ecosystem water-use efficiency among Douglas-fir forest, aspen forest and grassland ... Schaberg P.G., Wilkinson R.C., Shane J.B., Donnelly J.R. & Cali P.F..
Plant, Cell and Environment (2010) 33, 35–47
doi: 10.1111/j.1365-3040.2009.02049.x
Weather and climate controls over the seasonal carbon isotope dynamics of sugars from subalpine forest trees
pce_2049
35..47
JIA HU1, DAVID J. P. MOORE2 & RUSSELL K. MONSON1,3 1 Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO 80309, USA, 2Department of Geography, King’s College London, Strand, London, WC2R 2LS, UK and 3Cooperative Institute for Research in Environmental Sciences, University of Colorado, Boulder, CO 80309, USA
ABSTRACT We examined the environmental variables that influence the d 13C value of needle and phloem sugars in trees in a subalpine forest. We collected sugars from Pinus contorta, Picea engelmannii and Abies lasiocarpa from 2006 to 2008. Phloem and needle sugars were enriched in 13C during the autumn, winter and early spring, but depleted during the growing season. We hypothesized that the late-winter and early-spring 13C enrichment was due to the mobilization of carbon assimilated the previous autumn; however, needle starch concentrations were completely exhausted by autumn, and we observed evidence of new starch production during episodic warm weather events during the winter and early-spring. Instead, we found that 13C enrichment was best explained by the occurrence of cold night-time temperatures. We also observed seasonal decoupling in the 13C/ 12 C ratios of needle and phloem sugars. We hypothesized that this was due to seasonally-changing source-sink patterns, which drove carbon translocation from the needles towards the roots early in the season, before bud break, but from the roots towards the needles later in the season, after bud break. Overall, our results demonstrate that the 13C/12C ratio of recently-assimilated sugars can provide a sensitive record of the short-term coupling between climate and tree physiology. Key-words: carbon isotope; conifer; discrimination; mountain ecosystem; sugars; temperature.
INTRODUCTION Carbon isotope analyses of plant materials, such as sugars, starches, bulk leaf tissue and wood, have provided insight into plant water use efficiency across daily to decadal timescales (Brugnoli et al. 1988; Farquhar, Ehleringer & Hubick 1989; Dupouey et al. 1993; Ferrio & Voltas 2005). The ability to use plant carbon isotope composition (expressed as d 13C) to address a broad range of questions in biogeochemistry and plant ecophysiology is made possible by the discrimination against 13CO2 during diffusion and carboxylation in leaves (Farquhar, O’Leary & Berry 1982). Even at the Correspondence: J. Hu. Fax: +1 303 492 8699; e-mail: jia.hu@ colorado.edu © 2009 Blackwell Publishing Ltd
global scale, the theory underlying 13CO2 discrimination by leaves has been used to partition global CO2 uptake into its component terrestrial and ocean sinks (Keeling et al. 1995; Fung et al. 1997; Battle et al. 2000). Despite the broad use of 13C/12C ratios in biogeochemical studies, there remain significant gaps in our knowledge about how climate and weather affect carbon isotope discrimination (Bowling, Pataki & Randerson 2008). Filling these gaps is critical to the development of improved models aimed at predicting the consequences of future climate change. One approach that has been developed in recent years, and shows promise towards understanding the influence of climate on isotope discrimination, is the analysis of d 13CR, the carbon isotope ratio of ecosystem-respired CO2, as obtained from the intercept of ‘Keeling plots’ (Flanagan et al. 1996; Buchmann, Kao & Ehleringer 1997; Bowling et al. 2002; Pataki et al. 2003). Theoretically, this ratio should reflect the combined respiratory influences of heterotrophic and autotrophic components of the ecosystem, including those associated with soil, roots, leaves and stems. Variation in the d 13CR has been shown to correlate with short-term variation in weather (Bowling et al. 2002; Fessenden & Ehleringer 2003; McDowell et al. 2004; Lai et al. 2005; Alstad et al. 2007; Schaeffer et al. 2008) and longer-term variation in climate (Pataki et al. 2003; Ponton et al. 2006), and it has been assumed that this correlation is driven by influences on photosynthetically produced sugars, which are used as substrates for respiration (Högberg et al. 2001; Keel, Siegwolf & Korner 2006; Bowling et al. 2008). Recent studies have begun to focus on the sugars themselves, and to discern the short-term climate and weather influences on their carbon isotope composition (Pate & Arthur 1998; Cernusak et al. 2003; Keitel et al. 2003; Scartazza et al. 2004). However, these studies have been limited to temperate forests, dominated by deciduous trees, where cold or freezing temperatures were not considered. Given the importance to establishing links between climate and d 13CR, there is a need for a broader set of studies of climate-isotope interactions in additional ecosystems and including additional plant functional groups. We had two goals in conducting the studies described here: first, we aimed to examine the climate variables that most influence the d 13C values of needle and phloem sugars (d 13Cns and d 13Cps, respectively), and to determine if 35
36 J. Hu et al. dynamics in d 13Cns and d 13Cps correlate with changes in those climate variables at the time scales typical of changing weather systems. Second, we aimed to isolate possible causes of a pattern of 13C enrichment of both d 13Cns and d 13Cps that we observed during the autumn, winter and spring periods. This pattern of 13C enrichment is likely unique to plants that are capable of CO2 assimilation during periods when cold weather persists. In the subalpine forest ecosystem, the dominant trees exhibit the evergreen growth habit, and a large portion of annual carbon uptake occurs in the early spring when periods of cold weather occur frequently (Sacks, Schimel & Monson 2007). Overall, our studies provide a test of established paradigms concerning climate–isotope interactions when framed at time scales not typically explored, and within the context of seasonal climate forcings not typically considered.
