Leaf wax n-alkane δD values of field-grown barley reflect leaf water δD values at the time of leaf formation

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Geochimica et Cosmochimica Acta 74 (2010) 6741–6750 www.elsevier.com/locate/gca

Leaf wax n-alkane dD values of field-grown barley reflect leaf water dD values at the time of leaf formation Dirk Sachse a,⇑, Gerd Gleixner b, Heinz Wilkes c, Ansgar Kahmen d,e a

DFG-Leibniz Center for Surface Process and Climate Studies, Institut fu¨r Erd- und Umweltwissenschaften, Universita¨t Potsdam, Germany b Max-Planck-Institut fu¨r Biogeochemie, Jena, Germany c Helmholtz Centre Potsdam GFZ German Research Centre for Geosciences, Potsdam, Germany d University of California at Berkeley, Institute of Integrative Biology, Berkeley, CA, USA e Institute for Plant Sciences, ETH Zurich, Switzerland Received 16 March 2010; accepted in revised form 19 August 2010; available online 27 August 2010

Abstract Leaf wax n-alkanes from barley (Hordeum vulgare) from a field in Switzerland exhibited changes in dD values on the order of 20& over a growing season, while source water (soil water) and leaf water varied by 40&. Additionally the seasonal variability in dD values of leaf wax n-alkanes of different barley leaves can only be found across different leaf generations (i.e. leaves that were produced at different times during the growing season) while n-alkane dD values did not vary significantly within a leaf generation. Interestingly, dD values of n-alkanes correlated best with the dD values of leaf water at midday of the sampling day but showed no significant correlation with soil water (e.g. precipitation) dD values. These results provide empirical evidence that leaf wax dD values record leaf water enrichment, and therefore integrate the isotopic effects of precipitation and evapotranspiration. Our results show that leaf wax n-alkane dD values from grasses are ‘locked in’ early during leaf development and hence record the environmental drivers of leaf water enrichment, such as vapor pressure deficit (VPD). Our data have important implications for the interpretation of paleorecords of leaf wax dD. We suggest that leaf wax n-alkane dD values from sedimentary records could be used to estimate changes in the degree of leaf water enrichment and hence VPD. Ó 2010 Elsevier Ltd. All rights reserved.

1. INTRODUCTION The compound-specific hydrogen isotope composition (dD) of lipid biomarkers in marine and lake sediments is increasingly being used to reconstruct paleohydrologic changes over geological timescales (Huang et al., 2002; Pagani et al., 2006; Sachs et al., 2009). This is because lipids derived from aquatic organisms record the hydrogen isotope composition of their water source (Sauer et al., 2001; Huang et al., 2004; Sachse et al., 2004; Zhang and Sachs, 2007). Isotopic fractionation during lipid synthesis is ⇑ Corresponding author. Address: DFG-Leibniz Center for

Surface Process and Climate Studies, Institut fu¨r Erd- und Umweltwissenschaften, Universita¨t Potsdam, Karl-LiebknechtStr. 24, Haus 27, 14476 Potsdam-Golm, Germany. Tel.: +49 331 977 5841; fax: +49 331 977 5700. E-mail address: [email protected] (D. Sachse). 0016-7037/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.gca.2010.08.033

mainly influenced by the biosynthetic pathway used (Sessions et al., 1999; Sauer et al., 2001; Chikaraishi and Naraoka, 2003; Chikaraishi et al., 2004, 2009; Zhang and Sachs, 2007). Recently, it has also been shown that the type of metabolism of bacteria (i.e. heterotroph, phototroph) can result in widely different dD lipid values (Zhang et al., 2009). In general, however, the dD values of lipids from photosynthesizing organisms with only one possible hydrogen source (water) can in most cases be related to changes in the dD values of the organisms water source. In contrast to aquatic organisms, the water that is used for lipid biosynthesis in leaves of terrestrial plants is subject to pre-biosynthetic enrichment of the heavy isotope (deuterium) (Sachse et al., 2004, 2006; Smith and Freeman, 2006; Feakins and Sessions, 2010). The environmental factors and plant physiological processes that are responsible for the enrichment of deuterium in leaf water are reasonably well understood (Farquhar, 1989; Barbour et al., 2004;

