Responses of foliar δ 13 C, gas exchange and leaf morphology to reduced hydraulic conductivity in Pinus monticola branches

Transcription

1 Tree Physiology 21, Heron Publishing Victoria, Canada Responses of foliar δ 13 C, gas exchange and leaf morphology to reduced hydraulic conductivity in Pinus monticola branches LUCAS A. CERNUSAK 1,2 and JOHN D. MARSHALL 1 1 Department of Forest Resources, University of Idaho, Moscow, ID , USA 2 Present address: Environmental Biology Group and CRC for Greenhouse Accounting, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, ACT 2601, Australia Received December 5, 2000 Summary We tested the hypothesis that branch hydraulic conductivity partly controls foliar stable carbon isotope ratio (δ 13 C) by its influence on stomatal conductance in Pinus monticola Dougl. Notching and phloem-girdling treatments were applied to reduce branch conductivity over the course of a growing season. Notching and phloem girdling reduced leafspecific conductivity (LSC) by about 30 and 90%, respectively. The 90% reduction in LSC increased foliar δ 13 Cby about 1 (P < , n = 65), whereas the 30% reduction in LSC had no effect on foliar δ 13 C(P = 0.90, n = 65). Variation in the δ 13 C of dark respiration was similar to that of whole-tissues when compared among treatments. These isotopic measurements, in addition to instantaneous gas exchange measurements, suggested only minor adjustments in the ratio of intercellular to atmospheric CO 2 partial pressures (c i /c a ) in response to experimentally reduced hydraulic conductivity. A strong correlation was observed between stomatal conductance (g s ) and photosynthetic demand over a tenfold range in g s.although c i /c a and δ 13 C appeared to be relatively homeostatic, current-year leaf area varied linearly as a function of branch hydraulic conductivity (r 2 = 0.69, P < , n = 18). These results suggest that, for Pinus monticola, adjustment of leaf area is a more important response to reduced branch conductivity than adjustment of c i /c a. Keywords: carbon isotope ratio, intercellular CO 2 concentration, leaf area. Introduction Stable carbon isotope ratios (δ 13 C) of plant tissue provide useful information about photosynthetic gas exchange (Ehleringer 1991, Marshall and Zhang 1994, Brugnoli and Farquhar 2000). For plants with the C 3 photosynthetic pathway, discrimination against 13 C during photosynthesis is controlled by the CO 2 partial pressure within the leaf intercellular spaces relative to that in the surrounding atmosphere (c i /c a ) (Farquhar et al. 1982). The c i /c a is a function of supply and demand for CO 2 within leaf chloroplasts and can be related to photosynthetic water-use efficiency (water loss per unit carbon gain) (Farquhar and Richards 1984, Farquhar et al. 1989). Understanding the sources of variation in c i /c a and δ 13 C is important for predicting plant responses to environmental change (Ehleringer 1993a, Ehleringer and Cerling 1995, Marshall and Monserud 1996). In addition, variation in δ 13 C has been used to assess hydraulic limitations to maximum tree height (Yoder et al. 1994). The supply of CO 2 to chloroplasts is controlled by stomatal conductance (g s ). Stomatal conductance, in turn, is partly controlled by a plant s ability to conduct water from the soil to sites of evaporation in leaves (Sperry and Pockman 1993, Sperry et al. 1993, 1998, Bond and Kavanagh 1999). Excessive water tension in the soil-to-leaf hydraulic pathway can lead to hydraulic failure associated with xylem cavitation and embolism. Thus, when xylem tension is near the cavitation threshold, plants may respond by decreasing g s. Woody plants often exhibit large variations in δ 13 C, both within individuals and among taxa (e.g., Craig 1954, Francey et al. 1985, Leavitt and Long 1986, Gebauer and Schulze 1991, Yoder et al. 1994, Guehl et al. 1998, Martinelli et al. 1998). Meinzer et al. (1992, 1993) and Waring and Silvester (1994) suggested that some of this variation reflects variation in hydraulic conductivity. Subsequently, Panek (1996) presented a theory linking carbon isotope discrimination to conductance through the soil to leaf hydraulic pathway (expressed per unit leaf area). She noted that branches make a proportionally large contribution to the resistance along this pathway and suggested that δ 13 C should vary as a function of branch conductivity. Alternatively, plants may respond to variation in hydraulic conductivity by modifying the relationship between conducting area and leaf area, such that the balance between CO 2 supply and demand is unaffected by changes in hydraulic conductivity. Thus c i /c a and δ 13 C may be homeostatically maintained, whereas other parameters such as leaf area (Becker et al. 2000) and leaf morphology (Niinemets and Kull 1995) vary in response to alterations in hydraulic conductivity. To determine whether c i /c a and δ 13 C operate as homeostatic set points with respect to hydraulic conductivity, we investigated responses of foliar δ 13 C, gas exchange, leaf area, and

