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Complementary laboratory and field experiments showed that theinternal transport of carbon dioxide (CO2) in the xylemof trees is an important pathway for carbon movement. Carbon dioxidereleased by respiration dissolves in sap and moves upward in thetranspirational stream. The concentration of CO2 in xylemsap can be up to three orders of magnitude greater than that foundin the atmosphere. In the present experiments, diffusion outwardof a portion of xylem-transported CO2 caused a substantialoverestimation of the apparent rate of stem and branch respiration.Rates of CO2 efflux were linearly related to sap CO2 concentration.Direct manipulations of xylem sap CO2 concentration producedrapid and reversible changes in CO2 efflux from stemsand branches, in some cases quadrupling the rate of efflux. Theseresults demonstrated that apparent rates of stem and branch respirationof trees are in large part a by-product of the rate of CO2 diffusionfrom xylem sap.
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Temperature is considered the most important environmental factorcontrolling the rate of respiration (Amthor 1989)and is well correlated with respiration when measured in controlledconditions, but in field conditions, inconsistencies in the relationshipbetween temperature and rates of respiration are commonly found. Gunderson et al. (2000) observedthat leaf respiration rates in Acer saccharum Marsh. seedlingsgrowing in the field had almost no correlation with prevailing temperatures. Edwards & McLaughlin (1978) found thatdiurnal changes in stem respiration of Quercus alba L. and Liriodendrontulipifera L. trees were opposite the diurnal pattern of stemtemperature. Diurnal hysteresis, i.e. different rates of respiration measuredat the same temperature at different times during the day, has alsobeen observed (Negisi 1982; Lavigne 1987).Others have found good correspondence between stem temperature andrespiration (e.g. Maier, Zarnoch & Dougherty1998). Similarly, inconsistencies are apparent in the relationshipbetween stem respiration and nitrogen status (e.g. compare Lavigne & Ryan 1997; Stockfors& Linder 1998; Maier 2001).
Many explanations have been offered for the absence of consistentrelationships between respiration and environment, among and withintissue types, and between species. However, one possible cause thatis often overlooked is the transport of CO2 in xylemsap (Negisi 1979; Martin,Teskey & Dougherty 1994). Carbon dioxide is a constituentof the gas dissolved in xylem sap (Stringer &Kimmerer 1993). The carbon dioxide concentration of the gasphase of xylem sap ([CO2]) of treeshas been reported in the range 2–26% (MacDougal & Working 1933; Chase1934; Hari, Nygren & Korpilahti 1991; Levy et al. 1999). For example,xylem [CO2] ranged from 2 to 10% in Piceaabies, from 3 to 9% in Quercus robur L. andfrom 2 to 4% in Acer platanoides L. during the growingseason (Eklund 1990, 1993). Previous reportshave speculated that high xylem [CO2] mayhave an effect on measurements of respiration (Hari et al.1991; Levy et al. 1999).However, a link between xylem [CO2] and woodytissue CO2 efflux rates has never been established.
In the present study, the [CO2] ofxylem in trees was measured with microelectrodes. This techniquehad not been used previously for in situ measurements inplants and it allowed us to observe diurnal changes in xylem [CO2] for thefirst time. The main objective of the study was to establish a relationshipbetween xylem [CO2] and measurements ofwoody tissue respiration. We hypothesized that if xylem [CO2] changeddiurnally and/or seasonally, the rate of diffusion of CO2 fromstem to atmosphere would be altered. If this hypothesis was correct,then previous efflux-based estimates of respiration rates of woodytissues were in error because the assumption was that the CO2 hadevolved from locally respiring cells and had not been transportedthrough the xylem from other locations in the plant.
A series of field experiments was conducted during the growingseason in 1999, 2000 and 2001 at Whitehall Forest, a warm temperatemixed pine–hardwood forest near Athens, Georgia, USA. Measurementswere made on mature trees of white oak (Quercus alba L.),yellow-poplar (Liriodendron tulipifera L.) and loblolly pine(Pinus taeda L.) with stem diameters ranging from 15 to 70 cmmeasured at 1·7 m above ground. We chose thesespecies for their differing xylem structure: ring porous, diffuseporous and tracheid anatomy, respectively. In general, trees withring porous xylem have the highest proportion of large diameter xylemelements, smallest conducting area and fastest sap velocity. Treeswith tracheid xylem are characterized by the smallest diameter xylemelements, largest conducting area and slowest sap velocity. Thosewith diffuse porous xylem have a mixture of large and small vesselsand intermediate conducting area and sap velocity (Kramer& Kozlowski 1979). There was no green tissue apparentin the lower portion of the stems where measurements were made,indicating that photosynthetic refixation was not a factor in our measurements(Levy et al. 1999; Cernusak & Marshall 2000). Measurementswere made on all three species in 1999, but P. taeda waseliminated in 2000 and 2001 because resin flow damaged our sensorsand interfered with measurements.
