48 8 5P. coloratum 20 22 68 60 80 �10 1.2 5.7 0.8 24 �1 �81 5.4 3 26 �12 20.6 63 �15 43 �1 44 �1 5G. hirsutum 20 22 68 63 80 9 0.6 5.7 1.7 24 �3 �81 1.9 12 22 �7 20.1 66 �16 46 4 47 4 1N. tabacum 20 21 68 63 80 �10 0.7 14.7 0.7 25 �1 �81 3.4 7 24 �11 20.6 63 �8 42 10 53 13 12R. communis 20 21 68 61 81 �9 0.5 4.7 1.9 24 �4 �81 0.5 8 27 �17 26.5 62 �11 36 14 40 17 3

n-Alkanes (‰) Sterols (‰) Phytol (‰)

C27 C29 C31 C33 wt. avg. C28D5 C29D

5,22 C29D5 wt. avg.

d2H ebiochem ebiol d2H ebiochem ebiol d2H ebiochem ebiol d2H ebiochem ebiol d2H ebiochem ebiol d2H ebiochem ebiol d2H ebiochem ebiol d2H ebiochem ebiol d2H ebiochem ebiol d2H ebiochem ebiol

High temp. S. bicolor �182 �194 �152 �182 �194 �152 �180 �192 �150 �181 �193 �151 �269 �280 �242Z. mays �158 �175 �127 �166 �183 �135 �164 �181 �134 �163 �180 �132 �190 �206 �160 �203 �219 �174 �192 �209 �163 �195 �211 �165 �266 �281 �240P. coloratum �198 �204 �168 �195 �202 �166 �189 �195 �159 �194 �200 �165 �275 �280 �248G. hirsutum �223 �236 �195 �223 �236 �195 �354 �364 �330N. tabacum �120 �142 �82 �136 �157 �98 �128 �149 �90 �231 �250 �198 �232 �250 �198 �240 �259 �207 �235 �253 �201 �353 �369 �325R. communis �222 �234 �188 �209 �221 �174 �215 �227 �181 �339 �349 �310

Moderate temp. S. bicolor �183 �193 �153 �187 �196 �157 �183 �193 �153 �184 �194 �155 �268 �277 �242Z. mays �133 �140 �96 �146 �152 �109 �153 �159 �116 �144 �150 �108 �187 �193 �152 �204 �210 �170 �199 �205 �165 �197 �202 �162 �260 �265 �228P. coloratum �196 �201 �164 �196 �201 �165 �185 �190 �153 �192 �197 �161 �277 �282 �249G. hirsutum �225 �227 �189 �225 �227 �189 �349 �351 �319N. tabacum �137 �149 �102 �145 �157 �110 �141 �153 �106 �238 �249 �207 �240 �250 �209 �240 �251 �209 �239 �250 �209 �361 �370 �335R. communis �225 �229 �197 �206 �210 �177 �215 �219 �187 �335 �338 �311

Dif = High �Moderate S. bicolor 3 0 3 �1 �3 �1Z. mays �18.4 �29.4 �24.7 2 �9 �3 �7 �17 �12P. coloratum �2 �3 �4 2 1 1G. hirsutum 2 �8 �6 �5 �13 �11N. tabacum 12.7 3.9 16.2 5 �3 7 8 1 10R. communis 0 �8 6 �4 �10 1

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Y. Zhou et al. / Phytochemistry 72 (2011) 207–213 211

reflected significantly in either biological or biochemical isotopicfractionations during lipid biosynthesis.

4. Experimental

4.1. Growth experiment

Experiments were conducted in environment-controlledgrowth cabinets at the Australian National University. Seeds ofthree species of C3 plants (Gossypium hirsutum, Nicotiana tabacum,Ricinus communis) and three species of C4 plants (Sorghum bicolor,Zea mays, Panicum coloratum) were germinated in 10L plastic potsfilled with potting mix and fertilized with Osmocote (Osmocote,Sierra Horticultural Products, The Netherlands). Plants were wa-tered at 9 am and 2 pm from a sealed reservoir of Canberra tapwater (d2Hirg = �34‰). The temperature and relative humidity in-side the cabinets was continuously monitored and controlledwhilst mixing of air was achieved by forced circulation. To mini-mise change in the soil water isotopic composition due to evapora-tion from the soil surface, the soil was covered with a layer (5 cmthick) of 2–3 mm polyethylene beads. Growth conditions were setas follows: 12 h photoperiod (8 am to 8 pm), 70% relative humidity,400 ppm [CO2], 780 lmol photons m�2 s�1 for PAR (Photosynthet-ically Active Radiation). Day/night time temperatures for the hightemperature and ‘‘moderate’’ temperature cabinets were set at30 �C/18 �C (higher temperature treatment) and 20 �C/15 �C (mod-erate temperature treatment), respectively (Zhou, 2005, Ph.D.thesis).

4.2. Leaf water, soil water and vapour sampling, extraction andisotopic analysis

Leaf water was sampled between the 26th and 28th day ofgrowth corresponding to the period of fastest growth and biomassaccumulation. As chamber temperature and relative humidity, twoof the factors that determine the isotopic fractionation during leafevapotranspiration and the isotopic compositions of irrigationwater were relatively constant throughout the growth experiment,the sampled and measured leaf water is a better representation ofthe average isotopic composition of water used for biosynthesis ofthe lipid. Sampling was conducted at approximately 1 pm, 4 h afterthe morning watering (Roden et al., 1999). Fully expanded leavesfrom randomly chosen plants were either torn off (for C4 plants)or cut off with scissors (for C3 plants) as quickly as possible (within1 min) along the central main veins and placed in pre-chilledscrew-cap culture tubes and kept frozen until the water was ex-tracted by vacuum distillation using the method of Gan et al.(2002).