2 1216 CERNUSAK AND MARSHALL leaf morphology to reduced hydraulic conductivity in branches of Pinus monticola Dougl. (western white pine). To do this, we experimentally altered branch conductivity on trees growing at a common site, thereby avoiding complications associated with sampling trees exposed to large variations in soil water content and atmospheric vapor pressure deficit. Materials and methods Site description The study site is located at the University of Idaho Experimental Forest (46 50 N, W) at an altitude of 915 m a.s.l. The climate at the site is characterized by cool, mild winters and warm, dry summers. Mean annual temperature and precipitation are approximately 7 C and 720 mm, respectively. Most precipitation occurs between November and April, with the majority falling as snow. Soils on the site are deep, welldrained silt loams formed in volcanic ash. Sampled trees grew in a Pinus monticola plantation established after clear felling in At the time of the study in 1999, the trees averaged 4to5minheight. Selected study trees were apparently free of insects and disease and experienced minimal shading by neighboring trees. Experimental design Three treatments were applied to sample branches: notching, phloem girdling, and control. In the notching treatment, a razor blade was forced through the bottom half of each branch near the branch base. This effectively reduced the branch cross-sectional area by a factor of 0.5 in the plane encompassing the blade insertion. The razor blades were sterilized by rinsing with ethanol before insertion. Once inserted, the blades remained in place throughout the experiment, creating a permanent barrier to xylem water flux to the foliage distal to the point of insertion. In the phloem-girdling treatment, bark (periderm, cortex, and phloem) was removed from a 5-cm branch section near each branch base. This was achieved by inserting a razor blade through the bark until it met the hard, outer xylem. Two such incisions were made around the circumference of each branch and were joined by a straight line. The isolated section of bark was peeled away. The girdled branch sections were immediately wrapped in several layers of Parafilm to prevent desiccation. Parafilm was reapplied periodically during the experiment. The girdling treatment was intended to block phloem transport out of the branch, which was expected to result in assimilate accumulation in the foliage and feedback-inhibition of photosynthesis (Stitt 1991, Myers et al. 1999). Control branches were left untreated. A total of 25 trees were included in the experiment. On each tree, three branches within one whorl were selected from either the third, fourth, or fifth whorl from the treetop. The three selected branches had the most southerly exposures in the whorl. Each treatment was randomly applied to one of the three sample branches on each tree, for a total of 75 experimental branches. Treatments were applied between May 6 and 11, 1999, about 3 weeks before bud break occurred. Gas exchange Photosynthesis, g s, and c i /c a were measured on current-year foliage (foliage produced after the application of the treatments). Gas exchange measurements were performed with an LI-6200 portable photosynthesis system (Li-Cor, Inc., Lincoln, NE) equipped with a 0.25-l cuvette. Two or three needle fascicles (10 to 15 needles) were detached from each branch and placed in the cuvette. Gas exchange was measured within 5 min of needle detachment (Meng and Arp 1993). Gas exchange measurements were conducted between 0800 and 1100 h on August 3 and 4, Each of the three experimental branches was measured sequentially on each tree; thus there was no bias toward any of the treatments resulting from the time of measurement. Irradiance exceeded 1000 µmol m 2 s 1 of photosynthetically active radiation (PAR) during all measurements. We assumed that this irradiance was sufficient to saturate photosynthesis in Pinus monticola, as was the case for Pinus strobus L. (eastern white pine) (Maier and Teskey 1992). Projected leaf area of foliage measured for gas exchange was estimated from a regression equation relating needle length to projected leaf area (J.D. Marshall, unpublished data). Samples were frozen following gas exchange measurements and stored for determinations of needle length. Following needle length measurements, samples were air-dried at 70 C and weighed to the nearest 0.1 mg. Specific leaf area (SLA) of each sample was determined by dividing projected leaf area by leaf dry weight. Carbon isotope ratios The δ 13 C was determined for the foliage samples measured for gas exchange in early August as well as for current-year foliage sampled from treated branches in late August. All foliage samples were air-dried at 70 C and ground to a fine powder in a rotating ball mill. Subsamples of about 1 mg were combusted in an elemental analyzer (NC2500, CE Instruments, Milan, Italy); combustion products were swept by a helium carrier gas and continuous-flow interface into an isotope ratio mass spectrometer (Delta Plus, Finnigan MAT, Bremen, Germany) operated by the Idaho Stable Isotope Laboratory, University of Idaho. All carbon isotope ratios are expressed in delta notation relative to the Pee Dee Belemnite standard. The analytical precision of the analyses, based on repeated measurements of a working standard (Idaho flour), was ± 0.08 (standard deviation). Foliar carbon and nitrogen concentrations were determined from peak areas obtained from mass spectrometric measurements. Although we applied our treatments well before bud break occurred, it is possible that current-year foliage was partially constructed from stored photosynthate. If this were the case, whole-tissue δ 13 C would not provide an accurate measure of the response of isotopic discrimination to the experimental treatments, whereas δ 13 C of dark-respired CO 2 should provide TREE PHYSIOLOGY VOLUME 21, 2001