Xylem [CO2] was measured in situ usingthe microelectrode technique described by McGuire& Teskey (2002). We used a commercially available CO2 microelectrode (ModelMI-720; Microelectrodes, Inc., Bedford, NH, USA) consisting of aglass pH electrode and an Ag–AgCl reference electrode enclosedin a replaceable plastic housing with a gas-permeable Teflon membranetip. The housing is filled with NaHCO3/NaClelectrolyte solution. With the housing installed, the glass microelectrodeis 3 mm in diameter and 30 mm long. The sensorhas a total length of 84 mm and body diameter of 6 mm.The electrode develops a measurable voltage that is proportionalto [CO2].
Microelectrodes were calibrated with humidified compressed gasof known [CO2] and equations were developed toconvert millivolt output to [CO2] (%).To compensate for measurement temperature, an empirically derivedcorrection equation was applied to the results (McGuire& Teskey 2002).
To measure [CO2] of xylem sap insitu, 4 mm diameter holes were drilled 30 mminto the stem at a height of 1·5 m. A microelectrodewas inserted into each hole and sealed at the stem surface withflexible putty adhesive (Blu-tack; Bostik Australia Pty. Ltd, Thomastown,Victoria, Australia). The microelectrode measured [CO2] ofgas in the headspace of the hole, which is proportional to the concentrationof all products of CO2 dissolved in xylem sap (Hari et al. 1991; Levy et al. 1999). Gas concentration(%) was converted to total dissolved carbon ([CO2*],mmol L−1) using equations based onHenry's Law (Butler 1991; McGuire& Teskey 2002). The dissolution of CO2 intobicarbonate and carbonate ions increases with increasing pH; therefore,the pH of sap must be considered when calculating [CO2*].Based on pH measurements of xylem sap expressed from twigs witha pressure chamber (PMS Instruments, Corvallis, OR USA) during thegrowing season in 2000 and 2001, we estimated that average sap pHwas 6·0 in both species, and this value was used in ourcalculations.
Stem temperature was measured with thermocouples placed in 2 mmdiameter holes at the same depth and within 20 mm of themicroelectrodes. For some experiments, stem CO2 effluxand sap velocity were also measured. Efflux was measured with aninfrared gas analyser (IRGA) (Model 6252; Li-Cor Inc., Lincoln,NE, USA) using a 135 or 82 cm2 fan-stirred polyvinylchloride (PVC) cuvette sealed onto the stem 40 mm belowthe microelectrodes. Compressed air of known [CO2] (350–375 p.p.m) wassupplied at 500 mL min−1 tothe cuvette. The IRGA was operated in open configuration and effluxrate was calculated using standard procedures (Long& Hallgren 1985). Sap velocity was measured with thermaldissipation sensors (Model TDP-80; DynaMax Corp., Houston, TX, USA) installed10 cm above the microelectrodes (Granier1987). All sensors were protected from solar radiation bywrapping the stem with 40 cm wide reflective bubble insulation (Reflectix;Reflectix Inc. Markelville, IN, USA). Sensor data were collectedsimultaneously every 10 s and averaged and recorded every600 s using a datalogger (Model 21X; Campbell Scientific,Inc., Logan, UT, USA). Data were processed and analysed using systat 2000,SigmaPlot 2001 (SPSS Inc., Chicago, IL, USA), and Microsoft Excel2000 (Microsoft Inc., Redmond, WA, USA).
In 2000 we conducted experiments to determine the effect of sap [CO2*] onCO2 efflux of woody tissue. We manipulated sap [CO2*] ofdetached branch segments in the laboratory at a constant temperature(25 °C). Air at 2 and 10%[CO2] wasbubbled from compressed gas cylinders into two 20 L reservoirsfor 4 h. We used tubing to connect a reservoir to one endof a 40 cm segment of a 25 mm diameter detachedbranch of Q. alba or L. tulipifera. Water flowed continuouslythrough the branch segment under pressure from a 1 m headat a rate of ∼0·5 mL min−1.The branch segment was sealed into a 30 cm long tubularfan-stirred PVC cuvette. Approximately 5 cm of the branch segmentprotruded from each end of the cuvette. Radial CO2 effluxinto the cuvette was measured with an IRGA using the same procedureas described for in situ measurements. To measure sap [CO2*],a CO2 microelectrode was inserted radially 3 mminto the xylem at the outflow end of the branch segment. When sap [CO2*] andCO2 efflux stabilized, the branch was connected to theother reservoir until restabilization occurred.