METHODS We made our observations during the growing seasons of 2006 and 2007, as well as the winter/spring period of 2007/ 2008 at the Niwot Ridge AmeriFlux site. The site is located in a subalpine forest just below the Continental Divide (elevation 3050 m), in the Front Range of the Colorado Rocky Mountains, USA. The dominant tree species include Pinus contorta (lodgepole pine), Abies lasiocarpa (subalpine fir) and Picea engelmanii (Engelmann spruce). Generally, lodgepole pine dominates the ecosystem at the lower elevations of the site, whereas Engelmann spruce and subalpine fir dominate the ecosystem at higher elevations. Mean annual temperature is 1.5 °C and average precipitation is 800 mm, about 60% of which is in the form of winter snow (measured from the 10-year mean). However, in the summer, convective storms also bring rain to the site; generally beginning in late July. Characteristics of the site and associated carbon and water fluxes have been described in previous papers (Monson et al. 2002, 2005; Turnipseed et al. 2002; Moore et al. 2008).
Meteorological measurements We obtained values for air temperature, relative humidity and atmospheric saturation vapor pressure deficit (VPD) from 30-min averaged measurements at 21.5-m height on the main AmeriFlux tower (http://urquell.colorado.edu/ data_ameriflux/). Precipitation was measured using a heated tipping bucket rain/snow gauge (Campbell Scientific, Logan, UT, Met One Model 385). We measured volumetric soil moisture (q) at two depths: 5 cm and 15 cm, using time-domain reflectometry probes (Campbell Scientific, models CS615 and CS616). Soil water potential measurements were made using a soil water potential sensor (Campbell Scientific, model 257), which provided an integrated signal from the upper 15 cm of soil. Snow water equivalent (SWE) values for the Niwot Ridge LTER C1 site were obtained from the SNOTEL database (http:// www.co.nrcs.usda.gov/snow/snow/) for 2006 and 2007. The SWE values refer to cumulative snow for the season for
each respective day and incorporate responses to gain from snowfall and to loss from snowmelt.
Simplified Photosynthesis EvapoTranspiration Model (SIPNET) In order to determine if the trees were photosynthesizing during the first spring collection periods (April 10, 2006 and March 15, 2007), we used SIPNET to determine net ecosystem productivity (NEP), gross primary productivity (GPP) and ecosystem respiration (RE), where NEP = GPP - RE. The SIPNET model, based on the Photosynthesis-EvapoTranspiration (PnET) family of models (Aber & Federer 1992; Aber et al. 1995; Aber, Reich & Goulden 1996), was simplified for our purposes by decreasing the number of free parameters and run time (we used the model simplifications described by Braswell et al. 2005; Sacks et al. 2006, 2007). SIPNET contains two vegetation carbon pools and an aggregated soil carbon pool and simulates the carbon dynamics between these pools and the atmosphere. The vegetation pool is split into leaves and wood, where ‘wood’ refers to the combined pool of boles, branches and roots. The model performs two time steps per day: day and night. The lengths of the day and night-time steps in the model varied seasonally to account for changes in day length; fluxes were appropriately scaled for these changes in the length of the time steps. Both NEP and ET (evapotranspiration) observations were used to parameterize the SIPNET model. Using the protocol and parameter starting values detailed in Moore et al. (2008), we estimated 17 of the 32 model parameters using a variation of the Metropolis algorithm (Metropolis et al. 1953) modified by Hurtt and Armstrong (1996). The remaining 15 parameters were held constant at values estimated from the literature and from field studies at the Niwot Ridge site because they were difficult to estimate independently from the eddy flux data or because they had little or no effect on modeled NEE. The scheme used to assess the maximum likelihood outcomes of the model are discussed in detail in Braswell et al. (2005), Sacks et al. (2006) and the parameter values used are listed in Moore et al. (2008). In this data assimilation and analysis, GPP was constrained by the entire NEP flux record obtained from the tower-based measurements from 2005 through 2008.