3 δ 13 C RESPONSE TO REDUCED CONDUCTIVITY 1217 a measure of the isotopic composition of more recently fixed photosynthate. We collected dark-respired CO 2 from whole current-year shoots in late August The procedure used for CO 2 collection and isotopic analysis has been described (Cernusak et al. 2001). Briefly, current-year shoots were harvested and transported to the laboratory; on the same day, shoots were placed in a 1-l cuvette attached to the Li-Cor LI The closed system was then scrubbed free of CO 2 and allowed to refill with respired CO 2. When the CO 2 concentration within the system reached 360 µmol mol 1, a 1-ml air sample was extracted with a gas-tight, locking syringe (VICI Precision Sampling, Baton Rouge, LA). Samples of about 400 µl of air were injected into an automated trace-gas condensing device (Precon, Finnigan MAT, Bremen, Germany). The condensate was swept by a helium carrier gas through a 50-m PORO- PLOT Q column, which separated N 2 O from CO 2, and into the isotope ratio mass spectrometer. The analytical precision, based on repeated injections of a standard gas (ISU-720C, Oztech Trading Corp., Dallas, TX; δ 13 C = ), was ± 0.2. Hydraulic conductivity Hydraulic conductivity (K h ) was determined for a randomly selected subsample of the treated branches (six trees, 18 branches) in early September Branch sections of approximately 50 cm in length were excised near the branch bases. For notched and girdled branches, the section included the region of treatment. Once in the laboratory, the branch sections were recut under water and the bark removed to prevent resin from bleeding into and occluding the severed tracheids. The K h was measured as described by Sperry et al. (1988). The flow rate of purified water (filtered to 0.2 µm and acidified to ph 2 with HCl) through branch sections was determined gravimetrically. Flow was generated by pressure from a hydraulic head and K h was calculated as the water flux divided by the pressure gradient multiplied by the branch length. Branch sections were kept underwater during flow measurements to prevent evaporation from the wood surface. The flow rate under zero hydraulic head was measured and subtracted from the pressurized flow rate as described by Stiller and Sperry (1999). To estimate projected leaf area distal to the branch sections, we determined SLA of five needle fascicles on each branch as described for the gas exchange measurements. Specific leaf area was then used to convert whole-branch leaf dry mass to projected leaf area. Leaf-specific hydraulic conductivity (LSC) was calculated as the quotient of K h and whole-branch projected leaf area. We analyzed treatment-related variation in measured parameters by analysis of variance. Individual branches were considered to be independent experimental units; thus we assumed branches to be autonomous with respect to fluxes of carbon and water. When treatment was a significant term in the analysis of variance, individual treatments were compared by Tukey s method for pair-wise comparisons. All statistical analyses were performed with SYSTAT 9.0 (SPSS Inc., Chicago, IL). Results Leaf structure Leaves produced after the experimental manipulations varied in structure among treatments. Bud break occurred at the study site on about May 27, Needle length was measured on August 3 and 4, 1999 and again between August 26 and 28, Current-year foliage increased in length between the two measurement periods for all three treatments (P< for control, P<0.001 for notched, and P = 0.03 for girdled); however, the increase was less for girdled branches than for control or notched branches (Figure 1A). On both measurement dates, needle length of girdled branches was less than needle length of control branches (P<0.001 for early August, P< for late August), whereas needle lengths of control and notched branches did not differ (P = 0.83 for early August, P = 0.27 for late August). Patterns of change in SLA between early and late August were opposite to those in needle length (Figure 1B). Specific leaf area was unchanged between early and late August for control (P = 0.18) and notched (P = 0.21) branches, but de- Figure 1. Needle length (A) and specific leaf area (B) of Pinus monticola foliage collected in either early or late August Treatments were applied in late May Error bars represent one standard error. TREE PHYSIOLOGY ONLINE at