In July and August 2001, field experiments were conducted todetermine the effect of xylem sap [CO2*] onstem CO2 efflux. To manipulate sap [CO2*]insitu, water enriched with CO2 was introduced intothe xylem. A length of 6 mm inside diameter tubing wastightly sealed into a hole drilled into a Q. alba or L.tulipifera stem. The tubing was connected to a 9 Lreservoir of water at 20%[CO2]. Tensionin the xylem pulled the water at an average flow rate of 300 mL h−1 fromthe reservoir into the tree, where it combined with the sap. Theresponse to this manipulation was observed over a 24 hperiod. Sap [CO2*], stem CO2 efflux,stem temperature and sap velocity were measured with the sensorsand cuvette placed on the stem in the same configuration as describedpreviously for field measurements. The infusion tubing was insertedinto the stem 10 cm below the cuvette.
Xylem sap [CO2*] changed diurnally, typically increasing atnight and decreasing during the day (Fig. 1a).This pattern was well correlated with diurnal changesin sap velocity (equation below). During the day, transient increasesin sap [CO2*] correspondedwith small decreases in sap velocity caused by passing clouds. Meansap [CO2*] was 5·2 mmol L−1,with a minimum and maximum of 3·7 and 6·2 mmol L−1.Measurements of stem CO2 efflux (Fig. 1c) werewell correlated with both stem temperature (Fig. 1b) andxylem sap [CO2*]. The linearregression equations for these relationships were: [CO2*] = 5·7 − 18·8V, R2 = 0·71; CO2 efflux = −2·3 + 1·3T, R2 = 0·91;CO2 efflux = 5·6 + 1·0[CO2*], R2 = 0·73,where V is sap velocity and T is stem temperature.
On other days, CO2 efflux corresponded more closely withxylem sap [CO2*] than stemtemperature (Fig. 2). Measurementsof sap [CO2*], stem temperatureand stem CO2 efflux were made continuously over threedays. Stem CO2 efflux and sap [CO2*] followedthe same pattern over time, which was opposite the diurnal patternof stem temperature. On these days, stem CO2 effluxwas poorly correlated with stem temperature (R2 = 0·13),but was better correlated with sap [CO2*] (CO2 efflux = −1·81 + 1·12[CO2*], R2 = 0·65).
For the two species we measured, Q. alba and L. tulipifera,mean daily xylem sap [CO2*] averaged4·0 and 3·2 mmol L−1,respectively (Table 1).Mean and diurnal xylem [CO2] and sap [CO2*] variedsubstantially during the growing season. Mean daily sap [CO2*] rangedfrom 0·9 to 7·4 mmol L−1 in Q. alba,and 1·1 to 8·3 mmol L−1 in L. tulipifera.Although it is likely that these differences are related in someway to weather conditions or changes in physiological activity ofthe trees, mean sap [CO2*] was poorlycorrelated with mean daily temperature or time of year.
Table 1. Mean(standard deviation), minimum and maximum daily average xylem [CO2] andsap [CO2 *] of Q.alba and L. tulipifera trees measured throughout thegrowing season in 2000 and 2001 at Whitehall Forest. Tree diametersranged from 15 to 70 cm
From these data (Figs 1and 2, Table 1),it was apparent that the [CO2] of xylemwas much higher than that of ambient air, and exhibited diurnalvariation that was well correlated with rates of stem CO2 efflux.These observations suggested to us that if CO2 was diffusingfrom the xylem through the bark into the atmosphere, then this process couldbe largely responsible for the inconsistencies and unexplained variationthat is common among studies of woody tissue respiration. To demonstratethis effect, we changed the xylem sap [CO2*] inexcised branch segments of Q. alba and L. tulipifera andmeasured CO2 efflux from the segments under constanttemperature conditions in the laboratory. The results of these experimentsshowed a clear correspondence between sap [CO2*] andCO2 efflux (Fig. 3).When water enriched with 10% CO2 gas flowed throughthe branch segment, measured sap [CO2*] increasedfrom an initial value of 2·3 to 4·2 mmol L−1,which produced a corresponding change in CO2 efflux (i.e.apparent respiration) from 0·6 to 1·4 µmol m−2 s−1.After 16 h, the water supply was changed from 10%[CO2] to2%[CO2], causing a rapid drop in CO2 efflux from the branch that wasproportional to the decrease in [CO2*] inthe xylem sap. This experiment was repeated several times (Table 2) with similar results. In all cases, when CO2-enriched waterwas infused into branches, there was a rapid increase in xylem sap [CO2*] anda proportional increase in CO2 efflux from the branch,and this effect was reversible. On average, in Q. alba branches,the 10% CO2 treatment increased stem [CO2*] by5·1 mmol L−1, whichin turn tripled the rate of CO2 efflux from the branch.In L. tulipifera, the treatment produced a smaller averageincrease in [CO2*] (3·6 mmol L−1),but a greater increase in CO2 efflux (from 0·4to 1·5 µmol m−2 s−1),almost quadrupling the initial value. The differences in the responsesof the two species were probably due to differences in the effectivenessof the water infusion treatment in ring porous and diffuse porous xylem,as well as differences in the ability of CO2 to diffuse throughthe cambium and bark layers into the air.
