Leaf water deuterium enrichment shapes leaf wax n-alkane δD values of angiosperm plants I: Experimental evidence and mechanistic insights

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Geochimica et Cosmochimica Acta 111 (2013) 39–49 www.elsevier.com/locate/gca

Leaf water deuterium enrichment shapes leaf wax n-alkane dD values of angiosperm plants I: Experimental evidence and mechanistic insights Ansgar Kahmen a,⇑, Enno Schefuß b, Dirk Sachse c a Institute of Agricultural Sciences, ETH Zurich, Switzerland MARUM – Center for Marine Environmental Sciences, University of Bremen, Germany c DFG-Leibniz Center for Surface Process and Climate Studies, Institute of Earth and Environmental Sciences, University of Potsdam, Germany Available online 10 September 2012 b

Abstract Leaf wax n-alkanes of terrestrial plants are long-chain hydrocarbons that can persist in sedimentary records over geologic timescales. Since meteoric water is the primary source of hydrogen used in leaf wax synthesis, the hydrogen isotope composition (dD value) of these biomarkers contains information on hydrological processes. Consequently, leaf wax n-alkane dD values have been advocated as powerful tools for paleohydrological research. The exact kind of hydrological information that is recorded in leaf wax n-alkanes remains, however, unclear because critical processes that determine their dD values have not yet been resolved. In particular the effects of evaporative deuterium (D)-enrichment of leaf water on the dD values of leaf wax n-alkanes have not yet been directly assessed and quantified. Here we present the results of a study where we experimentally tested if and by what magnitude evaporative D-enrichment of leaf water affects the dD of leaf wax n-alkanes in angiosperm C3 and C4 plants. Our study revealed that n-alkane dD values of all plants that we investigated were affected by evaporative Denrichment of leaf water. For dicotyledonous plants we found that the full extent of leaf water evaporative D-enrichment is recorded in leaf wax n-alkane dD values. For monocotyledonous plants we found that between 18% and 68% of the D-enrichment in leaf water was recorded in the dD values of their n-alkanes. We hypothesize that the different magnitudes by which evaporative D-enrichment of leaf water affects the dD values of leaf wax n-alkanes in monocotyledonous and dicotyledonous plants is the result of differences in leaf growth and development between these plant groups. Our finding that the evaporative D-enrichment of leaf water affects the dD values of leaf wax n-alkanes in monocotyledonous and dicotyledonous plants – albeit at different magnitudes – has important implications for the interpretation of leaf wax n-alkane dD values from paleohydrological records. In addition, our finding opens the door to employ dD values of leaf wax n-alkanes as new ecohydrological proxies for evapotranspiration that can be applied in contemporary plant and ecosystem research. Ó 2012 Elsevier Ltd. All rights reserved.

1. INTRODUCTION The hydrogen and oxygen isotopic composition of plants (dD and d18O, respectively) contains hydrological ⇑ Corresponding author. Address: Institute of Agricultural Sci-

ences, ETH Zu¨ rich, Universita¨tsstrasse 2, LFW C55.2, 8092 Zu¨ rich, Switzerland. E-mail address: [email protected] (A. Kahmen). 0016-7037/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.gca.2012.09.003

information e.g., on the isotopic composition of precipitation or on plant water relations and have thus become important sources of information for environmental research (Dawson et al., 2002). To date most studies have employed the d18O values of plant cellulose to obtain hydrological information from plant materials (Barbour, 2007; Kahmen et al., 2011a). With the development of compound specific stable hydrogen isotope analysis (Burgoyne and Hayes, 1998; Hilkert et al., 1999) it is now possible to

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analyze the dD values of plant compounds that can provide hydrological information that is complementary to the sort of information that can be provided by cellulose d18O. The analysis of leaf wax n-alkane dD is particularly promising in this respect (Sachse et al., 2012). Leaf wax n-alkanes are long-chain hydrocarbons with 25–35 carbon atoms that are vital components of waxy plant cuticles (Eglinton and Hamilton, 1967). What makes leaf wax n-alkanes unique is that they are abundant in leaves, soils, sediments and even in atmospheric dust and that they can persist in the environment over millions of years (Eglinton and Eglinton, 2008). In addition, dD values of leaf wax n-alkanes (and n-alkanoic acids) obtained from lake surface sediments or soils show tight correlations with measured and modeled dD values of precipitation (Sachse et al., 2004; Hou et al., 2008; Rao et al., 2009; Polissar and Freeman, 2010; Luo et al., 2011; Garcin et al., 2012). With this exceptional combination of properties, leaf wax n-alkane dD values have been suggested as powerful new proxies that can provide hydrological information across spatial and temporal scales that cannot be assessed with conventionally used plant materials such as cellulose d18O (Sauer et al., 2001; Schefuß et al., 2005, 2011; Pagani et al., 2006; Tierney et al., 2008). Despite the enormous potential, the interpretation of leaf wax n-alkane dD values remains associated with uncertainties because critical processes that control their hydrogen isotopic composition are not yet fully understood. In general, the dD values of leaf wax n-alkanes are determined by the isotopic composition of water that is available for the biosynthesis of leaf wax n-alkanes (here referred to as the “biosynthetic water pool”) and the net biosynthetic fractionation (ebio) that accounts for various biochemical hydrogen isotope fractionations during biosynthesis of n-alkanes from the biosynthetic water pool (Fig. 1) (Sachse et al., 2006; Smith and Freeman, 2006). ebio can range from 150 to 170& but is believed to be rather constant for a given species (Sessions et al., 1999). In contrast to ebio, the hydrogen isotopic composition of the biosynthetic water pool can be quite variable for a given species. The biosynthetic water pool is primarily determined by the dD values of plant source water, which equals in most cases dD values of precipitation (Sachse et al., 2012). In addition, several recent studies have indicated that the evaporative deuterium (D)-enrichment of soil and in particular leaf water also exerts a strong influence on the dD values of the biosynthetic water pool (Smith and Freeman, 2006; Pedentchouk et al., 2008; Sachse et al., 2009, 2010; Feakins and Sessions, 2010; Polissar and Freeman, 2010; McInerney et al., 2011). Not only precipitation dD values but also leaf water evaporative D-enrichment should thus shape the dD values of leaf wax n-alkanes. The relative contributions of precipitation and D-enriched leaf water to the biosynthetic water pool are, however, poorly understood and it has even been questioned if leaf water evaporative D-enrichment is large enough to be a potential important source of variation in leaf wax n-alkane dD values (Hou et al., 2008). In addition, it has been doubted that the contribution of D-enriched leaf water to the pool of biosynthetic water is sufficient in mag-