4 1218 CERNUSAK AND MARSHALL creased for girdled branches (P <0.0001). On both sampling dates, SLA of girdled branches was less than that of controls (P < for early and late August), whereas SLA of notched branches did not differ from controls (P = 0.96 for early August, P = 0.87 for late August). Current-year foliage of girdled branches also differed in chemical composition from controls (Table 1). Foliar nitrogen and carbon concentrations, expressed per unit mass, were less for girdled branches than for control branches. Foliage of notched branches, on the other hand, did not differ in nitrogen or carbon concentration from that of control branches. Gas exchange Maximum photosynthetic rates of current-year foliage were strongly related to g s for all treatments combined (Figure 2). The relationship between g s and photosynthesis was approximately linear for g s values ranging from 20 to near 150 mmol H 2 Om 2 s 1 ; more than 90% of the data fell within this range. The intercept of the linear portion of this relationship was approximately zero. Maximum photosynthetic rates ranged from less than 2 to more than 12 µmol CO 2 m 2 s 1. Photosynthetic rates of girdled branches were less than half those of controls; whereas those of notched branches did not differ from controls (Figure 3A). Mean g s of notched branches was similar to that of controls (Figure 3B), whereas mean g s of girdled branches was substantially less than the control value. We observed no difference among treatments in c i /c a as measured by instantaneous gas exchange (Figure 3C). Figure 2. Light-saturated photosynthetic rates plotted against stomatal conductance for Pinus monticola foliage measured in early August 1999 between 0800 and 1100 h local time at irradiances exceeding 1000 µmol m 2 s 1. Measurement of each treatment spanned the full range of measurement times. branches did not differ from that of controls (Figure 4B). In general, the δ 13 C of dark respiration was enriched over that of whole tissue by about 2. This difference did not vary among treatments (Figure 4C). Stable isotope ratios Whole-tissue δ 13 C of current-year foliage collected in late August was higher (less negative) by 0.88 for girdled branches than for controls. In contrast, notched branches did not differ from controls (Figure 4A). The δ 13 C of foliage collected in early August did not differ from that collected in late August for control or notched branches (P = 0.74 for control branches, P = 0.56 for notched branches). In contrast, foliar δ 13 Cofgirdled branches increased by 0.15 between early and late August (P = 0.002). Values shown in Figure 4 are those measured in late August. Variation among treatments in δ 13 C of respired CO 2 was similar to that of whole tissues. The δ 13 C of dark respiration from girdled branches was 0.90 heavier than that from control branches, whereas δ 13 C of dark respiration from notched Table 1. Nitrogen and carbon concentrations ([N] and [C], respectively) of current-year Pinus monticola foliage. Treatments were applied to branches in late May 1999, and foliage samples were collected in late August Means within a row followed by different letters are significantly different at P < Values in parentheses are one standard error. Control Notched Girdled [N] (mg g 1 ) 14.6 (0.19) a 14.5 (0.26) a 8.9 (0.25) b [C] (mg g 1 ) 484 (1.75) a 485 (1.81) a 477 (2.23) b Figure 3. Net photosynthesis (A), stomatal conductance (B), and c i /c a ratio (C) for current-year foliage of treated Pinus monticola branches. Measurements were conducted in early August Bars within a panel followed by different letters are different at P<0.05. Error bars represent one standard error. TREE PHYSIOLOGY VOLUME 21, 2001

5 δ 13 C RESPONSE TO REDUCED CONDUCTIVITY 1219 between current-year leaf area on the main axis of sample branches and K h of the measured sections at the bases of branches (Figure 6). Note that in this analysis, K h was expressed independently of the amount of subtending leaf area. Figure 4. Whole-tissue carbon isotope ratio for foliage collected in late August (A), carbon isotope ratio of CO 2 respired in late August (B), and the difference between carbon isotope ratios of whole-tissue and respired CO 2 (C) for current year foliage of treated Pinus monticola branches. Bars within a panel followed by different letters are different at P<0.05. Error bars represent one standard error. Foliar δ 13 C decreased with increasing g s for all treatments combined (data not shown). The relationship between the two parameters was curvilinear and was best described by an exponential decay function (δ 13 C = exp( 0.009g s ); r 2 = 0.41, n = 59). In addition, foliar δ 13 C declined linearly with SLA for all treatments combined. The two were related by the equation δ 13 C = SLA (r 2 = 0.39, P< , n = 65). Hydraulic conductivity Leaf specific hydraulic conductivity (LSC) of control branches was ± kg m 1 s 1 MPa 1 (mean ± SE). The LSC of notched branches was reduced by about 30% relative to controls (LSC = ± kg m 1 s 1 MPa 1 ), with the difference between the two being moderately significant (P = 0.07, n = 18). For girdled branches, LSC was reduced by more than 90% when compared with the control value (LSC = ± kg m 1 s 1 MPa 1 ), and the difference was highly significant (P <0.0001, n = 18). The δ 13 C of current-year foliage was negatively related to LSC when compared across