Table 2. Mean(standard deviation) xylem sap [CO2 *] and CO 2 effluxof detached 25 mm diameter branch segments of Q. alba and L.tulipifera measured in the laboratory. Temperature was constant at25 °C. Branch segments were infused with waterenriched with 10% CO2. Initial values are priorto infusion. Final values are maximums observed after several hoursof 10% CO2 infusion. Δ is the differencebetween initial and final values
The parallel change in CO2 efflux and xylem sap [CO2*] wasevidence that CO2 transported in the xylem could diffusefrom branches and affect measurements of respiration. However, thebark of branches is thinner than the bark of the main stem, andxylem sap is normally under tension, whereas in this test it wasunder slight pressure. To remove these limitations, we conducteda modification of this experiment in situ using large treeswith well-developed bark. In the first trial, conducted on a Q.alba tree, sap [CO2*],stem CO2 efflux, and air and stem temperatures were initiallymonitored for 26 h (Fig. 4).During this time, a close correlation between stem CO2 effluxand sap [CO2*] was apparent(Fig. 4 inset, CO2 efflux = −1·67 + 1·50[CO2*], R2 = 0·96).Efflux was also well correlated with xylem [CO2] (R2 = 0·93).When water containing 20%[CO2] wasinfused into the xylem, a rapid and parallel increase in sap [CO2*] andstem CO2 efflux was observed. Similar to the laboratoryexperiment, CO2 efflux more than doubled from the highestvalue of the previous day. This response was not related to temperature.Air temperature decreased slightly from day 1 to day 2, and stemtemperature was nearly constant because the test was conducted ona large tree (70 cm diameter) with high thermal mass thatwas located in a shaded position under an intact forest canopy.At the end of the experiment, when the tree had taken up all of thewater in the reservoir, sap [CO2*] andstem CO2 efflux decreased.
The experiment was repeated in a smaller L. tulipifera tree(20 cm diameter) in a more open canopy position. Sap [CO2*],stem CO2 efflux and air and stem temperature were monitoredfor 24 h (Fig. 5).During this time, a positive correlation was again observed betweenstem CO2 efflux and sap [CO2*] (Fig. 5 inset, CO2 efflux = 4·87 + 1·16[CO2*], R2 = 0·50),as well as xylem [CO2] (R2 = 0·67).Efflux was not as closely correlated with [CO2*] asin the previous experiment, presumably due to the effect of diurnal changesin stem temperature (Fig. 5b)on the equilibrium between [CO2] and [CO2*] (Butler 1991). Similar to the previous experiment,when infusion into the stem began, a rapid increase in both sap [CO2*] andstem CO2 efflux was observed. Sap [CO2*] increasedfrom 3·7 to 7·5 mmol L−1 and stem CO2 efflux increased from 8·6 to 18·3 µmol m−2 s−1,more than doubling the apparent rate of respiration. Diurnal airand stem temperatures were nearly identical on these two days (Fig. 5b),so temperature cannot explain the change in efflux from day 1 today 2.
Mean daily xylem [CO2] measuredin this study ranged from 1·6 to 15·9%,which is similar to reports using other techniques (MacDougal& Working 1933; Chase 1934; Hari et al. 1991; Eklund 1993; Levy et al.1999). The approaches used in previous studies limited samplingto once-daily or longer-term observations. In the present study,the microelectrode approach made it possible to sample at a muchgreater frequency, so the diurnal pattern of xylem sap [CO2*] was revealed.Daily fluctuations in sap [CO2*] wereoften large, and corresponded closely to diurnal changes in therate of CO2 efflux from the stem. When sap [CO2*] wasexperimentally manipulated in the laboratory and the field, we found adirect relationship between [CO2*] andCO2 efflux from the stem, strongly suggesting that theincrease in efflux was caused by the change in [CO2*] inthe xylem sap.