Fig. 1. Conceptual model summarizing the key drivers of leaf wax n-alkane dD values. In the model, dD values of leaf wax n-alkanes are driven by the dD values of the biosynthetic water pool and ebio. The dD values of biosynthetic water are influenced by the dD values of the plant’s source water and leaf water evaporative deuterium enrichment (DD). ebio is the difference between the dD values of the biosynthetic water pool and leaf wax n-alkane dD values. eapp is the observed apparent fractionation between the dD values of leaf wax n-alkanes and the dD values of the plant’s source water. eapp is a composite fractionation including leaf water Denrichment and ebio and is thus marked black. Since we assume ebio to be a species-specific constant in our model, within species variations in eapp should reflect variations in the dD of the biosynthetic water pool. Note that the arrows and Y-axis are not to scale. Modified after Sachse et al. (2006, 2012).

nitude to influence the dD values of leaf wax n-alkanes (McInerney et al., 2011). Given these uncertainties it remains unclear if leaf wax n-alkane dD values simply reflect hydrological signals derived from the dD values of precipitation or if leaf wax n-alkane dD values are also shaped by Denriched leaf water and contain thus additional information on plant physiological processes such as evapotranspiration. To resolve these uncertainties and to develop a mechanistic foundation for the robust interpretation of the dD values recorded in leaf wax n-alkanes we report here the results of an experimental study in which we tested if and by what magnitude leaf water evaporative D-enrichment influences the dD values of leaf wax n-alkanes in different angiosperm plant species. For our study we grew five plant species that belong to different plant functional types in climate-controlled growth chambers and carefully monitored xylem water, leaf water, water vapor and leaf wax n-alkane dD values of these plants. Our experimental approach allowed us to quantify the effects of leaf water evaporative D-enrichment on leaf wax n-alkane dD values and provides thus the mechanistic basis for a robust interpretation of leaf wax n-alkane dD values. In a companion paper, we validate the experimental findings that we present here with an extensive field survey and employ a global leaf water model to discuss the general relevance of our findings for the global application of leaf wax nalkane dD values as environmental proxies (Kahmen et al., 2013).

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2. MATERIALS AND METHODS 2.1. Experimental setup To determine if and to what extent leaf water evaporative D-enrichment influences the dD of leaf wax n-alkanes we grew a dicotyledonous legume (Phaseolus vulgaris (bean)), a dicotyledonous annual plant (Helianthus annuus (sunflower)), a dicotyledonous perennial woody plant (Populus balsamifera (poplar)) and two monocotyledonous grass species, a C4 grass (Zea mays (maize)) and a C3 grass (Triticum aestivum (wheat)) in two climate-controlled growth chambers. We grew four replicate plants of each species in each of the two chambers. To ensure that all photosynthates, including leaf wax n-alkanes, were assimilated under the experimental conditions the plants were grown from seeds or 20 cm leafless cuttings (poplar) in 2.5 l PVC pots beginning on February 1st 2011. To create significant differences in leaf water evaporative D-enrichment, one chamber had a constant low relative humidity with mean daily values ranging between 27.8% and 47.2% and a mean for the duration of the experiment of 36.0% (“dry chamber”) (Fig. 2). The other chamber had a constant high relative humidity with mean daily values ranging between 68.3% and 75.8% and a mean for the duration of the experiment of 71.2% (“wet chamber”) (Fig. 2). Mean daily air temperature in the dry chamber varied between 20.9 °C and 24.8 °C and averaged at 23.9 °C for the duration of the experiment (Fig. 2). Mean daily air temperature in the wet chamber varied between 21.2 °C and 26.5 °C and averaged at 25.3 °C for the duration of the experiment (Fig. 2). Light intensity did not differ between the dry and the wet chamber and were constantly held at 500 lmol m2 s1. Humidity

70 30 60

25 20

50 40 30

Relative humidity (%)

Air temperature (°C)

80

01 1 /2 /0 3

18

/0 3 10

02

/0 3

/2

/2

01 1

01 1

01 1 /2 /0 2

22

/0 2 14

06

/0 2

/2

/2

01 1

01 1

20

Relative humidity wet cham ber (%) Relative humidity dry cham ber (%) Air tem perature wet cham ber (°C)