all treatments combined (Figure 5). In addition, we observed a strong, linear relationship Discussion We tested the hypothesis that foliar δ 13 C varies in response to reduced hydraulic conductivity in Pinus monticola branches. As predicted by theory (Panek 1996), δ 13 C increased with decreasing LSC; however, the increase in δ 13 C was small. A more than 90% reduction in LSC led to an average increase in foliar δ 13 C of about 1, whereas a 30% reduction in LSC had no effect on foliar δ 13 C. Both isotopic and instantaneous measurements suggested only minor adjustments in c i /c a over a large range of hydraulic conductivities, despite large changes in g s. Although c i /c a and δ 13 C appeared to be fairly homeostatic parameters, leaf area and leaf morphology were more plastic. In particular, current-year leaf area varied linearly as a function of branch conductivity. Thus, it appears that, for Pinus monticola, adjustment of leaf area may be a more likely response to alterations in branch conductivity than adjustment of c i /c a. Relationships between foliar δ 13 C and branch conductivity have been investigated previously over a naturally occurring climate gradient in Oregon, USA (Panek and Waring 1995, Panek 1996). The shape of the relationship between δ 13 C and LSC that we observed (Figure 5) is similar to that reported by Panek (1996). However, the range of δ 13 C values was slightly less in our study. We induced variation in LSC by experimentally treating branches at a common site, whereas in the Oregon study sample trees were exposed to varying degrees of soil water stress and atmospheric vapor pressure deficit, in addition to variation in LSC. Because LSC decreased with increas- Figure 5. Whole-tissue carbon isotope ratio of current-year Pinus monticola foliage collected in late August plotted against the leaf-specific hydraulic conductivity (LSC) of the supporting branch. Data are for all treatments combined. Conductivity was measured in early September. Conductivity values are expressed as LSC The equation relating δ 13 C to LSC is y = 24.8x with r 2 = 0.61, P < , and n = 18. TREE PHYSIOLOGY ONLINE at

6 1220 CERNUSAK AND MARSHALL Figure 6. Variation in current-year leaf area on the main terminus of Pinus monticola branches expressed as a function of branch hydraulic conductivity (K h ). Both conductivity and leaf area were measured in early September. Data are for all treatments combined. Conductivity values are expressed as K h Leaf area is related to conductivity by the equation y = x with r 2 = 0.69, P < , and n = 18. ing aridity across the Oregon transect, stomatal constraints arising from factors other than LSC may have contributed to the observed variation in δ 13 C. Branch length influences hydraulic conductance because it is a component of the path length between soil and leaf. If other parameters are held constant, an increase in branch length should cause a decrease in leaf-specific conductance; the decrease should be proportional to the amount of added resistance caused by the additional branch length relative to the total resistance from soil to leaf. Observations of increasing foliar δ 13 C with increasing branch length support the hypothesis that branch conductance influences foliar δ 13 C (Waring and Silvester 1994, Panek and Waring 1995, Walcroft et al. 1996, Warren and Adams 2000). However, a relationship between branch length and foliar δ 13 C has not always been observed (Heaton and Crossley 1995, Livingston et al. 1998, Warren and Adams 2000). A lack of response of foliar δ 13 C to variation in branch length could result from reductions in leaf area that compensate for the additional resistance conferred by increased path length. Although more data are necessary to determine the extent to which such compensation occurs, the relationship between current-year leaf area and K h observed in this study (Figure 6) suggests the possibility. This suggestion is further supported by observed reductions in the amount of leaf area per unit sapwood area with increasing tree height (Schafer et al. 2000). Instantaneous measurements of c i /c a did not differ significantly among treatments, whereas δ 13 C, an integrated measure of c i /c a, did. The instantaneous measurements were taken over 30 s. In contrast, the respired CO 2 δ 13 C values should integrate over time scales of hours to days, and whole-tissue δ 13 C values should integrate over time scales of weeks to months (the period over which the leaf tissue was formed). A longer time period of integration should lead to a higher signal to noise ratio, as any high-frequency variation in c i /c a will be averaged out to a greater extent. The coefficients of variation for the three estimates confirm this suggestion. For the control treatment, coefficients of variation were 7.7, 2.4, and 1.6% for instantaneous c i /c a, respired CO 2 δ 13 C, and whole-tissue δ 13 C, respectively. Thus, the power to detect differences among treatments was greater for the isotopic data than for the instantaneous data. Additionally, instantaneous c i /c a was measured in mid-morning when the atmospheric vapor pressure deficit was relatively low. As the vapor pressure