Analysis of d 13Cns, d 13Cps and d 13C of bulk needle tissue We extracted sugars every 14 d during the growing season from phloem and needle tissues following the protocol of Gessler, Rennenberg & Keitel (2004). The same trees were tagged and used for sequential collections. We collected samples from six trees for each species and the trees were distributed broadly along a 100 m transect that ran east and west of the main flux tower. Phloem sugars were collected by cutting two or three 2-cm bark disks from the tree, 1.5 m from the base, peeling a thin layer of phloem from the core, © 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 35–47
Sugar carbon isotope ratios 37 rinsing with distilled water, placing the core into 2 mL of 15 mm sodium hexametaphosphate solution for 5 h, followed by pipetting the solution into vials and freezing at -20 °C. The sodium hexametaphosphate solution prevented callus formation while allowing phloem sugars to diffuse into the solution. Needles were collected from the upper tree crown and immediately frozen in liquid nitrogen. The needles included both current year and previous year needles, and were collected between 1000 h to 1200 h during each collection to avoid any diel patterns associated with changes in needle sugar and starch concentrations. Following bud break, we also compared the carbon isotope composition of current year needle sugars against previous year needle sugars to explore differences between needle sugars produced from needles differing in age class. In the lab, the needles were freeze-dried for 3 d, and then ground to a fine powder using liquid nitrogen and a mortar and pestle. Sugars were extracted from 150 mg of ground needle tissue using 150 mg of polyvinylpolypyrrolidone and 2 mL of distilled water combined in a vial, incubated at 10 °C for 1 h and then boiled for 2 min. The samples were centrifuged at 12,000 g for 10 min, the supernatant was decanted and frozen at -20 °C, and then the supernatant was freeze-dried. The supernatant was considered as the soluble fraction, which consists mainly of sugars, but other water-soluble compounds such as organic acids and amino acids were also present. Dried phloem and needle sugar samples were loaded into sample tins and weighed prior to isotope analysis. All samples were analyzed for 13C/12C ratios at the Center for Stable Isotope Biogeochemistry at the University of California, Berkeley. The 13C/12C ratio is expressed using d notation (d 13C) with units of parts per thousand (‰).
Starch concentration Starch was extracted from needles collected from March 15, 2007 to April 15, 2008. We followed the extraction protocol of Schulze et al. (1991), where starch content was assumed to be proportional to the amount of free glucose observed following starch hydrolysis. Briefly, an initial extraction procedure was performed to remove soluble sugars using 1 mL of 80% ethanol with 50 mg of dried leaf material. The extracted leaf residue was allowed to air dry overnight. Water was then added to the leaf material and autoclaved for 2 h at 121 °C to force the starch into solution. This solution was centrifuged and the supernatant containing the soluble starch was removed. Finally, two hydrolases were used to degrade starch into glucose: amyloglucosidase (Apergillus niger) and alpha-amylase (Bacillus sp.). The degraded glucose samples were kept frozen until further analysis. Glucose concentrations were analyzed using highperformance anion-exchange chromatography-PAD with a Beckman model 126 HPLC (Fullerton, CA, USA), a Dionex CarboPak-PA1 (Cedar Rapids, IA, USA) separation column fitted with a CarboPack-PA1 guard column and a Dionex ED40 pulsed amperometric detector. Each © 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 35–47
sample was eluted with 0.2 M NaOH isocratic at flow rate of 1.0 cm3min-1. Glucose standards were purchased from Sigma (St. Louis, MO, USA). Starch concentrations are expressed as a percentage of dry leaf biomass that is glucose derived from starch.
Correlation between d 13Cns, d 13Cps and environmental variables In our studies of climate controls over d 13Cns and d 13Cps, we chose to focus on a set of environmental variables that have been demonstrated in other studies to influence either bulk leaf tissue d 13C or respired d 13CR. These variables included soil moisture (q), air temperature, soil temperature and vapor pressure deficit (VPD). Daily soil moisture and soil temperature were averaged over a 24-h period, daytime temperature and VPD were averaged between 10:00 to 17:00, and night-time temperature was averaged between 24:00 to 6:00. We also explored potential lags in the response of d 13Cns and d 13Cps to environmental variables by correlating the d 13C value of recently fixed sugars with environmental variables averaged for 1–5 d prior to the collection dates.
Statistical analysis In order to test for differences in d 13Cns and d 13Cps among the three species, we ran a repeated measure in anova (SAS proc mixed, The SAS Institute, Version 9.2, Cary, NC), with species, specific tree ID and date as class variables. The interaction between species and date was also examined. To test for a relationship among d 13Cns, d 13Cps and various abiotic factors (VPD, soil moisture, soil temperature, daytime and night-time temperatures), we ran a simple linear regression (SAS, proc reg) from 1 d to 5 d before the collection dates. Significance was assigned in all analyses for P < 0.05.
RESULTS Meteorological measurements The two study years of 2006 and 2007 exhibited markedly different winter and summer climates. During 2006, cumulative SWE for the winter was 43.9 cm, about 10% lower than the previous 9-year mean (49.1 cm) (from 1997 to 2005). The maximum SWE in 2006 was 12.8 cm, which occurred on March 27 (Fig. 1a). During the month of June, the site received lower-than-normal rain and soil water potential reached a seasonal minimum by the end of June (Fig. 1e). A large rain event on July 8, however, rehydrated the soils. Small rain events throughout the remainder of the growing season had little effect on soil water potential at 15 cm. Overall, in 2006, the site received 24 cm of summer rain, which was 22% lower than the 9-year mean (30.8 cm). In contrast, cumulative SWE in 2007 was 57.4 cm, about 17% higher than the previous nine-year mean. The maximum SWE in 2007 was 17.9 cm, which occurred on
38 J. Hu et al.
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Figure 1. Meteorological measurements made from the Niwot Ridge AmeriFlux tower site for 2006 and 2007. (a,b) Snow water equivalent (SWE), (c,d) precipitation, (e,f) soil water potential (WP) measured between 0–15 cm depth, and (g,h) average monthly daytime air temperature (open circle, solid line) and night-time air temperature (filled circle, dashed line).