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Cernusak et al., 2005; Farquhar and Cernusak, 2005; Farquhar et al., 2007; Kahmen et al., 2008). Briefly, the enrichment of leaf water in deuterium depends on changes in the vapor pressure gradient between the leaf and the atmosphere (mainly a function of relative humidity and temperature), the difference in the isotopic composition between source water (soil water) and water vapor surrounding the leaf and leaf physiological properties such as transpiration rate (Kahmen et al., 2008, 2009). The degree of leaf water enrichment in deuterium can vary strongly over diurnal and seasonal timescales (Cernusak et al., 2002). It remains, however, unknown if and how the variability in leaf water dD is imprinted on the isotope composition of higher plant lipids. Two recent studies have shown significant seasonal variability in the isotope composition of leaf wax n-alkanes from deciduous and coniferous tree leaves (Pedentchouk et al., 2008; Sachse et al., 2009), suggesting that n-alkanes in leaves of these trees are continuously produced during the growing season and thus record the seasonal variability of leaf water dD values. As such, the isotopic composition of leaf wax n-alkanes, which are ultimately deposited in the sedimentary record, should reflect a seasonal integral of the leaf water evaporative enrichment in deuterium and it’s corresponding environmental drivers. It is critical to note, however, that these previous studies can only speculate on the temporal integration of lipid dD values, as the variability of the biosynthetic source water, leaf water, was not directly determined in these studies. The cuticular wax layer is typically established in the early developmental stages of a leaf (Jetter and Schaffer, 2001). However, systematic changes in the wax composition over time, at least partly due to de-novo synthesis of wax compounds, have also been reported (Jetter et al., 2006). Such changes can follow the natural course of leaf development and senescence or can be induced by environmental factors. Decreasing humidity and temperature for instance both resulted in compositional changes of the wax lipids (a shift from alkanes and ketones to alcohols and aldehydes) in Brassica species (Baker, 1974). Drought conditions have been observed to lead to an increase in total wax load of roses by 8–14% (Jenks et al., 2001). Reduced light intensities have been observed to result in chain length decrease of various wax compounds in barley leaves (Giese, 1975). For the quantitative interpretation of leaf wax lipid dD values in sedimentary records it is important to understand the temporal integration with which leaf wax lipid dD values record the variability of leaf water dD values. The aim of the study that we present here was therefore to improve the understanding of processes that leaf wax n-alkane dD values in grass leaves over seasonal timescales and estimate the ‘temporal integration’ of higher plant lipid dD values. Specifically, our goals were to: (a) determine the seasonal variability of leaf wax nalkane dD values of field-grown barley plants (Hordeum vulgare), (b) compare the variability in n-alkane dD values with the variability in environmental water (soil water) and biosynthetic source water (leaf water) dD,

(c) determine the ‘biosynthetic integration time’ of grassderived leaf wax n-alkane dD values.

2. MATERIALS AND METHODS 2.1. Study site and sampling We conducted our experiment on a 1.55 ha agricultural field located near the town of Oensingen (Canton Solothurn, Switzerland, 47°170 11.100 N, 7°440 01.500 E, 452 m a.s.l.) at the northern edge of the Swiss Plateau in 2005. The soil at the site is a fluvisol composed of 42% clay, 33% silt and 25% sand. The mean annual temperature (1964–1991) is 8.4 °C with a mean annual precipitation of 1000 mm that is well distributed over the whole year. The field has been cultivated with varying crop types in a long-term crop rotation system. We collected leaf water, soil water and atmospheric water vapor approximately every week from the beginning of the life cycle of the barley plants in early April (plant height 5 cm) until plant senescence just before harvest at the end of June. For soil water, we collected between 5 and 10 g soil from the rooting zone of the plants at 5 cm soil depth. For leaf water, we collected the uppermost fully developed leaf from 8 to 10 neighboring individual plants and combined these leaves into a single bulk sample. To obtain an estimate of the spatial variability of soil water dD and leaf water dD within the barley field, we collected all samples in five replicates (n = 5) that were randomly located within the barley field. We collected a total of three different leaf generations over the course of our study, as plants grew in height during our experiment and developed new upper leaves (generation 1: first and second sampling; generation 2: third and fourth sampling; generation 3: fifth to ninth sampling). To account for the diurnal isotopic variability in soil and leaf water, we collected leaf and soil samples twice on every sampling day, once in the morning predawn and once at midday at 13:00 h. To characterize the isotopic composition of atmospheric vapor, we collected water vapor samples in four replicates using a cryo-trap. Air was pumped at a flow rate of 35 L h1 through glass U-tubes (12 mm inner diameter, 200 mm length) that were submerged in liquid nitrogen/ethanol slush. After vapor collection, the U-tubes were sealed, the frozen water thawed and transferred into 2 ml crimp cap vials (Kahmen et al., 2008). Bulk leaf water and soil water was obtained from leaves and soil samples using cryogenic vacuum distillation (West et al., 2006) conducted at the Paul Scherrer Institut (PSI) in Villigen, Switzerland. For these extractions leaf or soil samples were heated in the extraction vials to 80 °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 2 h per sample. After extraction, the U-tubes with the frozen water were removed from the extraction line, sealed, the water thawed and transferred into 2 mL crimp cap vials that were stored at 20 °C prior to isotope analysis.