deficit increased during the day, treatment related differences in c i /c a would have likely become more pronounced. We observed a strong correlation between photosynthesis at saturating irradiance and g s in treated branches (Figure 2). The relationship was such that c i /c a was held nearly constant across experimental treatments (Figure 3C). A similar relationship between photosynthesis and stomatal conductance was observed in the C 3 plant species Eucalyptus pauciflora Sieber ex A. Spreng. and Gossypium hirsutum L. under wide-ranging experimental conditions (Wong et al. 1978, 1979). More recently, analyses of photosynthesis in the leaves of several C 3 species have indicated increasing inhibition of photosynthetic metabolism with decreasing leaf water potentials (Kanechi et al. 1996, Escalona et al. 1999, Tezara et al. 1999). These reductions in photosynthetic demand for CO 2 in water-stressed leaves appear to be caused by a slower regeneration of ribulose bisphosphate leading to inhibition of ribulose bisphosphate carboxylase/oxygenase activity (Tezara et al. 1999). We suggest that correlated reductions in photosynthetic demand for CO 2 and g s limit the response of foliar δ 13 C to perturbations in hydraulic conductivity. It was recently reported that photosynthetic capacity was strongly related to LSC in seven conifers and 16 angiosperm species (Brodribb and Feild 2000). Similarly, in a laboratory experiment, photosynthesis and g s varied linearly with LSC in Pinus ponderosa Dougl. ex Laws. seedlings (Hubbard et al. 2001). Correlated reductions in g s and photosynthesis may be a general response to reduced hydraulic conductivity among taxa. If this is the case, only subtle shifts in δ 13 C should be expected in response to variation in LSC. Phloem girdling caused a marked decrease in xylem hydraulic conductivity. The girdling treatment was initially intended to reduce phloem export, which was expected to reduce photosynthetic demand while maintaining g s. Clearly the treatment did not have the desired effect. It is possible that air was trapped against the xylem when the girdled sections were wrapped with Parafilm. If the xylem was nicked when the bark was cut away, it would have provided an opening through which air could enter the xylem conduits. Although the Parafilm was replaced regularly during the experiment, it may have developed leaks allowing continued air entry. Alternatively, we considered the possibility that fungi invaded the girdled sections. However, attempts to culture and isolate fungi at the conclusion of the experiment were unsuccessful. Because we do not know the mechanism by which girdling reduced conductivity, we are unsure of when the loss of conductivity TREE PHYSIOLOGY VOLUME 21, 2001

7 δ 13 C RESPONSE TO REDUCED CONDUCTIVITY 1221 occurred. However, differences in leaf area and structure suggest that it may have occurred early in the experiment. In contrast to phloem girdling, notching had a relatively minor effect on hydraulic conductivity. This result is consistent with a previous experiment in which foliar gas exchange and LSC showed no response to notching in branches of young and old Pinus ponderosa (Hubbard et al. 1999). Hubbard et al. (1999) suggested that there was redundancy in the sapwood of their pine branches. Because we treated each branch at only a single point along its axis, it is also possible that the branches formed a smaller portion of the total resistance along the pathway from soil to leaf than we anticipated. Foliage from phloem-girdled branches differed from controls in both carbon and nitrogen concentration (Table 1). The decrease in carbon concentration may have resulted from starch accumulation in the foliage because phloem was removed from the branch bases, thus disrupting the pathway for sugar transport out of the branches. An excess accumulation of starch could have decreased the photosynthetic demand of the foliage, thus decreasing foliar δ 13 C relative to controls. However, there was no correlation between foliar δ 13 C and carbon concentration (P = 0.33, n = 65), or between photosynthesis and carbon concentration (P = 0.12, n = 58). Therefore, we suggest that photosynthetic demand was not limited by high starch concentrations. Different biochemical fractions in leaves frequently have different isotopic signatures (Brugnoli and Farquhar 2000). For this reason, it is often preferable to extract cellulose when comparing δ 13 C among tissues that may have different biochemical compositions. The difference in carbon concentration between foliage of girdled and control branches suggests possible differences in tissue composition. Although we chose to compare whole-tissue δ 13 C rather than cellulose δ 13 C, we also compared the δ 13 C of dark respiration. Assuming similar substrates for respiration, the measurement of dark-respired CO 2 should be as effective as extracting cellulose in removing variation associated with differences in tissue composition. Measurements of whole-tissue δ 13 C and respired CO 2 δ 13 C yielded similar patterns among treatments. Foliar nitrogen concentration is frequently related to photosynthetic capacity (Field and Mooney 