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April 27, 2007 (Fig. 1b), and the larger snow pack in 2007 began melting a week later than in 2006. During July and August of 2007, the site also received more rain than in 2006, and the soils remained wetter during the later parts of the growing season (Fig. 1f). The rainstorms in 2007 were more evenly spread across the summer, as opposed to the single large storm in July of 2006. Overall, in 2007, the site received 31.2 cm of rain, which was 1% higher than the previous 9-year mean. The late-winter and spring air temperature regimes were different between 2006 and 2007, consistent with the differences in snow-melt dynamics. Mean daytime air temperatures for January, February and March were warmer in 2007 than in 2006 (Fig. 1g,h) (P < 0.001). However, mean daily air temperature in April, May and June were cooler in 2007 than in 2006 (Fig. 1g,h) (P < 0.001). During spring
(March–June), night-time air temperatures often dropped below freezing. In both years, during May, June and July, mean daytime air temperatures increased progressively to reach a maximum in August. This same pattern was also observed in the night-time air temperature record.
SIPNET In 2006, SIPNET modelled NEP became positive (positive carbon uptake by the entire forest) on April 8, and GPP commenced on April 7 (Fig. 2a,c). In 2007, NEP fluctuated between positive and negative values, but after March 4, NEP was consistently positive (Fig. 2b). In 2007, GPP also fluctuated between positive and negative values starting in January, but after February 18, 2007, GPP remained consistently positive (Fig. 2d). By convention, GPP is often used © 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 35–47
Sugar carbon isotope ratios 39
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in the ecosystem flux literature to mean the difference between daily net photosynthesis and daily mitochondrial respiration in autotrophic tissues; thus it is possible to be negative in sign when day and night needle respiration is estimated by the model to exceed daytime net photosynthesis. In 2006, both NEP and GPP increased quickly during spring; however, in 2007, although NEP and GPP became positive earlier, the seasonal increase in the magnitude of GPP and NEP was slow to develop, compared with 2006. In 2006, ecosystem respiration (RE) from January to April in 2006 was lower than that for 2007. In both years, RE began to increase during the same period as NEP and GPP (in May), but maximum RE was reached later in the growing season than NEP or GPP.
Carbon isotope ratios Variation in d 13Cns value exhibited individual significant effects due to date (P < 0.0001) and species (P < 0.0001) for © 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 35–47
Figure 2. (a,b) Net ecosystem productivity (NEP) as estimated from the Simplified Photosynthesis EvapoTranspiration Model (SIPNET) for 2006, 2007 and 2008. (c,d) Gross Primary Productivity (GPP, black line) and ecosystem respiration (RE,, grey line) as estimated from SIPNET, where NEP = GPP – RE. (e,f) Extracted needle sugars d 13C (d 13Cns) from subalpine fir (open circle, n = 6), lodgepole pine (shaded triangle, n = 6) and Engelmann spruce (dark square, n = 6) collected in 2006, 2007, and 2008.
both 2006 and 2007. For the seasonal pattern, in 2006 and 2007, all three species had the least negative d 13Cns values at the beginning of the season, and the d 13Cns values became more negative as the season progressed (Fig. 2e,f). In 2006, the d 13Cns of all three species reached a minimum asymptote during June. In 2007, no clear asymptote was reached and the d 13Cns values fluctuated from week to week. However, in both 2006 and 2007, the most negative d 13Cns for all three species occurred around the same time in August. In our comparison of current year d 13Cns versus previous year d 13Cns, we found no difference in the carbon isotope ratio of needle sugars in needles produced in two different years. The d 13Cns for current year needles was not significantly different from the d 13Cns for previous year’s needles on two sampling dates (June 28 and July 12) for any of the three tree species (data not shown). Phloem sugars were only analyzed for 2007 because we were still working on a phloem collection method during
40 J. Hu et al.
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Figure 3. Phloem sugars d 13C (d 13Cps) of subalpine fir (open circle, n = 6), lodgepole pine (shaded triangle, n = 6) and Engelmann spruce (dark square, n = 6) collected in 2006, 2007 and 2008. All the trees sampled for phloem sugars were the same trees sampled for leaf sugars.
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2006. Variation in the d 13Cps value exhibited a significant effect due to date (P < 0.001), but not species (P = 0.09). However, there was a significant combined interaction between species and date (P < 0.001). Similar to leaf sugar data, phloem sugars were enriched with 13C during the early part of the growing season (Fig. 3). After the snowpack began to melt and the soil moisture levels increased (between the May 11 and May 23 collection dates), d 13Cps began to decrease and exhibited the most negative values by June 1.This minimum in d 13Cps occurred almost 2 months before the minimum in d 13Cns values. For the remainder of the growing season, d 13Cps increased, resulting in maximum values on November 29. Phloem sugars were, on average, more enriched in 13C than needle sugars for all 18 collection dates in 2007. Subalpine fir d 13Cps values were higher by 2.16‰, lodgepole pine d 13Cps values were higher by 1.46‰, and Engelmann spruce d 13Cps values were higher by 1.71‰. A significant correlation was observed between d 13Cns and d 13Cps (P = 0.0001, R2 = 0.13) (Fig. 4), though variance between the values was high and the correlation only explained 13% of the total variance.