Seasonal origin of leaf wax n-alkane dD values in grasses

Meteorological parameters (such as temperature, relative humidity, vapor pressure deficit, VPD) were continuously monitored at the site on a small tower (2 m high) and are presented in Fig. 1. 2.2. Determination of leaf and soil water dD values Soil water and leaf water samples were analyzed for their dD values using a modified thermal conversion/elemental analyzer system connected to an isotope ratio mass spectrometer (IRMS) (TC/EA + DeltaplusXL, Finnigan MAT, Bremen, Germany) at Max-Planck Institute for Biogeochemistry in Jena (Gehre et al., 2004). 2.3. Preparation of leaf samples and biomarker identification and quantification The dried leaf samples (from which the leaf water had been removed, see above) were ground. Lipid extraction was performed individually on three single leaves (0.18– 0.45 g) from three different plant individuals per sampling

relative humidity [%]

Air temperture [°C]

170

160

150

140

130

120

110

100

Day of Year

30

air temperture

25 20 15 10 5 0 100 90 80 70 60

relative humidity

evapotranspiration [mm/m-2/s]

50

evapotranspiration

20 15 10 5 0

VPD

VPD [kPa]

12

6743

date to obtain a measure of variability among different plant specimens. Soluble organic matter was extracted using an accelerated solvent extractor (ASE200, Dionex Corp., Sunnyvale, USA) with dichloromethane/methanol mixture (9:1) at 100 °C and 103 bar (=1500 psi) for 5 min in three cycles (33 ml cells, 60% flush volume). The total extract was separated on a silica gel column into two fractions: hydrocarbons (solvent: n-hexane) and other compounds (solvent: methanol). Constituents of the hydrocarbon fraction were identified and quantified using a GC-FID (Agilent GC6890N, Agilent, Santa Clara, CA, USA) equipped with a DB5ms column (30 m, ID:0.32 mm, film thickness: 0.5 lm, Agilent, Palo Alto, USA) by comparison to an external n-alkane standard mixture (n-C14 to n-C36). 2.4. Determination of biomarker dD values One to two microliters of aliphatic hydrocarbon fraction dissolved in n-hexane were injected into a HP6890N GC (Agilent Technologies, Palo Alto, USA), equipped with a HP Ultra 1 column (50 m, ID: 0.2 mm, film thickness: 0.33 lm, Agilent). With injection the PTV injector was heated at 700 °C/min from 80 to 300 °C and held at this temperature for the remaining run. The injector was operated in splitless mode. The oven was maintained for 1 min at 80 °C then heated at 10 °C/min to 150 °C, then at 3 °C/min to 300 °C and held for 25 min at the final temperature. The column flow was held constant at 1.0 ml/min throughout the run. The eluting compounds were transferred via a GC-C/ TC III combustion interface (ThermoFisher Scientific, Bremen, Germany) to a high-temperature conversion furnace operated at 1440 °C (Hilkert et al., 1999) and quantitatively converted to H2, which was introduced into an isotope ratio mass spectrometer (IRMS) (Delta V plus, ThermoFisher Scientific, Bremen, Germany) for compound-specific analysis of dD values at GFZ Potsdam. Three replicate measurements were performed on each sample. All dD values were normalized to the VSMOW scale using a laboratory standard mixture of n-alkanes (n-C14 to n-C34) with known dD values, previously normalized to standard mixtures obtained from A. Schimmelmann (University of Indiana). After the measurement of two samples (6 GC runs), the standard mixture was measured three times. If necessary a drift correction was applied (Werner and Brand, 2001). The Hþ 3 factor was determined once a day and stayed constant within the analytical error of the instrument at 3.43 (SD = 0.05; n = 13) during the measurement period, indicating stable ion source conditions.

10 8

3. RESULTS AND DISCUSSION

6 4

3.1. dD of source water (soil water and leaf water)

2 170

160

150

140

130

120

110

100

0

Day of Year

Fig. 1. Meteorological measurements (as daily mean values) from the Onesingen site over the studied growing season of the grasses.