1986). Ideally, such comparisons should rely on measurements expressed on a similar basis. In this study, the expression of nitrogen concentrations per unit leaf area led to a weak relationship with photosynthetic rates (r 2 = 0.13, P<0.01, n = 58), and no relationship with foliar δ 13 C(P = 0.71, n = 65). A lack of correlation between area-based photosynthesis and nitrogen concentration was also reported for several evergreen conifer species grown in Wisconsin, USA (Reich et al. 1995). We suggest that the response of foliar δ 13 C to reduced LSC in girdled branches was not dampened by a decrease in photosynthetic capacity resulting from low foliar nitrogen concentrations. This suggestion is supported by the results of the notching treatment showing that the reduction in LSC occurred independently of any change in foliar nitrogen concentration; a 30% reduction in LSC had no effect on foliar δ 13 C. In conclusion, the experimental data supported the hypothesis that foliar δ 13 C varies as a function of hydraulic conductivity in Pinus monticola. However, the response of foliar δ 13 Cto reductions in LSC appeared to be moderated by the strong correlation between stomatal conductance and photosynthetic demand for CO 2. This result reinforces the notion that c i /c a is an important set point for photosynthetic gas exchange (Wong et al. 1979, Ehleringer 1993a, 1993b, Ehleringer and Cerling 1995). In contrast, leaf area may be more plastic in its response to reduced hydraulic conductivity. Future studies may profit from determining the extent to which variation in leaf area compensates for reductions in hydraulic conductivity resulting from increased branch length or other long-term losses in conducting capacity. Acknowledgments L.A.C. was supported by a fellowship from the Stillinger Foundation at the University of Idaho. We are grateful to R. Brandon Pratt and R. Alan Black for assistance with hydraulic measurements, and to George Newcombe for assistance in attempting to culture fungi. We thank Drs. Margaret Barbour, Graham Farquhar, Katy Kavanagh, Jeff Miller and Jeanne Panek for critical comments on earlier versions of the manuscript. References Becker, P., F.C. Meinzer and S.D. Wullschleger Hydraulic limitation of tree height: a critique. Funct. Ecol. 14:4 11. Bond, B.J. and K.L. Kavanagh Stomatal behavior of four woody species in relation to leaf-specific hydraulic conductance and threshold water potential. Tree Physiol. 19: Brodribb, T.J. and T.S. Feild Stem hydraulic supply is linked to leaf photosynthetic capacity: evidence from New Caledonian and Tasmanian rainforests. Plant Cell Environ. 23: Brugnoli, E. and G.D. Farquhar Photosynthetic fractionation of carbon isotopes. In Photosynthesis: Physiology and Metabolism. Eds. R.C. Leegood, T.C. Sharkey and S. von Caemmerer. Kluwer Academic Publishers, The Netherlands, pp Cernusak, L.A., J.D. Marshall, J.P. Comstock and N.J. Balster Carbon isotope discrimination in photosynthetic bark. Oecologia 128: Craig, H.A Carbon-13 variations in Sequoia rings and the atmosphere. Science 119: Ehleringer, J.R C/ 12 C fractionation and its utility in terrestrial plant studies. In Carbon Isotope Techniques. Eds. D.C. Coleman and B. Fry. Academic Press, San Diego, pp Ehleringer, J.R. 1993a. Gas-exchange implications of isotopic variation in arid-land plants. In Plant Responses to Water Deficit. Eds. H. Griffiths and J. Smith. BIOS Scientific Publishers, London, pp Ehleringer, J.R. 1993b. Carbon and water relations in desert plants: an isotopic perspective. In Stable Isotopes and Plant Carbon Water Relations. Eds. J.R. Ehleringer, A.E. Hall and G.D. Farquhar. Academic Press, San Diego, pp Ehleringer, J.R. and T.E. Cerling Atmospheric CO 2 and the ratio of intercellular to ambient CO 2 concentrations in plants. Tree Physiol. 15: Escalona, J.M., J. Flexas and H. Medrano Stomatal and nonstomatal limitations of photosynthesis under water stress in field-grown grapevines. Aust. J. Plant Physiol. 26: Farquhar, G.D. and R.A. Richards Isotopic composition of plant carbon correlates with water-use efficiency in wheat genotypes. Aust. J. Plant Physiol. 11: TREE PHYSIOLOGY ONLINE at

8 1222 CERNUSAK AND MARSHALL Farquhar, G.D., J.R. Ehleringer and K.T. Hubick Carbon isotope discrimination and photosynthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40: Farquhar, G.D., M.H. O Leary and J.A. Berry On the relationship between carbon isotope discrimination and the intercellular carbon dioxide concentration in leaves. Aust. J. Plant Physiol. 9: Field, C. and H.A. Mooney The photosynthesis nitrogen relationship in wild plants. In On the Economy of Plant Form and Function. Ed. T. Givnish. Cambridge Univ. Press, New York, pp Francey, R.J., R.M. Gifford, T.D. Sharkey and B. Weir Physiological influences on carbon isotope discrimination in huon pine (Lagarostrobos franklinii). Oecologia 66: Gebauer, G. and E.