41

and temperature settings were held constant in the chamber day and night. Lights in the chambers were turned on at 8:00 h in the morning and turned off at 20:00 h in the evening. Plants in both chambers were irrigated with water, which had a hydrogen isotopic composition of +27&. The isotope composition of the irrigation water was enriched in D compared to local tap water (70&). We decided to use D-enriched irrigation water based on Craig-Gordon leaf water model predictions to maximize the difference between leaf water dD in the wet and the dry chamber. The D-enriched irrigation water was produced in a 500 l bucket, which we filled with tap water. We added deuterated water (99 atom% D, Sigma Aldrich) to the tap water in bucket to generate water with dD values of +27&. dD values of the irrigation water were monitored throughout the experiment and remained constant over time (27 ± 1&). To avoid evaporative D-enrichment of soil water in the pots, we covered the soil in each pot with aluminum foil. We punched 15 holes into the foil of each pot to allow irrigation water to infiltrate the soil but covered the foil with a 2 cm layer of coarse quartz gravel (3 mm diameter) to avoid evaporation through the holes in the aluminum foil. 2.2. Leaf and xylem water collection To determine the dD values of xylem water and leaf water all plants were harvested at midday on March 16th 2011, 44 days after the experiment started. To determine if leaf water dD values were consistent in the dry and in the wet chamber during the time when the leaves that we used for n-alkane analysis developed and matured, we also collected leaves for leaf water extraction at midday (i.e. in the middle of the light period) on March 1st 2011 and March 9th 2011 29 and 37 days after the beginning of the experiment, respectively. All harvested leaves had their mid-vein removed and the leaf blade was stored in 10 ml exetainer vials. The vials were kept frozen at 20 °C until leaf water extractions. To determine the dD values of xylem water, we sampled the root crowns of all five plant species as described by Barnard et al. (2006). Leaf and xylem water was cryogenically extracted using a method described by West et al. (2006). In brief, leaf and xylem samples were heated in the extraction vials to 95 °C. The evaporated water was collected in glass U-tubes that were submerged in liquid nitrogen. The extraction line was subject to a vacuum of 0.03 hPa. Extractions were performed for at least two hours per sample. After extraction, the U-tubes with the frozen leaf water were removed from the extraction line, sealed, the water thawed and transferred into 2 ml GC crimp cap vials. 2.3. Water vapor collection

Air tem perature dry cham ber (°C)

Fig. 2. Mean daily relative humidity and air temperature in the wet and in the dry chamber for the duration of the experiment. As a result of plant growth and associated increasing transpiration rates the dry chamber became increasingly humid at the end of the experiment.

To determine the dD values of atmospheric vapor in the two growth chambers, we collected two replicate atmospheric vapor samples in each chamber at 9:00, 12:00, 15:00 and 18:00 on March 1st 2011, March 9th 2011, and March 14th 2011 (29, 37 and 43 days after the beginning

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of the experiment) using cryo-traps. A cryo-trap consisted of a 1 m long PE tube (inner diameter 5 mm) that was coiled three times and the coils submerged in a dry ice/ethanol slush (80 °C). To collect atmospheric vapor, air was pumped at a flow rate of 10 l/h through the loops of the cryo-trap. After vapor collection, the loop traps were sealed, the frozen water thawed and transferred into 2 ml GC crimp cap vials. Before we used the cryo-traps in our experiments, we tested their performance in the lab. These tests showed that the trap removes >99.9% of all atmospheric vapor and that no isotope fractionation effects occurred during vapor collection (K. Simonin, unpublished data). 2.4. Stable water isotope analysis All water samples were analyzed for dD values using the high-temperature carbon reduction method by coupling a high-temperature elemental analyzer (TC/EA; Finnigan MAT, Bremen, Germany) to a DeltaplusXP isotope ratio mass spectrometer via a ConFlo III interface (Finnigan MAT, Germany; (Werner et al., 1999)) at the Institute for Agricultural Sciences, ETH Zurich, Switzerland. Water was injected automatically with a GC PAL autosampler (CTC, Zwingen, Switzerland) equipped with a 10 lL gastight syringe. The measurement of dD values was done on the H2 peaks while measurements of d18O values were simultaneously performed on CO peaks generated by a single injection (Peak-Jump from hydrogen to oxygen cup configuration). d18O data are not reported here. The positioning of the samples and laboratory standards in a measurement series for all hydrogen isotope ratio measurements as well as post-run off-line calculations like offset-, memory effect- and drift corrections for assigning the final dD values on the V-SMOW scale have been performed according to Werner and Brand (2001). All data were normalized to V-SMOW/SLAP, assigning consensus values of 0 and 428& for dD to V-SMOW and SLAP reference waters, respectively (Coplen, 1988). The precision of the lab internal standard was 0.5& during the analysis of the data that are reported here. 2.5. n-Alkane sampling, extractions, identification, quantification and isotope analysis To determine the dD of leaf wax n-alkanes we collected 2–3 fully mature leaves from each replicate plant in both chambers on March 16th, 44 days after the experiment started. The collected leaves had developed during March 1st 2011 and March 10th 2011 (days 29 and 38 of the experiment), the period of time during which leaf water dD was monitored. We removed the mid veins of the leaves and dried the leaves for 48 h at 60 °C. Dried leaf samples were ground to a fine powder using a ball mill (Retsch, Du¨ sseldorf, Germany). For n-alkane extractions, 0.50–0.75 g of ground plant material was immersed in 30 ml of a 9:1 dichloromethane/methanol (DCM/MeOH) mixture and placed in an ultrasonic bath for 15 min in the organic geochemical laboratory at University of Potsdam. The DCM/ MeOH mixture containing the compounds of interest was