3.1.1. Soil water Soil water sampled at dawn did not differ in it’s isotopic composition from samples collected at midday (see Fig. 2). Soil water dD values also closely matched the mean monthly dD values of precipitation, collected during the

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100

120

Day in Year 130 140 150

160

170

180

leafwater midday leafwater pre-dawn soil water (midday) soil water (dawn) GNIP (Suhr)

20 10 0

leaf water midday

Δ

-10

leaf water dawn

L max

Δ

56.4 ± 10.5‰

-20

L min

29.7 ± 5.8‰

-30

soil water meteoric water

-40 -50

-150

-70

nC29 nC31 nC33 CWA

-80

-170 -180 -190 -200 -210 -220

2

leaf 1 generation

3

ε

-160

-230

-alkane D [‰] vs VSMOW

-60

app

-155 ± 21‰

ε

bio

-175±15‰ to -192 ± 6‰ mean: -184‰

lipid D/H

n

D water [‰] vs VSMOW

110

-240 -250 100

110

120

130 140 150 Day in Year

160

170

180

Fig. 2. dD values of soil water and leaf water and n-alkanes of barley grass leaves over the growing season (CWA is the concentration weighted average dD value). Shaded boxes indicate leaf generations. Error bars represent standard deviation among the two to three replicate leaf samples (for n-alkanes) and the five replicate water samples (leaf water, soil water). GNIP data are mean monthly values, see text. Right panel: schematic view of the relationships between the H-sources (water) and product (n-alkanes) after Sachse et al. (2006). Values are average seasonal values, for e derived from concentration weighted n-alkane dD values only for newly developed leaves (leaf age 0 weeks). Note that an estimated mean ebio of 184& agrees well with previous estimates for grasses of 181& (Smith and Freeman, 2006).

same time at the GNIP network station in Suhr (ca. 30 km E of the site) (data courtesy of Schweizerisches Bundesamt fu¨r Umwelt (BAFU), Nationale Grundwasserbeobachtung (NAQUA)). This indicates no evaporative enrichment of the soil water at 5 cm soil depth. For the following discussion and calculations we therefore used the dD values of soil water sampled at midday (Table 1). The stable hydrogen isotopic composition of soil water increased over the progression of the growing season by almost 40& from 78& in April to 40& in late June. Roughly, soil water dD values increased over the season (Fig. 1), following the typical seasonal pattern in precipitation dD values for temperate climates (Dansgaard, 1964). 3.1.2. Water vapor Since water vapor can exert a major control on the isotopic composition of the leaf water (via the isotopic difference between source water and water vapor) we also analyzed its isotopic composition at pre-dawn and at midday (Table 1). The isotopic composition of water vapor at pre-dawn showed strong positive correlation with soil water (r2 = 0.7, p = 0.005) and was depleted by between 47& and 84& relative to the soil water, suggesting that the water vapor at dawn is in equilibrium with soil water and therefore local precipitation. In contrast to the pre-dawn values, dD values of water vapor at midday showed reduced variability over the season and did not correlate with dD values of soil water. Only midday air temperature showed a significant correlation with dD of water vapor at midday (r2 = 0.5,

p = 0.031), suggesting that air temperature among other undetermined factors has an effect on the isotopic composition of water vapor at the site in Oensingen. 3.1.3. Leaf water dD values of leaf water at pre-dawn as well as leaf water at midday showed a general increase over the two-month period of about 40&. This increase was similar in magnitude to the increase that we observed for soil water dD at the same time (Fig. 2). Although dD values of leaf water at pre-dawn were enriched in deuterium between 13& and 43& relative to the soil water, we observed a strong correlation between soil water and leaf water pre-dawn dD values (r2 = 0.7, p = 0.006). The leaf water enrichment above source (soil) water at pre-dawn, which we termed minimal enrichment (DLmin), was positively correlated with the atmospheric vapor pressure deficit (VPD) measured at dawn (r2 = 0.7, p = 0.022; data not shown). We therefore conclude that the hydrogen isotope composition of leaf water at pre-dawn, which represents the least deuterium enriched source water for lipid biosynthesis, is controlled by the hydrogen isotope composition of soil water and an evaporative enrichment in deuterium (DLmin) of on average 29.7 ± 5.8&. The hydrogen isotope composition of leaf water at midday represents the highest possible enrichment in deuterium of the water used for biosynthesis in leaves (DLmax). dD values of leaf water at midday did not show any significant correlation with soil water (r2 = 0.3, p = 0.119). Midday

71.4

51.5

39.2

April

May

June

DLmax

43.5 64.5 18.3 48.7 75.3 66.2 67.9 70.4 59.8 49.4

DLmin

33.2 28.5 13.0 25.8 25.7 42.6 26.0 31.1 41.3

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leaf water enrichment (DLmax) followed the patterns of deuterium evaporative enrichment that has been described in previous studies and mechanistic leaf water models (Barbour et al., 2004; Farquhar et al., 2007; Kahmen et al., 2008), and was largely a function of atmospheric VPD (r2 = 0.7, p = 0.014) with leaf transpiration rate and the hydrogen isotope composition of atmospheric water vapor being also important. 3.2. Changes in leaf wax n-alkane concentration and composition over the growing season

*

Data courtesy of the Swiss Federal Agency for the Environment (Bundesamt fu¨r Umwelt BAFU, Nationale Grundwasserbeobachtung NAQUA).