date, while in 2007, the best correlation was obtained between d 13Cns and daytime air temperature 3 d prior. In both 2006 and 2007, we found a significant relationship between soil temperature and d 13Cns for 1–5 d prior to the collection date, but the temperature 3 d prior to the collection date yielded the strongest relationship (Fig. 5c,d, Table 1). In 2006, there was no significant relationship between d 13Cns and soil moisture, even for values up to 5 d prior to collection date (Table 1). However, in 2006, when soil moisture measurements during the period of spring snowmelt were removed (q > 0.30 m3m-3), there was a significant negative relationship between soil moisture and the d 13Cns (fir R2 = 0.58, pine R2 = 0.76, spruce R2 = 0.75, P < 0.01 for all three species; data not shown in Table 1). In 2007, we found a significant, positive relationship between soil moisture and d 13Cns for daily moisture values both 1 and 2 d
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Correlation between d 13Cns, d 13Cps and environmental variables d
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The strongest correlation between the d 13Cns and the five environmental factors (daytime air temperature, night-time air temperature, soil temperature, soil moisture, VPD) occurred for daytime and night-time air temperature for both 2006 and 2007, although only the results for daytime temperature are shown. In both years, there was a significant negative relationship between daytime temperature and d 13Cns for all three species when regressed against temperatures for 1–5 d prior to the collection date (Fig. 5a,b, Table 1). In 2006, the best correlation was estimated for d 13Cns and daytime air temperature 2 d before the collection
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Figure 4. Correlations between d 13C needle sugar (d 13Cns) and d 13C phloem sugar (d 13Cps) for year 2007. There was a positive relationship between these two variables (P < 0.0001, R2 = 0.13). The line represents a 1:1 relationship. © 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 35–47
Sugar carbon isotope ratios 41 –24 (a)
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Figure 5. The relationship between d 13C needle sugar (d 13Cns) and daytime air temperature (°C), and d 13Cns and soil temperature (°C). Fir d 13Cns and regression lines are represented by open circles and solid lines, respectively, pine d 13Cns are represented by shaded triangles and dotted lines, and spruce d 13Cns are represented by shaded squares and dashed lines. In both years, as air and soil temperatures increased, d 13Cns became more negative. (a) Year 2006: fir R2 = 0.66, pine R2 = 0.62, spruce R2 = 0.58. (b) Year 2007: fir R2 = 0.36, pine R2 = 0.40, spruce R2 = 0.55. (c) Year 2006: fir R2 = 0.34, pine R2 = 0.39, spruce R2 = 0.31. (d) Year 2007: fir R2 = 0.17, pine R2 = 0.15, spruce R2 = 0.21. The regression analysis for all relationship was significant to P < 0.05.
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before the collection date (P < 0.001). However, the correlation coefficients were low (R2 < 0.09 for all three species) and the sign of the relationship was opposite to the relationship we found in 2006 (Table 1). In 2006, there was a significant negative relationship with respect to atmospheric VPD, 2 and 3 d before the collection date, but the VPD 2 d before the collection date yielded the strongest relationship (Table 1). In 2007, the negative relationship between d 13Cns and VPD was significant 1 d before the collection date, but the R2-values were low (R2 < 0.08 for all three species) (Table 1). Because we found no significant species difference in d 13Cps, we combined all the species d 13Cps values to regress against environmental variables. We found a significant negative relationship between d 13Cps and daytime air temperature from 1 to 5 d before the collection date, although the R2 values were low (Table 1). There was also a highly significant negative relationship between d 13Cps and soil temperature 5 d before the collection date (R2 = 0.84, P < 0.0001). We also found a significant correlation between d 13Cps and VPD for 1–5 d prior to the collection dates, but the R2-values were also low. We found no significant relationship between d 13Cps and soil moisture for 1 through 5 d prior to the collection date. © 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 35–47
Starch concentration Needle starch concentrations were lowest during the fall and winter period and highest during the early spring just prior to bud burst (Fig. 6). On January 2, 2008, the average starch concentration for all three species was only 0.1%, while on May 24, 2007, the average starch concentration for all three species was 9.4%. We found a significant effect of date on starch concentration (P < 0.0001). After reaching a minimum the previous autumn, starch concentrations began to increase again sometime between January 1 and February 1, and increased consistently through the remainder of the winter and into the spring. We also found a significant difference in starch concentrations among species. Lodgepole pine had higher starch concentrations than the other two species, but all three species were significantly different from one another (P < 0.0001). We also found an interaction between species and date (P < 0.001).