then filtered to remove suspended material. The so-obtained total lipid extracts (TLE) were evaporated and later dissolved in 1.5 ml of n-hexane. 5a-Androstane (10–40 lg) was added to each sample as an internal quantification standard, depending on plant species. To purify the n-alkanes for GC–MS and GC–IRMS measurements, solid phase extraction (SPE) columns filled with 1.5 g silica gel. TLEs dissolved in n-hexane were loaded onto the columns. The fraction containing n-alkanes was eluted with 8 ml of n-hexane. Fractions were collected in test tubes, evaporated, redissolves in 1 ml hexane and transferred into GC vials for analysis. The polar compounds retained on the SPE column were eluted with 12 ml of a 1:1 DCM/MeOH mixture and archived. The fractions containing n-alkanes were analyzed using an Agilent 7890A Gas Chromatograph equipped with a 5975C Series Mass Spectrometric Detector (MSD) system and an additional FID detector connected through a splitter valve at the Institute for Earth and Environmental Sciences at the University of Potsdam. The GC was equipped with an Agilent J&W HP-5ms column (30 m long, 0.25 mm diameter, 0.25 lm film thickness). The GC–MSD/FID system was equipped with a PTV injector, set at an initial injection temperature of 70 °C for 0.85 min. After injection, the injector was heated with a rate of 720 °C/min to a temperature of 300 °C for another 2.5 min. The temperature program of the GC started at 70 °C, held for 2 min and was then heated to 320° at a rate of 12 °C/min, the final temperature was held for another 15 min. n-Alkanes were identified using their mass spectra and retention times of an external n-alkane standard mixture. Quantification was done using the peak areas of the FID trace of the GC– MSD/FID in relation to the peak areas of the internal standard (5a-androstane). Hydrogen isotope compositions of n-alkanes were analyzed on a Trace GC coupled via a pyrolysis reactor to a MAT 253 mass-spectrometer at MARUM, University of Bremen, Germany. Isotope values were measured against calibrated H2 reference gas. dD values are reported in & V-SMOW. The H3-factor varied from 5.30 to 5.37 over the measuring period (5.34 ± 0.02). All samples were run in triplicate. An n-alkane standard (Mix A3 from A. Schimmelmann) of 15 externally calibrated n-alkanes was measured in triplicate every three samples. Since Mix A3 dD values measured against the calibrated reference gas where identical to the certified values within the analytical uncertainty of the IRMS, we decided against an additional correction of the measured values. Additionally, the dD values of the internal standard (5a-androstane) were used to monitor the precision of the analysis. It had a standard deviation of ±1.7 (n = 34) over the measurement period. 2.6. Data analysis The magnitude of the effect of D-enriched leaf water on the dD of leaf wax n-alkanes depends on the influence of the D-enriched leaf water on the biosynthetic water pool (Fig. 1). To quantify the contribution of leaf water to the biosynthetic water pool for the individual species in our experiment we first determined concentration-weighted

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43

mean (CWA) leaf wax n-alkane dD value (dDn-ALK) to get a single n-alkane dD value for each species at a treatment by calculating: 33 X ðdDk  conc:k Þ dDn-ALK ¼ ð1Þ conc:tot k¼25 where dDk are dDn-C25 –dDn-C33 , conc.k are the concentration of n-C25–n-C33 alkanes in lg per g leaf material and conc.tot is the total n-alkane concentration in lg per g leaf material. To quantify the influence of D-enriched leaf water on the dD value of the biosynthetic water pool, we determined the apparent fractionation (eapp, Eq. (2)) between concentration-weighted mean leaf wax n-alkane dD values (dDnALK) and plant source water, which in our case equals xylem water (dDxy): eapp ¼

ðdDn-ALK þ 1Þ 1 ðdDxy þ 1Þ

ð2Þ

Since delta values are typically reported in per mil, this implies a factor of 1000 in the equation. In essence, eapp is a measure of the deviation of leaf wax n-alkane dD values from source water dD values and is influenced by the isotopic composition of the biosynthetic water pool and the net biosynthetic fractionation, ebio (Fig. 1). We assume that ebio for a given species did not vary between the two treatments in our experiment since key environmental variables that could have affected ebio such as temperature or light intensity were held constant in the two chambers (Sessions, 2006; Yang et al., 2009; Zhou et al., 2011). Any variations in eapp between the dry and the wet chamber should thus be caused by variations in the dD value of the biosynthetic water pool (Fig. 1). If related to variations in the degree of mean leaf water evaporative D-enrichment above source water (DD, Eq. (3)), the slope of the relationship between leaf water DD and eapp should indicate the degree to which leaf water D-enrichment influences the dD values of the biosynthetic water pool and the intercept should provide a value for the biosynthetic fractionation. Mean DD was calculated as DD ¼