0.5 2.6 1.9 0.8 1.0 2.3 1.2 2.5 1.9 0.8 111.6 99.0 105.5 91.8 95.2 89.6 92.0 110.0 77.9 88.0 4.1 5.0 2.4 3.9 3.6 3.2 1.4 3.4 1.2 3.4 1.1 1.8 1.4 1.3 0.9 2.6 1.9 3.6 1.3 3.6 29.0 18.6 24.0 1.9 8.3 8.2 11.9 21.0 16.9 7.8 1 2 3 4 5 6 7 8 9 10

13.04.05 22.04.05 04.05.05 12.05.05 20.05.05 27.05.05 03.06.05 10.06.05 16.06.05 24.06.05

103 112 124 132 140 147 154 161 167 175

69.9 83.2 40.9 42.7 64.2 69.8 55.9 47.8 39.4 n.d.

2.4 1.4 4.1 2.0 3.1 3.2 1.2 4.0 0.6 1.1 0.8 2.0 1.1 2.9 1.5 2.9 2.7 1.6 38.5 51.8 29.0 20.0 38.2 30.2 27.8 16.4 0.9 n.d.

121.5 156.3 104.6 111.8 134.5 113.6 107.2 108.9 86.1 n.d.

2.4 1.5 2.4 2.5 1.6 1.3 1.0 2.2 1.9

69.5 78.0 41.5 44.7 62.3 69.8 52.4 46.1 40.5 39.7

dD* Stdev dD Stdev dD Stdev dD Stdev Stdev dD Stdev dD

dD

Mean monthly precipitation Vapor midday Soil water midday Leaf water midday Vapor dawn Soil water dawn Leaf water dawn DOY Sampling date

Table 1 dD values of leaf water, soil water and water vapor at dawn and midday. Values were obtained from five replicate samples, standard deviation is therefore representative of natural heterogeneity; DOY – day of year; mean monthly precipitation dD values are taken from the GNIP measurement site at Suhr, Switzerland (30 km from Oensingen).

Seasonal origin of leaf wax n-alkane dD values in grasses

The major n-alkanes detected in the analyzed barley leaves (listed in the order of increasing concentration) were n-C29, n-C31 and n-C33, with significantly smaller contributions from n-C35, n-C25 and n-C27 (see Fig. 3). Overall n-alkane concentrations increased to their maximum concentrations of more than 50 lg/g leaf dry weight in the second sampling week, before they decreased to the minimum concentration of below 30 lg/g dry weight in the fourth week. This pattern was mainly due to the increase and subsequent decrease in the concentration of n-C29, n-C31 and n-C33, while the other n-alkanes showed relatively constant levels. From the fifth to the 10th week a slow increase in overall n-alkane concentrations to 50 lg/g leaf dry weight was observed. While the concentration of n-C33 stayed almost constant, all other n-alkanes showed increases in concentration, most notably n-C31, which reached almost the same concentration as n-C33 by the end of the studied period. The n-alkanes with shorter chain-lengths increased in concentration over the measurement period, expressed in a decrease of the average chain length index (ACL) from 31.5 to 30.8. 3.3. Changes in leaf wax n-alkane dD values over a growing season We analyzed the dD values of the three major n-alkanes n-C29, n-C31 and n-C33. Low concentrations of the other compounds did not permit meaningful isotope analyses. The variability of the triplicate IRMS measurement was small (typical standard deviations were 4–5&) and always within the variability of the replicate leaf samples, see Table 2. In the subsequent discussion we therefore use the mean dD values from the three analyzed plant samples and present the standard deviation (see Table 2) to account for the natural heterogeneity among replicate samples. In general n-alkane dD values increased over the twomonth growing period by about 20&. The seasonal variability of dD values was similar for the three different n-alkanes (Fig. 2), with more negative dD values for the longer chain length compounds. The seasonal variability of the apparent hydrogen isotope fractionation eapp (between soil water dD and n-alkane dD values) was up to 40& and similar for all three analyzed n-alkanes. The observed seasonal range in n-alkane dD variability is in accord with previous observations from individual trees (Pedentchouk et al., 2008) and tree stands (Sachse et al., 2009). No significant correlation between soil water dD or predawn leaf water dD values and the dD values of the three

cummulative n-alkane concentration [ng/g dry weight]

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60000

50000

40000 n-C25 n-C27 n-C29 n-C31 n-C33 n-C35

30000

20000

10000

0 100

110

120

130 140 150 Day in Year

160

170

180

Fig. 3. Concentration of n-alkanes over the sampling period. Error bars on top of each box denote the respective standard deviation of the compound among the three replicates analyzed.