DISCUSSION Seasonal changes in climate and day-to-day changes in weather at this subalpine forest site influenced the carbon
42 J. Hu et al. Table 1. P-values and R2-values from regression analysis among d 13C needle sugar (d 13Cns), d 13C phloem sugar (d 13Cps), and air temperature, soil temperature, soil moisture (q) and vapour pressure deficit (VPD), for 1 through 5 d prior to sample collections in 2006 and 2007. The three species were combined for d 13Cps because there were no species’ differences. Asterisks denote level of significance (*P < 0.05, **P < 0.01, ***P < 0.001) Air temperature (°C)
2006 d 13Cns
2007 d 13Cns
2007 d 13Cps
Species
1 d prior
2 d prior
3 d prior
4 d prior
5 d prior
Fir Pine Spruce Fir Pine Spruce All species
R2 = 0.19*** R2 = 0.18*** R2 = 0.18*** R2 = 0.09*** R2 = 0.09*** R2 = 0.14*** R2 = 0.13***
R2 = 0.66*** R2 = 0.62*** R2 = 0.58*** R2 = 0.05* R2 = 0.05* R2 = 0.09** R2 = 0.07***
R2 = 0.35*** R2 = 0.50*** R2 = 0.37*** R2 = 0.36** R2 = 0.40** R2 = 0.55*** R2 = 0.09***
R2 = 0.1** R2 = 0.24** R2 = 0.15*** R2 = 0.1** R2 = 0.09** R2 = 0.17*** R2 = 0.09***
R2 = 0.08* R2 = 0.17*** R2 = 0.17*** R2 = 0.15*** R2 = 0.1** R2 = 0.2** R2 = 0.13***
Soil temperature (°C)
2006 d 13Cns
2007 d 13Cns
2007 d 13Cps
Species
1 d prior
2 d prior
3 d prior
4 d prior
5 d prior
Fir Pine Spruce Fir Pine Spruce All species
R2 = 0.33*** R2 = 0.39*** R2 = 0.32*** R2 = 0.15*** R2 = 0.15*** R2 = 0.21*** R2 = 0.09***
R2 = 0.34*** R2 = 0.39*** R2 = 0.31*** R2 = 0.14*** R2 = 0.15*** R2 = 0.09** R2 = 0.08***
R2 = 0.34*** R2 = 0.38*** R2 = 0.30*** R2 = 0.17*** R2 = 0.15*** R2 = 0.09** R2 = 0.09***
R2 = 0.32*** R2 = 0.37*** R2 = 0.3*** R2 = 0.17*** R2 = 0.15*** R2 = 0.09** R2 = 0.08***
R2 = 0.30*** R2 = 0.36*** R2 = 0.28*** NS R2 = 0.18** R2 = 0.07* R2 = 0.84***
Soil moisture (m3 m–3)
2006 d 13Cns
2007 d 13Cns
2007 d 13Cps
Species
1 d prior
2 d prior
3 d prior
4 d prior
5 d prior
Fir Pine Spruce Fir Pine Spruce All species
NS NS NS R2 = 0.09** R2 = 0.05* R2 = 0.05* NS
NS NS NS R2 = 0.09** R2 = 0.05* R2 = 0.05* NS
NS NS NS R2 = 0.08** NS NS NS
NS NS NS R2 = 0.07* NS NS NS
NS NS NS NS NS NS NS
VPD (kPa)
2006 d 13Cns
2007 d 13Cns
2007 d 13Cps
Species
1 d prior
2 d prior
3 d prior
4 d prior
5 d prior
Fir Pine Spruce Fir Pine Spruce All species
NS NS NS R2 = 0.08** R2 = 0.06* R2 = 0.05* R2 = 0.17***
R2 = 0.31*** R2 = 0.30*** R2 = 0.26*** NS NS NS R2 = 0.05***
R2 = 0.19*** R2 = 0.25*** R2 = 0.23*** R2 = 0.04* NS NS R2 = 0.015*
NS NS NS R2 = 0.08** NS NS NS
NS NS NS NS NS NS R2 = 0.03**
isotope ratios of both needle and phloem sugars. The fact that we found correlations between weather and the d 13C value of sugars supports the assumptions of past studies that climate dynamics are transmitted to dynamics in d 13CR, the carbon isotope ratio for ecosystem respiration, through sugar substrates (Högberg et al. 2001; Bowling et al. 2008). Of particular note in our results were: (1) climate appears to impose a seasonal trend on the sugar carbon isotope ratio with more enrichment by 13C during the autumn-winterspring period, which decreases as the growing season progresses, (2) seasonal dynamics in temperature appear to
explain more of the overall growing season variance in sugar isotope values than soil moisture, (3) short-term dynamics in weather have the potential to affect sugar carbon isotope ratios with a lag of a few days, consistent with the lags observed in past studies of weather effects on d 13CR (Bowling et al. 2002; Lai et al. 2005; Schaeffer et al. 2008), and (4) the seasonal patterns of 13Cns and 13Cps differed, suggesting that sugars from needles and phloem were decoupled, particularly during the period just before and during bud burst and the subsequent phase of rapid shoot growth. © 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 35–47
Sugar carbon isotope ratios 43
Figure 6. Starch concentrations for fir (open circles), pine (shaded triangles) and spruce (dark squares) for 2007 and 2008, expressed as percent starch of dry needle mass. Starch concentrations began to increase from April to June 2007, and then decreased quickly following bud burst (around June 20, 2007). Starch contents continued to decrease and remained low for fall 2007 and winter 2007/2008. All three species were significantly different from one another (P < 0.001).