-D. Schulze Carbon and nitrogen isotope ratios in different compartments of a healthy and declining Picea abies forest in the Fichtelgebirge, NE Bavaria. Oecologia 87: Guehl, J.M., A.M. Domenach, M. Bereau, T.S. Barigah, H. Casabianca, A. Ferhi and J. Garbaye Functional diversity in an Amazonian rainforest of French Guyana: a dual isotope approach (δ 15 N and δ 13 C). Oecologia 116: Heaton, T.H.E. and A. Crossley Carbon isotope variations in a plantation of Sitka spruce, and the effect of acid mist. Oecologia 103: Hubbard, R.M., B.J. Bond and M.G. Ryan Evidence that hydraulic conductance limits photosynthesis in old Pinus ponderosa trees. Tree Physiol. 19: Hubbard, R.M., M.G. Ryan, V. Stiller and J.S. Sperry Stomatal conductance and photosynthesis vary linearly with plant hydraulic conductance in ponderosa pine. Plant Cell Environ. 24: Kanechi, M., N. Uchida, T. Yasuda and T. Yamaguchi Nonstomatal inhibition associated with inactivation of Rubisco in dehydrated coffee leaves under unshaded and shaded conditions. Plant Cell Physiol. 37: Leavitt, S.W. and A. Long Stable-carbon isotope variability in tree foliage and wood. Ecology 67: Livingston, N.J., D. Whitehead, F.M. Kelliher, et al Nitrogen allocation and carbon isotope fractionation in relation to intercepted radiation and position in a young Pinus radiata D. Don tree. Plant Cell Environ. 21: Maier, C.A. and R.O. Teskey Internal and external control of net photosynthesis and stomatal conductance of mature eastern white pine (Pinus strobus). Can. J. For. Res. 22: Marshall, J.D. and R.A. Monserud Homeostatic gas-exchange parameters inferred from 13 C/ 12 C in tree rings of conifers. Oecologia 105: Marshall, J.D. and J. Zhang Carbon isotope discrimination and water-use efficiency in native plants of the north-central Rockies. Ecology 75: Martinelli, L.A., S. Almeida, I.F. Brown, M.Z. Moreira, R.L. Victoria, L.S.L. Sternberg, C.A.C. Ferreira and W.W. Thomas Stable carbon isotope ratio of tree leaves, boles and fine litter in a tropical forest in Rondonia, Brazil. Oecologia 114: Meinzer, F.C., G. Goldstein and D.A. Grantz Carbon isotope discrimination and gas exchange in Coffee during adjustment to different soil moisture regimes. In Stable Isotopes and Plant Carbon Water Relations. Eds. J.R. Ehleringer, A.E. Hall and G.D. Farquhar. Academic Press, San Diego, pp Meinzer, F.C., N.Z. Saliendra and C.H. Crisosto Carbon isotope discrimination and gas exchange in Coffea arabica during adjustment to different soil moisture regimes. Aust. J. Plant Physiol. 19: Meng, F.R. and P.A. Arp Net photosynthesis and stomatal conductance of red spruce twigs before and after twig detachment. Can. J. For. Res. 23: Myers, D.A., R.B. Thomas and E.H. DeLucia Photosynthetic responses of loblolly pine (Pinus taeda) needles to experimental reduction in sink demand. Tree Physiol. 19: Niinemets, Ü. and O. Kull Effects of light availability and tree size on the architecture of assimilative surface in the canopy of Picea abies: variation in needle morphology. Tree Physiol. 15: Panek, J.A Correlations between stable carbon-isotope abundance and hydraulic conductivity in Douglas-fir across a climate gradient in Oregon, USA. Tree Physiol. 16: Panek, J.A. and R.H. Waring Carbon isotope variation in Douglas-fir foliage: improving the δ 13 C climate relationship. Tree Physiol. 15: Reich, P.B., B.D. Kloeppel, D.S. Ellsworth and M.B. Walters Different photosynthesis nitrogen relations in deciduous hardwood and evergreen coniferous tree species. Oecologia 104:24 30 Schafer, K.V.R., R. Oren and J.D. Tenhunen The effect of tree height on crown level stomatal conductance. Plant Cell Environ. 23: Sperry, J.S. and W.T. Pockman Limitation of transpiration by hydraulic conductance and xylem cavitation in Betula occidentalis. Plant Cell Environ. 16: Sperry, J.S., F.R. Adler, G.S. Campbell and J.P. Comstock Limitation of plant water use by rhizosphere and xylem conductance results from a model. Plant Cell Environ. 21: Sperry, J.S., N.N. Alder and S.E. Eastlack The effect of reduced hydraulic conductance on stomatal conductance and xylem cavitation. J. Exp. Bot. 44: Sperry, J.S., J.R. Donnelly and M.T. Tyree A method for measuring hydraulic conductivity and embolism in xylem. Plant Cell Environ. 11: Stiller, V. and J.S. Sperry Canny s compensating pressure theory fails a test. Am. J. Bot. 86: Stitt, M Rising CO 2 levels and their potential significance for carbon flow in photosynthetic cells. Plant Cell Environ. 14: Tezara, W., V.J. Mitchell, S.D. Driscoll and D.W. Lawlor Water stress inhibits plant photosynthesis by decreasing coupling factor and ATP. Nature 401: Walcroft, A.S., W.B. Silvester, J.C. Grace, S.D. Carson and R.H. Waring Effects of branch length on carbon isotope discrimination in Pinus radiata. Tree Physiol. 16: Waring, R.H. and W.B. Silvester Variation in foliar δ 13 C values within the crowns of Pinus radiata trees. Tree Physiol. 14: Warren, C.R. and M.A. Adams Water availability and branch length determine δ 13 C in foliage of Pinus pinaster. Tree Physiol. 20: Wong, S.-C., I.R. Cowan and G.D. Farquhar Leaf conductance in relation to assimilation in Eucalyptus pauciflora Sieb. ex Spreng. Plant Physiol. 62: Wong, S.-C., I.R. Cowan and G.D. Farquhar Stomatal conductance correlates with photosynthetic capacity. Nature 282: Yoder, B.J., M.G. Ryan, R.H. Waring, A.W. Schoettle and M.R. Kaufmann Evidence of reduced photosynthetic rates in old trees. For. Sci. 40: TREE PHYSIOLOGY VOLUME 21, 2001