ðdDLW þ 1Þ 1 ðdDxy þ 1Þ

ð3Þ

where dDLW is the mean dD value of the plants’ bulk leaf water collected three times throughout the experiment (Fig. 3). As above, since delta values are typically reported in per mil, this implies a factor of 1000 in the equation. 3. RESULTS 3.1. Leaf water dD values in the dry and wet chamber The climatic conditions in the two growth chambers caused bulk leaf water of the five plant species to be D-enriched in the dry compared to the wet chamber (Table 1, Fig. 3). Despite slight variations we found that leaf water dD values in both chambers were remarkably consistent for the time when the leaves that we used for n-alkane extractions developed between day 29 and day 44 of the experiment (Table 1, Fig. 3). When averaged over the time of leaf development leaf water of the investigated five plant

Fig. 3. Midday leaf water dD values for five plant species on day 29, 37 and 44 of the experiment. Gray dots indicate values from the dry chambers; black dots indicate values from the wet chambers. Values shown are means of four replicate plants. Numeric values including standard deviations are presented in Table 1.

species was D-enriched between 91& and 104& in the dry compared to the wet chamber (Figs. 3 and 4). Xylem water was also slightly D-enriched in the dry compared to the wet chamber by between 9& and 12& (Table 1, Fig. 4). This enrichment could have been caused by evaporative Denrichment of soil water or by the contamination of xylem water with D-enriched phloem water that cannot always be separated completely from the xylem when collecting the samples (Barnard et al., 2006). In either case soil water evaporative D-enrichment was an order of magnitude less than the observed leaf water evaporative D-enrichment between the two treatments and did therefore not compromise the goal of this experiment. We also observed large differences in atmospheric vapor dD values between the two chambers, which were consistent throughout the experiment (on average 131 ± 5& in the wet chamber and 71 ± 22& in the dry chamber). We suspect that these differences were caused by the automated humidification (wet chamber) and aridification (dry chamber) of the air in the chambers. The strongly D-depleted water vapor (relative to plant source water of +27&) caused leaf water in the wet chamber to be depleted in D compared to the plant’s source water. This is, because the dD of leaf water is not solely driven by atmospheric humidity but can also be strongly affected by the dD of atmospheric vapor, in particular at high atmospheric humidity (Kahmen et al., 2008; Sachse et al., 2009). These differences in vapor dD values between the two chambers despite identical source water dD values illustrate that in artificial settings such as growth chamber experiments it cannot be automatically assumed that vapor dD is in equilibrium with source water dD. It is thus imperative to assess dD of atmospheric vapor in the experimental system if leaf water dD is to be mechanistically understood or if leaf water dD values are not directly measured but derived from models.

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Table 1 Leaf water and xylem water dD values of all five plant species grown in the wet and in the dry climate controlled growth chambers on days 29, 37 and 44 after the beginning of the experiment. Values are the mean of four replicate plants, sd = one standard deviation of the mean. Treat.

Leaf water day 29 Leaf water day 37 Leaf water day 44 Xylem water day 44

Wet Dry Wet Dry Wet Dry Wet Dry

Bean

Sunflower

Poplar

Wheat

Maize

Mean (&)

sd

Mean (&)

sd

Mean (&)

sd

Mean (&)

sd

Mean (&)

sd

37 66 35 69 19 75 18 31

5 2 3 5 3 3 2 1

38 58 36 68 22 73 19 29

1 1 5 1 3 2 2 2

36 58 30 64 35 60 18 27

2 2 4 2 4 8 1 1

43 53 39 59 39 50 21 29

1 1 2 2 2 1 1 1

34 59 24 68 13 68 21 31

11 5 8 2 4 3 2 1

3.2. Leaf wax n-alkane dD values in the dry and wet chamber The five plant species contained n-C25, n-C27, n-C29, nC31, and n-C33 alkanes in different quantities (Table 2). The growth conditions in the dry and the wet chamber had no effect on abundance or average chain length distribution of the n-alkanes in the five species (Table 2). All nalkanes were D-enriched in the dry chamber compared to n-alkanes from leaves grown in the wet chamber in all five plant species (Table 3, Fig. 4). For the dicotyledonous species the relative CWA D-enrichment of leaf wax n-alkanes in the dry compared to the wet chamber varied between 104& and 123& and was thus in the same range as the D-enrichment of bulk leaf water in the dry compared to the wet chamber. The monocotyledonous grass species, however, showed a CWA D-enrichment of leaf wax n-alkanes in the dry compared to the wet chamber that varied between 29& and 66& and was thus lower than the Denrichment of bulk leaf water (Table 3, Fig. 4). eapp differed significantly between the wet and the dry chamber for all five plant species when tested with a oneway ANOVA where wet and dry chamber were used as treatment factors (Table 4, Fig. 5). For the dicotyledonous species the slope of the relationships between DD and eapp ranged between 0.83 and 1.06 with a mean of 0.96 ± 0.12 (Fig. 5). This suggests that D-enriched bulk leaf water contributes on average 96% to the biosynthetic water pool and that the dD values of leaf wax n-alkanes from dicotyledonous plant species record almost the full extent of bulk leaf water D-enrichment. For the monocotyledonous grass species the slope of the relationship between eapp and bulk leaf water DD was 0.18 and 0.68 (Fig. 5). This suggests that the biosynthetic water pool of grasses is also significantly influenced by D-enriched leaf water but that a much lower fraction of D-enriched bulk leaf water contributes to the biosynthetic water pool as compared to dicotyledonous plants. 4. DISCUSSION 4.1. Variability of leaf wax n-alkane dD values within a species and treatment The hydrogen isotope composition of different n-alkanes within a species and treatment was generally quite consistent with variations of less than 10& (Table 3). We found, however, a few exceptions, where individual n-alkanes