Climate imposes a seasonal trend on sugar carbon isotope ratio The enrichment with 13C in both needle and phloem sugars had already occurred by the beginning of spring in 2006 and was observed to occur progressively during the autumn and winter of 2007–2008 (Figs 2 & 3). We evaluated two competing explanations for these observations: (1) sugars collected in the autumn were enriched in 13C due to photosynthetic fractionation enhanced by dry soils or cold temperatures, and sugars collected in early spring were converted from 13C-enriched starch that was produced the previous autumn (Brugnoli et al. 1988; Gleixner et al. 1998; Tcherkez et al. 2004), and (2) sugars collected in the late winter and early spring were not produced through conversion from starch from the previous autumn, but represented recently-assimilated photosynthate that was enriched in 13 C due to climate conditions in the winter and spring. Our observations showed that starch reserves were lowest during the autumn (Fig. 6), with starch concentrations being near zero at the same time that needle and phloem sugars begin to show significant 13C enrichment. Furthermore, we also found starch concentrations to not increase again until the following winter and early spring, reaching maximum concentrations at the same time when d 13Cns began to become depleted in 13C. Thus, if the enriched 13C content of sugars collected during the winter and early spring reflected a signal carried over from starch produced the previous autumn, then individual sugar molecules (in both needles and phloem) would have to have relatively long turnover times (on the order of several months). Although we cannot rule this out, conventional wisdom would suggest that sugars turn over more frequently than that, even during the winter, and especially in the phloem. There is more support © 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 35–47
at present for the hypothesis that the sugars produced in late-winter and early spring reflect recent photosynthate that occurs during short ‘mild’ weather events in an otherwise cold climatic period (Freeland 1944; Schaberg et al. 1995; Strand et al. 2002). For example, we observed that needle starch concentrations began to increase during January and February of 2007, which should only be possible with active photosynthesis. The mean daytime temperature during these months met or exceeded 0 °C on 27 different dates, and in 8 of the days it exceeded 5 °C. Our past studies have shown that needles of the three dominant species at this site are capable of assimilating atmospheric CO2 at relatively high rates within the range of 0–5 °C (Huxman et al. 2003). Thus, we propose that during periodic mild winter weather events, needles of all three species assimilated CO2, storing part of it as starch in the needles, and transporting part of it in the phloem, during which time we detected 13C enrichment in extracted sugars. We conclude that the enrichment with 13C of needle and phloem sugars during the autumn, winter and early spring was due to the effects of cold temperatures. We observed a tight statistical correlation between both air temperature and soil temperature with variation in d 13Cns. In mountain forest ecosystems, night-time temperatures below 4 °C during the autumn and spring can cause a reduction in stomatal conductance the following day (Kaufmann 1982; Smith et al. 1984). During the autumn, in October and November, GPP was still relatively high (Fig. 2c,d), but night-time air temperatures regularly decreased to below 4 °C and may have decreased ci/ca, leading to an enrichment in 13C in needle sugars. During the transition from winter to early spring in February, March, April and May, we also observed significant rates of positive GPP in 2007, and night-time air temperatures continued to be below the 4 °C threshold on a regular basis (Fig. 1g,h). The fact that all three tree species are capable of photosynthesis at relatively low temperatures, combined with reduced stomatal conductance during the winter and early-spring period (see Huxman et al. 2003; Monson et al. 2005), leads us to the conclusion that significant quantities of sugars were produced, but at relatively low ci/ca ratios, resulting in 13C enrichment.
Temperature explains more of the seasonal variance in sugar isotope ratio than soil moisture We had hypothesized that the enrichment in 13C during autumn was due to either low moisture or low temperature conditions. During autumn in both years, we found 13 C enrichment occurring despite persistent precipitation events and high soil moisture levels. These results were inconsistent with our original hypothesis. Schaeffer et al. (2008) found a significant correlation between soil moisture and d 13CR at Niwot Ridge, but only when soil moisture levels were less than 0.15 m3m-3. Similarly, we observed only a significant correlation between d 13Cns and soil moisture when soil moisture levels were below 0.30 m3m-3 in 2006.
44 J. Hu et al. The lack of correlation for all soil moisture levels appeared to be highly influenced by data from the early part of the growing season; i.e. the time during snow melt when soil moisture varied little, but temperature varied greatly. In 2007, we observed a positive relationship between d 13Cns and soil moisture, which was opposite to 2006. However, in 2007, soil water potential was higher throughout the growing season (Fig. 1e,f) and midday plant water potential demonstrated that all three species experienced higher water potentials through the season, compared with 2006 (data not shown). Furthermore, although the relationship was significant, the R2-values were very low (R2 < 0.09), suggesting that very little of the variance could be explained by soil moisture alone. Although we were unable to fully explain this positive relationship, other factors such as temperature and radiation may have played a role in complicating the relationship between d 13Cns and soil moisture (Gessler et al. 2001). From these results, we conclude that during autumn 13C enrichment of the sugars was due to a combination of cold air and soil temperatures, both of which potentially limited hydraulic conductance within the trees and forced CO2 assimilation to occur at low needle ci/ca ratios (Smith et al. 1984; DeLucia 1986; Day, Delucia & Smith 1989; Day, Heckathorn & Delucia 1991; Schaberg et al. 1995). SIPNETmodelled NEP and GPP indicated that during the enrichment of needle sugars in autumn and spring, NEP and GPP were both positive, indicating that the trees were actively photosynthesizing.
Short-term dynamics in weather affect sugar carbon isotope Past studies have noted the potential for short-term weather variation to affect d 13CR, which is presumably related to an effect on sugar d 13C (Pate & Arthur 1998; Bowling et al. 2002; Cernusak et al. 2003; Fessenden & Ehleringer 2003; Keitel et al. 2003; McDowell et al. 2004; Lai et al. 2005; Schaeffer et al. 2008). Our results support these observations by directly demonstrating the sensitivity of d 13Cns to changes in weather. The tight coupling between weather and d 13Cns is best seen by comparing patterns during the springs of 2006 and 2007. In 2006, we observed an abrupt transition from winter to spring in mid-April, with forest GPP increasing rapidly over the span of a few days (Fig. 2c,d). The quick rise in GPP was accompanied by a quick and definitive decrease in the d 13Cns; presumably driven by warmer temperatures, increased CO2 assimilation rate, increased needle ci/ca ratios and progressive dilution of the 13C-enriched signal remaining from the winter and early spring. In the spring of 2007, forest GPP was episodically high during the late winter and early spring because of periodic warm air temperatures (Fig. 1h); however, the rates of GPP were lower during the months of April and May, compared with 2006. Presumably, the lower rates during this period were due to late-winter temperatures lingering relatively late in the spring. Consequently, during 2007, 13C enriched sugars were probably produced later into
the spring, and high rates of GPP at increased ci/ca were not available to dilute the winter 13C/12C signal as rapidly as in 2006. It is worth noting here that a previous study by Zarter et al. (2006) showed that in at least two of the species we studied, lodgepole pine and subalpine fir, episodic warm periods during the late winter are capable of stimulating photosynthesis (which is normally downregulated during the winter), and potentially permitting limited CO2 assimilation to occur.