n-alkanes was observed. Instead, we observed significant correlations of dD values of leaf water at midday with dD values of n-C31 (r2 = 0.5, p = 0.021) and n-C33 (r2 = 0.8, p < 0.001), while the relationship with n-C29 dD values was not significant (r2 = 0.2, p = 0.168). We also found significant relationships, when we compared eapp (the apparent fractionation between n-alkanes and soil water) with the maximum leaf water enrichment (DLmax) for n-C31 (r2 = 0.5, p = 0.027; data not shown) and n-C33 (r2 = 0.6, p = 0.011) alkane dD values (Fig. 4). The poor correlation of n-C29 dD values (and n-C29 eapp) with DLmax is largely caused by the more positive dD values of the sample collected in the early season (DOY 103). It could be that in the beginning of the season plants use a hydrogen source other than current leaf water for n-C29 such as stored substrates (i.e. deuterium enriched starch) (Damesin and Lelarge, 2003; Sessions, 2006). Evidently, when removing the DOY 103 data point from the statistical evaluation, the relationship between midday leaf water and n-C29 dD values and the relationship between eapp C29 and DLmax becomes significant (r2 = 0.5, p = 0.03 and r2 = 0.5, p = 0.036, respectively). The relationships between midday leaf water and n-alkane dD values and between eapp and DLmax provide the first empirical evidence that leaf wax n-alkanes are affected by the isotope composition of deuterium enriched leaf water. We urge caution, however, to conclude that dD values in leaf wax n-alkanes reflect the day-to-day variability of leaf water evaporative enrichment in deuterium. The data we show here originate from three different barley leaf generations that had developed over the course of the experiment (see Section 2). While leaf wax n-alkanes showed statistically significant differences in dD values across the different leaf generations, no significant differences were detected for n-alkane dD values within a given leaf generation (Figs. 2 and 5, one-way between-subject analysis of variance (ANOVA)). This suggests that dD values of the different leaf generations follow the overall seasonal trend in DLmax but that leaf wax n-alkane dD values remained constant within a given leaf generation although the dD values of the corresponding leaf water was yet highly variable. The variance across leaf generations was

significant below the 95% confidence level (p = 0.004) for the concentration weighted average dD values (as well as for the highest concentrated n-alkanes n-C31 (p = 0.036) and n-C33 (p = 0.004) but not for n-C29, see discussion above). In summary, these data suggest that once a leaf was established, little additional leaf wax n-alkanes were produced during the remaining life of the leaf. Essentially, the dD values of n-alkanes were ‘locked in’ early in the development of the leaf. The establishment of leaf wax n-alkane dD values early in the life of a leaf also explains, why the relationship between eapp C33 and DLmax was strongest when only the youngest leaves of the three leaf generations were included in the analyses (‘age 0 weeks’). This is, because only the leaf wax n-alkane dD values of these young leaves are affected by the current leaf water enrichment in deuterium (Fig. 4). In contrast, the relationship between eapp C33 and DLmax weakens substantially when all leaves of all three generations (ages 0–5 weeks) were included in the analyses. The results we show here are in agreement with previous studies that have shown the strongest increase in total wax abundance as well as the majority of changes in the chemical composition of plant leaf waxes occurs during early development of leaves (Jetter and Schaffer, 2001; Richardson et al., 2005; Jetter et al., 2006) but remains relatively constant in mature leaves, at least in the absence of environmental stress. Our suggestion that leaf wax dD values of grasses are ‘locked in’ early during the growth of the leaf, should result in a 1:1 or ‘ideal relationship’ between leaf water dD and leaf wax dD values for the newly developed leaves (age 0 weeks). This is because a change in leaf water dD would directly be transferred into leaf wax dD with the same magnitude. An ‘ideal relationship’ results from a simple twopool relationship between dDsource and dDproduct where the slope (a) and intercept (e) derived fractionation factors for this relationship are equal (e.g. dDproduct = a  dDsource + e, where e = (a  1)  1000), see (Sessions and Hayes, 2005). If d values and not absolute ratios are used for calculations a slope of 1 is not to be expected (e.g. if a approaches unity, e would approach zero, see above), even for an ‘ideal relationship’. For the data we show here, the slope of the relationship between midday leaf water and dD of n-alkanes of newly developed leaves (age ‘0 weeks’) or a is 0.6, while the intercept, or e is 189 (Fig. 4) – and as such slope and intercept derived fractionation factors do not agree. This disagreement between slope (a = 0.6) and intercept (e = 189) derived fractionation factors implies, that a simple two-pool relationship (between leaf water midday dDleaf water midday and dDwax) cannot explain our observations. In fact, a slope of a = 0.6 and an intercept e = 189 suggests that dDwax is changing less than predicted from dDleaf water midday. A plausible explanation is that an additional, more deuterium depleted water pool may contribute to the biosynthetic source water for n-alkane biosynthesis. Two scenarios are conceivable: Temporal variability in leaf water dD. By comparing dDwax with dDleaf water midday values we do not account for the diurnal variability in the leaf water dD values. The