Vapour inside the cabinets was collected by pumping through adry ice-ethanol cold-trap at a rate of 2 L/min for 30–70 min. Mois-ture was sampled after watering around 9 am After 2 h to allow thegas exchange to stabilise, one sample was taken every 2 h until5 pm.

Soil water sampling was conducted in a similar way to leafwater sampling and on the same day as when the leaf water wassampled. A pre-chilled culture tube used for leaf water samplingwas pushed into the soil to a depth of 5 cm. Samples were thencapped and sealed with Parafilm and kept frozen until the waterwas extracted using vacuum distillation (Gan et al., 2002). To min-imise the inter-pot differences, for each species for a particulartemperature treatment, three soil and three leaf samples were ta-ken from three randomly choose pots. After water extraction, thewaters were then combined to produce an ‘‘average’’ sample.

Water isotopic composition was determined in the Environ-mental Biology stable isotope laboratory at the ANU according to

stuart-Williams et al. (2008). Hydrogen gas was produced fromthe water by reaction on chromium at 1050 �C. Approximately0.7 lL samples were injected into the reaction column using a Fi-sons Instruments AS800 autosampler. The H2 produced was carriedthrough a 1.5 m Porapak QS packed column with helium carrier. Asthe gas produced in the reaction is pure H2 no separation is neces-sary – the column serves mainly to shape the peak to providerepeatability during mass spectrometry. The dry hydrogen gaswas then analysed in an Isoprime stable isotope mass spectrome-ter. Reference gas was provided from the reference box and thedual-inlet bellows at different pressures to provide a small refer-ence peak before each sample and a large one after, permittingaccurate and updated calculation of the Hþ3 correction. Analyseswere typically performed in triplicate with calibrated laboratoryreference waters run approximately every 15 analyses. Precisionon the runs was typically 0.5‰ with the reference waters cali-brated to better than 1‰.

4.3. Gas exchange and boundary layer conductance measurements

Gas exchange measurement was conducted with a Li-Cor 6400(Li-Cor Inc., Lincoln, NE, USA). All measurements were conductedbefore harvesting on attached and fully expanded leaves near thecanopy top within the same time window (11 am to 4 pm) duringwhich leaf gas exchange behaviour was constant. Boundary layermeasurements were conducted using the method of Gan et al.(2002) inside the growth chamber. Leaf surface temperature (Tl)was measured with an infrared thermometer (Infracouple ModelM50, Mikron Instruments, Oakland, NJ, USA). Air temperature (Tair)and relative humidity (RHair) close to each species was measuredwith a hand-held Vaisala temperature and humidity probe (VaisalaInc., Helsinki, Finland).

4.4. Leaf harvesting, lipid extraction and separation

All leaves from three randomly chosen plants were harvestedwhen plants had been grown under constant conditions for 42–45 days and all seed reserves were effectively depleted. Extractionof leaf waxes and separation of lipids from waxes was according toGrice et al. (2008). Briefly, dried and powdered leaves were ex-tracted with dichloromethane (DCM):methanol (MeOH) (9:1, v/v)in a Soxhlet apparatus (48 h) to obtain both aliphatic and polar lip-ids. The extracts were then chromatographically separated on sil-ica gel columns. n-Alkanes were collected in the n-hexanefraction and dried before chromatographic and stable isotopicanalyses. The controlled experiment showed no significant isotopicfractionation during the extraction and drying procedure. Sterolextraction, separation and acetylation were conducted followingthe method of Chikaraishi and Naraoka (2005).

4.5. Gas chromatography/mass spectrometry (GC/MS) and hydrogencompound-specific isotope analyses (CSIA) of lipids

GC/MS analyses of n-alkanes were carried out using an HP 6890GC/MS. Details of instrumental and analytical conditions are de-scribed in Grice et al. (2008). GC/MS analyses of phytol and sterolswere carried out using an HP 6890 GC connected to an HP 5972AMSD. n-Alkanes, phytol and sterols were identified by comparisonof their retention times and mass spectra to those of standards.CSIA of hydrogen was performed on a Micromass IsoPrime massspectrometer interfaced to an HP 6890N GC. n-Alkane mixtureswere first chromatographically separated by GC and then pyroly-sed to H2 at 1050 �C on a chromium-based catalyst packed in aquartz tube located between the GC and the IRMS ion source.The Hþ3 correction was determined and is reported in detail byDawson et al. (2004). CSIA of H for individual acetylated sterols

212 Y. Zhou et al. / Phytochemistry 72 (2011) 207–213

and phytol was carried out by GC/pyrolysis/IRMS using a FinniganDelta plus XP interfaced with an HP 6890 GC according to Chikarai-shi and Naraoka (2005).

Hydrogen isotopic compositions are reported using the stan-dard d-notation relative to V-SMOW (Vienna Standard Mean OceanWater): d‰ = (RSAMPLE � RSTANDARD)/RSTANDARD � 1000 where RSAM-

PLE and RSTANDARD are the isotopic ratios (2H/1H) of sample and stan-dard, respectively. Average values of at least three runs weredetermined with an analytical error better than 3‰ for all lipidsand water analysed. Corrections were also made for the hydrogenatoms introduced to the sterols and phytol as a result ofacetylation.

Acknowledgements

Technical support from Geoff Chidlow, Kieran Pierce and SueWang are gratefully acknowledged. K.G. was supported by anARC QEII fellowship and ARC Discovery Project series. Y.Z.acknowledges a Ph.D. scholarship from the Australian NationalUniversity and a Grant-in-Aid (SKLLQG0916) from the State KeyLaboratory of Loess and Quaternary Geology, Chinese Academy ofSciences. G.F. acknowledges ARC Discovery Project support.

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