showed substantial (>10&) differences in dD values within a plant species and treatment. Since we included only chromatogram peaks that were in the linear range of the IRMS (between 1000 mV and 7000 mV) in our study, we conclude that the observed heterogeneities in n-alkane dD values within a plant and treatment were not caused by analytical errors but are of biological origin. Variation in the dD values of individual compounds within one plant sample have also been observed in other studies reporting dD values of more than one n-alkane (e.g. Sachse et al. 2006; Hou et al. 2007; Feakins and Sessions 2010; McInerney et al. 2011). The mechanism is unclear, but variations may originate in differences of the NADPH sourced hydrogen during biosynthesis (Schmidt et al., 2003; Zhang et al., 2009a). To account for the variability observed in different leaf wax n-alkane dD values in our further analysis, we calculated concentrationweighted average leaf wax n-alkane dD values for each species and treatment (CWA, Eq. (1)). This allows to generate a single n-alkane dD value that is representative for a plant species and treatment. 4.2. Effects of leaf water D-enrichment on leaf wax n-alkane dD values of monocotyledonous and dicotyledonous plants We observed strong differences in leaf water evaporative D-enrichment for all five species between the wet and the dry growth chambers (Figs. 3 and 4). These differences in leaf water evaporative D-enrichment had a strong influence on the dD values of leaf wax n-alkanes in both, dicotyledonous and monocotyledonous plants (Fig. 4). We quantified the effect of leaf water D-enrichment on leaf wax n-alkane dD values by assessing the contribution of leaf water to the biosynthetic water pool in the different plant species (Fig. 1 and Fig. 5). We found that in dicotyledonous plants, leaf water contributed on average 96% to the biosynthetic water pool while this contribution was only between 18% and 68% in grasses. Our data therefore suggest that leaf water evaporative D-enrichment influences the dD values of n-alkanes of all plants but that this effect is larger for dicotyledonous plants than for monocotyledonous plants. Differences in the magnitude by which leaf water Denrichment affects the dD values of leaf wax n-alkanes can be explained by fundamental differences in leaf growth

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150

Bean

n.a.

0 150

Sunflower

100

n.a.

50 0 150

Poplar

100

n.a.

50 0 150

Wheat

100 50

n.a.

Difference in δD values between dry treatment and wet treatment (‰)

50

n.a.

100

0 150

Maize

100

n.a.

50

Xy

le m

Le wa af ter w at er C 25 C 27 C 29 C 31 C 33 C W A

0

n

n

n

n

n

Fig. 4. Differences in xylem water, leaf water and leaf wax n-alkane dD values between the wet and the dry growth chamber for five different plant species. Data are the means of four replicate plants. Standard deviations for all dD values were below 9& and thus too small to be displayed in the figure. CWA: concentration weighted average leaf wax n-alkane dD values; n.a.: not analyzed due to low n-alkane concentrations.

and development between these two plant groups: In dicotyledonous plants leaf growth and expansion and thus leaf wax synthesis continues well after the initial unfolding of the lamina (Dale, 1988). Since the evaporative D-enrichment of leaf water in developing leaves can be in the same range as the evaporative D-enrichment of leaf water in fully matured leaves (Kahmen et al., 2011b), the biosynthetic water pool in developing leaves and consequently the dD values of n-alkanes are strongly influenced by D-enriched leaf water. Leaves of monocotyledonous grass species, in contrast, grow and expand at an intercalary meristem at

45

the base of a leaf blade (Kemp, 1980). This growth zone is covered by the sheath of the next oldest leaf and is therefore not directly subject to evaporative D-enrichment (Richardson et al., 2005). The mature parts of the leaf blade are, however, exposed to the atmosphere and the water in this part of the leaf blade becomes enriched in D. This Denriched leaf water diffuses towards the basal zones of leaf development (Helliker and Ehleringer, 2002), so that the biosynthetic water pool at the primary sites of leaf wax synthesis is a mix of non-enriched source water and D-enriched leaf water. The dD values of leaf wax n-alkanes from monocotyledonous grasses do therefore not record the full extent of bulk leaf water D-enrichment. This finding is consistent with previous reports that the dD values of leaf wax n-alkanes from grasses are generally D-depleted compared to dD values of leaf wax n-alkanes derived from dicotyledonous plants (Hou et al., 2007; Liu and Yang, 2008; McInerney et al., 2011; Sachse et al., 2012). With the experimental work that we present here, we argue that this difference can at least partly be explained by the fact that dicotyledonous plants record in their n-alkane dD values the full extent of leaf water D-enrichment while this effect is smaller for monocotyledonous plant species. Although leaf waxes of grasses are primarily synthesized at the intercalary meristem where the leaf grows and develops, a recent study has shown that grasses synthesize leaf waxes also on blades of fully developed leaves (Gao et al., 2012). This process seems to be particularly relevant, when plants are exposed to wind and rain, which cause abrasion of cuticle waxes that need to be re-generated (Baker and Hunt, 1986; Shepherd and Griffiths, 2006). Gao et al. (2012) suggest for example that n-C27–n-C31 leaf wax n-alkanes of wind-exposed leaves of the grass Phleum pretense are completely replaced within 71 and 128 days. The synthesis of these “secondary leaf waxes” occurs in parts of the grass leaf that are exposed to the atmosphere and thus subject to leaf water D-enrichment. The biosynthetic water pool utilized in the synthesis of such “secondary leaf waxes” will thus contain a larger fraction of D-enriched leaf water than the biosynthetic water pool at the intercalary meristem where the primary leaf waxes are synthesized. Leaf wax turnover and accumulation of “secondary” leaf wax n-alkanes on the blades of mature grass leaves will consequently have important effects on the dD values of the plant’s n-alkanes. The duration of the experiment that we describe here was much shorter than the turnover times reported by Gao et al. (2012) and leaf wax abrasion and regeneration was probably low due to the absence of wind and rain in the growth chambers. It is therefore likely that our study underestimated the effects that leaf water evaporative D-enrichment has on the dD values of leaf wax n-alkanes in grasses and that these effects are larger in grasses that grow in natural ecosystems, where grass leaves are subject to leaf wax abrasion and re-generation. Our data also show that the effect of D-enriched leaf water on the n-alkane dD was substantially larger for the C4 grasses than for C3 grasses. The observation is consistent with previously reported studies, in which C4 grasses exhibit on average more positive, i.e. D-enriched, dD values than C3 grasses (Smith and Freeman, 2006; Liu and Yang,