Table 2 dD values of n-alkanes from barley grass for the individual replicate plants and mean values per sampling date (DOY – day of year) and concentration weighted average n-alkane dD values. Sampling date

DOY

Sample Leaf Leaf age n-C25 Stdev n-C27 generation (weeks)

Stdev n-C29 Stdev n-C31 Stdev n-C33 Stdev Mean values per sampling (stdev of replicates)

Concentration weighted average dD

n-C29 Stdev n-C31 Stdev n-C33 Stdev 103

22.04.05

112

04.05.05

124

12.05.05

132

20.05.05

140

27.05.05

147

03.06.05

154

10.06.05

161

16.06.05

167

24.06.05

175

1Ma 1Mb 1Mc 2Ma 2Mb 2Mc 3Ma 3Mb 3Mc 4Ma 4Mb 4Md 5Ma 5Mb 5Md 6Ma 6Mb 6Mc 7Ma 7Mb 7Mc 8Ma 8Mb 8Mc 9Ma 9Mb 9Mc 10Ma 10Mb 10Md

1 1 1 1 1 1 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

0 0 0 1 1 1 0 0 0 1 1 1 0 0 0 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5

206 14

174 195

11 9

197.14 5.11

190 n.d. 195 207 204 212 n.d. n.d. 199 n.d. 197 n.d. n.d. 182 190 203 203 n.d. 196 191 192 192 193 n.d. n.d. n.d. 194 n.d. 197 200

7 5 2 5 3 6 – 7 6 7 4 2 2 3 17 2 5 6 9 7 7 4 3

217 n.d. 211 218 218 219 202 n.d. 209 196 205 n.d. 191 192 189 208 201 n.d. 201 184 200 n.d. 202 197 195 192 206 n.d. 208 213

1 3 8 7 4 5 5 6 7 12 1 7 8 5 7 3 9 3 4 6 1 13 4 2 2

218 n.d. 219 221 222 221 213 n.d. 213 204 210 209 201 192 196 209 208 n.d. 199 194 207 n.d. 197 n.d. n.d. n.d. 206 n.d. 201 205

2 1 5 1 1 6

193 4

214 4

219 1

212

208 4

218 1

221 0

218

4 4 3 0 5 1 6 6 5

199 n.a.

206 5

213 0

209

197 n.a.

201 6

208 3

204

186 5

191 1

196 4

192

203 0

204 5

208 0

206

193 2

195 9

200 6

197

4 6 6 7 4 6 7 1

192 1

199 3

197 n.a.

197

194 n.a.

198 8

206 n.a.

200

2 8

199 2

210 4

203 3

205

Seasonal origin of leaf wax n-alkane dD values in grasses

13.04.05

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D. Sachse et al. / Geochimica et Cosmochimica Acta 74 (2010) 6741–6750 -110 all: week 0:

-120

app app

nC33 = 0.5x - 189.9; r 2 = 0.6 nC33 = 0.6x - 189.4; r 2 = 0.99

app

nC 33 [‰]

-130 -140

0 1

-150

1 2

-160

0

3

5

-170

1

4

0

-180 -190 0

10

20

30

40 50 [‰]

60

70

80

L max

Fig. 4. Relationship between the apparent hydrogen isotope fractionation (eapp) between soil water and n-alkanes and the maximum leaf water enrichment (DLmax). Numbers correspond to the age of the leaf in weeks. The grey line is the linear regression through all samples, the black line is the linear regression through the week 0 samples only, respective regression equations are given.

majority of leaf waxes may not be synthesized from the most enriched midday leaf water but at least partly from more depleted leaf water earlier or later in the day. When assuming a mixture of 2/3 midday leaf water and 1/3 dawn leaf water the relationship between source water dD and n-C33 dD is characterized by dDn-C33 = 0.823  dDwater  186 (r2 = 0.99), which is klose to an ideal relationship between source water dD and n-C33 dD (with e = 186 a would be 0.814). Spatial variability in leaf water dD. In our analyses of bulk leaf water we did not account for any spatial variability in leaf water dD. It is, however, well established that leaf water in grasses exhibits a strong enrichment gradient from the base to the top of the leaf blade, with stronger enrichment at the leaf top (Helliker and Ehleringer, 2000, 2002). Leaf growth takes place through cell elongation at the base of the leaf and relies on photosynthate imported from ma-