Seasonal differences between d 13Cns and d 13Cps We observed a consistent offset in the d 13C value of needle sugars and phloem sugars (Fig. 4). The enrichment with 13 C of d 13Cps relative to d 13Cns has been observed in past studies, and it has been attributed to isotopic fractionation during phloem loading (Schleser 1992; Martinelli et al. 1998; Gessler et al. 2001, 2004; Damesin & Lelarge 2003). Fractionation may also occur diurnally (Tcherkez et al. 2004; Gessler et al. 2008), because the d 13C value of transitory starch formed in the leaves (the origin of phloem loaded sugars during the night) can be up to 4‰ greater than triose-P originating from the Calvin–Benson cycle (Gleixner et al. 1998). In addition to the consistent offset between d 13Cps and 13 d Cns, we observed uncorrelated (decoupled) seasonal dynamics in these values. For example, during late May of 2007, d 13Cps decreased abruptly, while d 13Cns began to decrease gradually over several weeks. We believe that changes in source/sink dynamics within the tree were responsible for this decoupled seasonal pattern.From March to early May, phloem sugars were enriched in 13C, and this enrichment was most likely due to phloem loading of enriched needle sugars during the winter and early spring (see Blechschmidt-Schneider 1990). The time when we detected the abrupt decrease in d 13Cps coincided with the time of rapid snowmelt in late May and early June (see Fig. 1). During this period, d 13Cns was also becoming depleted in 13C, as the newly assimilated sugars were diluting the 13C enrichment observed for winter/early spring needle sugars. It may be that as the snow melted quickly and root metabolism increased, the needle sugars depleted in 13C were quickly transported through the phloem (Schneider & Schmitz 1989; Kuhns & Gjerstad 1991) towards the roots, thus causing the abrupt decrease in d 13Cps. GPP was high during this period, but buds had not yet burst, thus limiting aboveground sink activity; this may have promoted the downward transport of recently-assimilated sugars during the snow melt period. Thus, during the snowmelt period the coupling between d 13Cps and d 13Cns may have been re-established. Shortly after this re-coupling, however, d 13Cps began to increase again, a pattern which was not observed in d 13Cns. This early-summer 13C enrichment in phloem sugars was correlated with the timing of bud burst, and may also coincide with a switch in the directional transport of sugars in the phloem of the main trunk; in this case, it may reflect the upward transport of stored carbon from the roots, which was assimilated the previous autumn with an © 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 35–47
Sugar carbon isotope ratios 45 enriched 13C content. The switching of phloem transport from the downward movement of sugars early in the summer (when needle photosynthesis rates are high but shoot growth has not yet occurred) to upward transport a couple weeks later of both recently-assimilated carbon and stored carbon from the previous autumn (when bud burst and rapid shoot growth occur), may explain the seasonal decoupling of d 13Cps and d 13Cns.
CONCLUSION We observed a novel pattern of 13C enrichment in the needle and phloem sugars of three subalpine forest tree species during the autumn, winter and spring. The most likely explanation of this enrichment is the occurrence of cold night-time temperatures during periods when daytime temperatures permit CO2 assimilation. The cold night-time temperatures likely force a reduction in stomatal conductance and cause CO2 to be assimilated at relatively low ci/ca ratios. Overall, we observed relatively high sensitivity of the d 13C value of needle sugars to variations in weather. This result provides direct support for past studies that have interpreted seasonal variation in the d 13C value of ecosystem respired CO2 as being due to weather effects on the d 13C value of sugar substrates. Finally, we observed seasonal decoupling between the d 13C values of needle and phloem sugars, which may be driven by seasonally changing sourcesink patterns in trees, which is reflected in the phloem d 13C value, but not in the needle d 13C value. Overall, our studies demonstrate that sugar carbon isotope signals provide a relatively sensitive record of short-term interactions between tree physiology and climate – a record that can provide unique insight into the effects of future climate change on ecosystem carbon budgets.
ACKNOWLEDGEMENTS This research was supported with funds from two grants from the U.S. National Science Foundation (Biocomplexity Program; Grant EAR 0321918 and Doctoral Dissertation Improvement Grant; Grant DEB 0709252). The authors would like to thank the following people for help with field and lab work: M. Richards, S. Love-Stowell, D. Koffler, J. Beauregard, K. Sencesqua, J. Monical, T. Rosenstiel and L. Scott-Denton, and a special thanks to D. Riveros-Iregui for helping with the logistics of international data communication. We would also like to thanks the reviewers of this manuscript for their helpful comments.
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