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Table 2 Concentration of individual leaf wax n-alkanes in the leaves of bean, sunflower, poplar, wheat and maize grown in the wet and in the dry climate controlled growth chambers in lg n-alkane per g leaf dry mass. Values are the mean of four replicate plants, sd = one standard deviation of the mean. Species

Bean Sunflower Poplar Wheat Maize

Treat.

n-C25

Wet Dry Wet Dry Wet Dry Wet Dry Wet Dry

n-C27

n-C29

n-C31

n-C33

Mean (lg/g)

sd

Mean (lg/g)

sd

Mean (lg/g)

sd

Mean (lg/g)

sd

Mean (lg/g)

sd

2 – 13 4 50 92 11 10 – 1

3 – 3 6 8 10 1 2 – 1

18 12 86 61 140 218 29 26 8 7

23 14 22 50 22 39 3 6 1 1

165 179 52 51 679 720 22 62 33 19

95 140 15 34 105 180 2 18 11 6

423 371 9 15 64 63 27 49 78 50

281 233 9 17 8 14 1 12 27 16

206 106 – – – – 45 53 36 9

220 75 – – – – 4 11 19 9

Table 3 dD values of individual leaf wax n-alkanes and the concentration weighted average leaf wax n-alkane dD values (CWA) of bean, sunflower, poplar, wheat and maize grown in the wet and in the dry climate controlled growth chambers. Values are the mean of four replicate plants, sd = one standard deviation of the mean. Species

Bean Sunflower Poplar Wheat Maize

Treat.

Wet Dry Wet Dry Wet Dry Wet Dry Wet Dry

n-C27

n-C25

n-C29

n-C33

CWA

Mean (&)

sd

Mean (&)

sd

Mean (&)

sd

Mean (&)

sd

Mean (&)

sd

Mean (&)

sd

– – 171 106 196 118 208 187 – –

– – 2 2 2 7 4 1 – –

– – 177 82 201 104 210 188 165 133

– – 2 3 3 6 5 1 2 7

210 124 181 85 200 98 208 200 178 128

3 1 3 5 2 6 3 3 5 5

204 122 176 79 199 99 208 174 185 128

3 6 4 3 3 4 4 4 5 3

203 119 – – – – 193 158 186 129

2 9 – – – – 5 4 4 6

206 122 178 83 200 101 204 180 183 128

2 5 3 3 2 6 5 4 5 4

Table 4 Apparent fractionation (eapp) for the five plant species grown in the wet and in the dry climate controlled growth chambers. eapp was calculated using Eq. (2). Values are the mean of four replicate plants, sd = one standard deviation of the mean. Statistical differences in eapp between the wet and the dry chambers for a given species were calculated using a one-way ANOVA. P-values of these analyses are presented in the table. Wet treatment

Bean Sunflower Poplar Wheat Maize

n-C31

Dry treatment

P-value

Mean (&)

sd

Mean (&)

sd

219 195 214 220 198

2 3 2 4 5

148 110 125 205 147

4 4 7 2 11

<0.001 <0.001 <0.001 0.001 <0.001

2008; McInerney et al., 2011; Sachse et al., 2012). Anatomical and biochemical differences between C3 and C4 plants have been suggested to explain differences between C3 and C4 grass leaf water (Helliker and Ehleringer, 2000) and nalkane dD values (Smith and Freeman, 2006; McInerney et al., 2011). Since the differences between C3 and C4

grasses that we report here are, however, based on only a single species from each group, we cannot ultimately judge if it is the influence of D-enriched leaf water on the biosynthetic water pool that can explain these differences. Our finding that leaf wax n-alkane dD values from both dicotyledonous and monocotyledonous plants are influenced by D-enriched leaf water differs from a recent conclusion of McInerney et al. (2011), who argued that the dD values of leaf wax n-alkanes of grasses are insensitive to leaf water evaporative D-enrichment. Since the influence of leaf water evaporative D-enrichment on the dD values of leaf wax n-alkanes from grasses that we observed here was quite variable, it is possible that McInerney et al. (2011) have investigated species in which the contribution of D-enriched leaf water to the pool of biosynthetic water was relatively small. McInerney et al. (2011) did, however, not measure leaf water dD directly but based their conclusion on model estimated leaf water dD, where some model variables, including the dD values of water vapor, had to be indirectly derived from d18O values. As shown above, dD values of water vapor in experimental systems with artificial humidification and/or dehumidification can be difficult to predict. While we cannot explain the different outcomes of McIner-