ture leaves in the early stage of leaf development. On the other hand, wax synthesis in barley leaves has been shown to commence only in the distal portion of this elongation zone (23–45 mm from the base of the leaf) (Richardson et al., 2005). If the majority of leaf waxes are formed in this lower portion of the leaf the corresponding leaf water may be less enriched in deuterium than the bulk leaf water that we measured here. Our finding that leaf wax dD values show little variation within a leaf generation and that thus the dD signal of leaf wax n-alkanes is determined early in the ontogenesis of a leaf is in contrast to observations on temperate, deciduous tree leaves, where leaf wax n-alkane production has been proposed to continue throughout the growing season (Gulz and Muller, 1992; Lockheart et al., 1997; Sachse et al., 2009). Physiological and life history differences between the studied barley plants and the previously studied temperate trees may be responsible for these contrasting observations. Grasses, such as barley produce new leaf generations throughout their vegetative phase and the older leaves are subject to senescence, whereas most deciduous trees produce leaves only once a year that remain until the end of the vegetation period. Ongoing leaf wax abrasion as well as periods of drought or other environmental stress may require leaf wax renewal year-round (Baker, 1974; Maffei et al., 1993; Jenks et al., 2001). If the timing of leaf development is as important for the establishment of leaf wax n-alkane dD values as suggested by our data, our results may partly explain the large variability in leaf wax lipid dD values in leaves of different plant species (Sachse et al., 2006; Hou et al., 2007). Samples from different tree and grass species may represent different leaf ages and therefore different time periods of the season with different degrees of leaf water enrichment. We suggest that the time of leaf development should be taken into account when comparing leaf wax lipid dD values with source water dD values from different plants as well as when studying seasonal variability. 4. CONCLUSIONS

-180

n-alkane D [‰] vs VSMOW

F = 13.8004; p = 0.004 -190

-200

-210

-220

-230

1

2 leaf generations

3

Fig. 5. Box and whisker plot of the concentration weighted average dD values of the different leaf generations, F ratio and pvalue correspond to the results of a one-way between subjects ANOVA and confirm that the variability of n-alkane dD values is significant only between the leaf generations but not among them.

In the present study we show for the first time that seasonal variability of leaf water enrichment over a growing season is recorded in leaf wax n-alkane dD values of barley plants. In contrast, the seasonal isotopic variability of soil water (e.g. precipitation) is not directly recorded. Since leaf water enrichment at the Oensingen site is mainly driven by VPD, which is determined by temperature and relative humidity, the variability of leaf wax n-alkane dD values records the changes of these parameters over the growing season. We, however, also show that leaf wax n-alkane dD values do not record the day-to-day variability of leaf water dD: while our data show significant variation of leaf wax dD values across generations, no such effects were found within an individual leaf generation. These results suggest that the hydrogen isotope composition of the barley derived leaf wax n-alkanes is determined by the leaf water present early during leaf development and that only little if any additional leaf wax n-alkanes are synthesized once a leaf is fully developed.

Seasonal origin of leaf wax n-alkane dD values in grasses

With regard to the interpretation of paleoclimate data our results give strong evidence that grass-derived leaf wax n-alkane dD values do not directly record dD values of precipitation, but rather a signal that is strongly modified by leaf water evaporative enrichment in deuterium. Hence, changes in leaf wax dD values in the sedimentary record should not be interpreted solely as changes in dD of precipitation, but dD values of leaf wax n-alkanes could also reflect changes in VPD or other parameters that are known to influence the isotopic composition of leaf water (Kahmen et al., 2008). Our data also suggest, that it is important to know the seasonal origin of the preserved signal, especially when the proxy is applied in climate regimes which are characterized by strong seasonal variations. The results we show here were obtained from a single species of an annual grass (barley). The generality of these results now needs to be tested in additional studies that include a larger species pool, ideally with plants from different life forms (evergreen vs. deciduous, trees vs. herbs) and different ecosystem types (mesic, arid, etc.). ACKNOWLEDGMENTS This research was supported by an Emmy-Noether Research Grant by the German Science Foundation (DFG) to Dirk Sachse (SA-1889/1-1). Ansgar Kahmen was supported by a Marie Curie Outgoing International Fellowship of the European Union (BiWaClim) and by the ESF programme SIBAE. We thank the Swiss Federal Agency for the Environment (Bundesamt fu¨r Umwelt BAFU, Nationale Grundwasserbeobachtung NAQUA) for providing the dD values of precipitation of the GNIP site at Suhr. We would like to thank Michael Gabriel (GFZ Potsdam) for help with lipid dD analysis and Nina Buchmann for providing access to the field site in Oensingen. Furthermore we would like to thank Francesca McInerney, Pratigya Polissar and one anonymous reviewer for the constructive comments, which helped to improve this manuscript.

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