Our findings call into question previous efflux-based respirationmeasurements of individual plant organs (leaf, stem, root) becauserates of respiration have been calculated under the assumption thatthe source of CO2 production is local, when it appearsthat the source cannot be known precisely. The high [CO2*] instem xylem sap, measured at 1·5 m above the soilsurface, coupled with the negative correlation between sap [CO2*] andsap velocity, provides evidence that CO2 evolved fromrespiring cells in the stem, and probably roots and soil, accumulatesin the sap and is relocated by mass flow. Farther along the transpirationalstream, a portion of this CO2 is released to the atmosphere,causing an overestimation of the rate of respiration. Xylem sap [CO2*] alsochanges substantially during a 24 h period, from low valuesduring the day to high values at night, in a pattern that is thereverse of air and stem temperatures. This fluctuation creates anadditional error in the estimation of respiration because it changesthe CO2 diffusion gradient from xylem to atmosphere,which in turn affects the rate of CO2 efflux from thestem throughout the day.
The error caused by the transport and diffusion of CO2 canbe substantial. On a day when there was little change in stem temperature(11 July 2001, Fig. 4),the diurnal increase in xylem sap [CO2*] was2·4 mmol L−1, witha corresponding increase of 67% in stem CO2 efflux.The error on that day was compounded by the minimum sap [CO2*] (4·9 mmol L−1),which also contributed to radial CO2 diffusion, suggestingthat the total magnitude of the error in the apparent rate of respirationwas well above 100%.
Although the [CO2*] ofxylem sap affects the rate of CO2 efflux through thebark, the majority of the CO2 remains in the xylem, evidentlybecause diffusion is limited by the cambium and bark layers. Differencesin the permeability of bark layers to CO2 may accountfor much of the high variation reported in rates of respirationof branches and stems (Sprugel 1990; Cernusak & Marshall 2000).
The fate of CO2 that remains dissolved in sap is unknown,but it is likely that much of it is delivered to the leaves whereit is refixed in photosynthesis. The capacity for fixation of CO2 transportedin xylem has been demonstrated in trees (Zelawski,Riech & Stanley 1970; Stringer &Kimmerer 1993) and crop plants (Hibberd& Quick 2002). Carbon delivered in sap is unaccountedfor in estimates of photosynthesis derived from the flux of atmosphericCO2 into leaves. Estimates of the contribution of CO2 deliveredin sap to the overall photosynthetic carbon gain of leaves varyfrom a few percent to as much as 9% (Hari et al.1991; Levy et al. 1999).Using the same approach, our calculations indicate that the maximum short-termcontribution may be as much as 15% in certain circumstances.This estimate was based on measured values in Q. alba ofxylem [CO2*], 24·8 mmol L−1,xylem sap pH, 6·9, and midday rates of leaf net photosynthesis, 13·6 µmol m−2 s−1 andtranspiration, 4·6 mmol m−2 s−1.
We speculate that there is a paradoxical but not inconsistentoutcome of the movement of carbon in the transpirational pathway.High xylem sap [CO2*] indicatesthat a large amount of CO2 that evolves from respiringcells does not escape to the atmosphere but remains in the xylem streamand is transported upward, leading to the conclusion that some stemand root tissues may actually be respiring more than is accountedfor by CO2 efflux measurements, even though efflux measurementscan also overestimate respiration at a ‘local’ tissuelevel.
Our findings have another implication. The effects of temperatureon rates of respiration at the cellular level are well established(Amthor 1989). However, our data show thatthe temperature effect may be overwhelmed against the large backgroundof CO2 efflux from the xylem. Thus, actual rates of stemand branch respiration in situ cannot be adequately predictedby temperature alone.
In conclusion, although the picture is not yet complete, a portionof the carbon captured from the atmosphere and then evolved by respirationof woody tissues is being transported, and possibly recycled, withintrees. Our measurements indicate that because respired CO2 istransported in the xylem and diffuses to the atmosphere at a different locationin the tree, respiration rates have been overestimated in some tissuesand underestimated in others. These findings suggest that we nowhave a great deal more to learn about respiration in trees.
We thank Reese Halter and Global Forest Institute for supportof this project. This is Global Forest publication GF-18-1999-12.
Received 7 February 2002;received inrevisedform 28 May 2002;accepted for publication 15 August 2002