A. Kahmen et al. / Geochimica et Cosmochimica Acta 111 (2013) 39–49

4.3. Differences in the biosynthetic fractionation (ebio) among different plant species

Dicotyledonous plants

εapp(‰)

-100

-150

-200

-250 -100

-50

0

50

Leaf water ΔD (‰) Sunflower: εapp=0.98x-145 Poplar: εapp =1.06x-160 Bean: ε app=0.83x-180

Monocotyledonous plants

εapp(‰)

-100

-150

-200

-250 -100

-50

0

47

50

Leaf water ΔD (‰) Maize: ε app =0.68x-169 Wheat: ε app=0.18x-209 Fig. 5. Relationship between the leaf water evaporative enrichment above source water (DD) and apparent fractionation eapp. The slope of the relationship indicates the fractional contribution of evaporatively-enriched leaf water to the pool of biosynthetic water. Grey lines indicate hypothetical 1:1 relationships between eapp and DD, where leaf water would contribute 100% to the biosynthetic water pool. Values for eapp were calculated using CWA n-alkane dD values. Data shown are the means of four replicate plants. For statistical differences in eapp for the individual species in the wet and the dry chamber see Table 4.

ney et al. (2011) and our study directly, our results suggest that the dD values of leaf wax n-alkanes from grasses are not generally insensitive to leaf water evaporative D-enrichment. Additional studies that quantify the average magnitude by which D-enriched bulk leaf water affects the dD values of both, C3 and C4 grasses are needed. The direct assessment of the hydrogen isotope composition of leaf and xylem water will be pivotal for such studies.

An unexpected discovery of our study is the difference in ebio among species. This difference was up to 65& among the different plant species in our experiment as indicated by the different intercepts in Fig. 5. ebio summarizes a suite of biochemical fractionations that occur during the biosynthesis of leaf wax n-alkanes. In particular NADPH-derived hydrogen additions to the carbon skeleton seem to be an important process that determines ebio because NAPDHderived hydrogen is strongly depleted relative to biosynthetic water (Luo et al., 1991; Schmidt et al., 2003; Zhang et al., 2009b). NADPH in plants is produced through different pathways, where the reduced hydrogen can have substantially different hydrogen isotopic compositions (Yakir, 1992; Schmidt et al., 2003; Sessions, 2006; Zhang et al., 2009a). Although we can currently only speculate about the reasons for the species-specific differences in ebio that we describe here, differential contributions from distinct NADPH sources to hydrogen in leaf wax n-alkanes could explain these differences. Several previous studies have reported substantial differences in leaf wax n-alkane dD values among different plant species that grew under similar environmental conditions (Sachse et al., 2006; Smith and Freeman, 2006; Feakins and Sessions, 2010; McInerney et al., 2011; Garcin et al., 2012). These differences have been attributed to differences in eapp, which includes differences in leaf water evaporative D enrichment and ebio (Fig. 1). Our finding of substantial differences in ebio among the five investigated species suggests that substantial differences in leaf wax n-alkane dD values among different plant species that grow under similar environmental conditions can be driven by species specific differences in ebio. 5. CONCLUSIONS Here we present the first experimental evidence showing that the evaporative D-enrichment of leaf water shapes the dD values of leaf wax n-alkanes in both, monocotyledonous and dicotyledonous plants. With the experimental approach that we chose here we were able to separate effects of leaf water dD values on leaf wax n-alkane dD values from other variables such as soil water evaporative Denrichment. Our finding has important implications for the interpretation of leaf wax n-alkanes in paleohydrological research and could in addition open the door to employ dD values of leaf wax n-alkanes as new ecohydrological proxies in plant and ecosystem research. Our results suggest, that leaf water evaporative D-enrichment and hence plant physiological processes such as evapotranspiration or their respective drivers (relative humidity, leaf to air vapor pressure deficit) can potentially be recorded in leaf wax n-alkane dD values. Our data predict that this effect is stronger for environments dominated by dicot plants compared to monocot-dominated ecosystems (i.e. grasslands). The evaporative D-enrichment of leaf water can, however, be quite variable depending on climate and leaf physiology so that the relevance of our experimental finding for natural

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ecosystems – where leaf water evaporative D-enrichment is possibly lower than in our experiment – remains unclear. We therefore address the importance of the effect that we have shown here in a companion paper (Kahmen et al., 2013), where we validate our experimental results with an extensive field study and employ a global leaf water model to discuss the relevance of our finding for the application of leaf wax n-alkane dD as environmental proxy world-wide. ACKNOWLEDGEMENTS The authors would like to thank John Hayes, Francesca McInerney and Brent Helliker for valuable comments on a previous version of this manuscript. We thank Annika Ackermann and Roland Werner for analyzing the liquid water samples at ETH Zurich and Johanna Menges for leaf wax n-alkane extraction and purification at UP. A.K. was supported by an ERC Starting Grant (279518 COSIWAX). D.S. was supported through an Emmy-Noether Research grant by the German Science Foundation (DFG SA-1889/ 1-1). E.S. was supported by a grant from the German Science Foundation (DFG Sche903/8-1).

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