Within-stem variation of respiration in Pseudotsuga. menziesii (Douglas-fir) trees. Research

Transcription

1 Reserch Within-stem vrition of respirtion in Pseudotsug Blckwell Science Ltd menziesii (Dougls-fir) trees Michele L. Pruyn 1, Brr L. Grtner 1 nd Mrk E. Hrmon 2 1 Deprtment of Wood Science nd Engineering/Deprtment of Forest Science, Forest Reserch Lortory, Richrdson Hll, Oregon Stte University, Corvllis, OR , USA; 2 Deprtment of Forest Science, Forest Science Lortory, Richrdson Hll, Oregon Stte University, Corvllis, OR , USA Summry Author for correspondence: M. L. Pruyn Tel: Fx: Emil: Michele.Pruyn@orst.edu Received: 2 Octoer 21 Accepted: 18 Jnury 22 A technique for mesuring in vitro respirtion ws investigted to understnd why rtes were higher thn those reported in vivo nd to elucidte trends within mture Pseudotsug menziesii (Dougls-fir) trees. Extrcted increment cores were divided into 3 rdil depths nd gs chromtogrph ws used to compre respirtion rtes rdilly nd verticlly within stems. Respirtion of inner rk ws 2 3 times greter thn spwood, nd 7% higher in outer thn inner spwood. Inner rk nd outer spwood relesed > % more CO 2 t treetops thn t ses. Trends were roust for CO 2 production on core dry-mss, volume, or totl cron sis. By contrst, CO 2 production on nitrogen sis showed lmost no significnt vrition. This in vitro technique provided n effective index for reltive differences in respirtion within tree stems. Discrepncies etween in vitro nd in vivo mesurements might e relted to the gseous environment in stems. The estimted within-stem grdients in respirtion were possily determined y enzyme quntity nd vilility nd could e useful in scling to whole-trees. Key words: stem respirtion, stem ertion, inner rk, spwood, Pseudotsug menziesii (Dougls-fir). New Phytologist (22) 1: Introduction In most studies, estimtes of whole tree nd stnd level respirtion rtes re otined from scling up smll smple mesurements cquired using infr red gs nlysis (IRGA) chmers t one or more position(s) on tree stems (Kinerson, 197; Ryn et l., 199; Edwrds & Hnson, 1996). Stem surfce re eneth the chmer hs often een used s n index of the mount of living tissue to ssocite with the mesured respirtion rte (Linder & Troeng, 1981; Mtyssek & Schulze, 1988). This rte cn then e extrpolted to the entire surfce re of the stem nd from there to the ecosystem level. However, spwood volume hs proved to e etter index for scling mintennce respirtion to whole trees or ecosystems (Sprugel & Benecke, 1991; Ryn & Wring, 1992), proly ecuse spwood volume is proportionl to the mount of living prenchym cells therein (Ryn, 199; Lrson, 199; Stockfors & Linder, 1998). Scling respirtion to the whole-tree level y spwood volume from mesurements t only one loction ssumes uniform respirtion rtes mong ll the stem prenchym cells. This ssumption is unlikely to e vlid ecuse spwood is not uniform in ge (numer of yers since the cells developed) or mturity (cmil ge t which cells were produced). Tissue ge nd mturity depend on rdil nd verticl position in the stem, oth of which my impct respirtion. Rdil trends in spwood respirtion hve een recorded from inner rk to the hertwood/spwood oundry for tissues extrcted just ove ground level in stems of Pinus rdit (Shin & Mcky, 1973), Frxinus nigr nd Acer rur (Goodwin & Goddrd, 19), nd in Pice ies nd other species (Møller & Müller, 1938). Rdil nd verticl trends in spwood respirtion were recorded for Pinus ponderos (Pruyn et l., 22). The impct of such within-stem vritions on whole-tree level respirtion rtes ws ddressed in P. ies (Stockfors, 2). Assuming constnt temperture-respirtion reltionship, New Phytologist (22) 1:

2 36 Reserch stem temperture ws mesured t multiple heights nd rdil depths to predict respirtion. Within-stem temperture vritions were oserved tht resulted in scling errors etween 2 nd 72% for single sunny dy nd 2 nd 8% for whole yer. Within-stem vrition in respirtion ws lso ccounted for y verging respirtion rtes (mesured vi IRGA) from vrious heights on stems (Lvigne, 1987; Ryn et l., 1996), or from different compss directions (Edwrds & Hnsen, 1996). In third pproch, the distriution of live cell volume in spwood smples ws estimted y using vitl stining techniques nd imge nlysis tools, which provided percentge of living tissue for scling respirtion rtes to the whole stem-level (Ryn, 199; Stockfors & Linder, 1998). Although these three methods re improvements for representing vrition of respirtion within spwood, multiple sources for inccurcy remin. First, the use of temperture to predict respirtion rtes my e prolemtic ecuse of temperture-independent vrition in CO 2 efflux from stems (Mrtin et l., 199). Second, the use of IRGA systems my sometimes underestimte respirtion ecuse ll spwood CO 2 production my not rech the stem surfce. Some respired CO 2 could e dissolved in the trnspirtion strem (Negisi, 197; Sprugel, 199; Levy et l., 1999), refixed y rk photosynthesis in some species (Nilsen, 199; Cernusk & Mrshll, 2), or stored within spwood prenchym cells (Lev-Ydun & Aloni, 199). Third, vitl stining techniques only indicte whether cells re live or ded, they do not directly convey the degree of respirtory ctivity mong the living cells. In previous study, we mesured rtes of CO 2 production under controlled lortory conditions of cores extrcted from mture Pinus ponderos (ponderos pine) stems. A primry ojective ws to quntify within-stem vrition of respirtion, nd then scle to the whole-tree level (Pruyn et l., 22). We lerned tht respirtion in spwood of those pine trees ws not homogenous nd tht scling to whole-trees resulted in rtes sustntilly higher thn those reported in situ from the literture. In the current study, we first exmined the sis for higher in vitro thn in vivo respirtion rtes of excised cores of Pseudotsug menziesii (Dougls-fir) y exploring potentil rtifcts of the method. Second, we determined whether rk-to-pith nd treetop-to-se trends in respirtory potentil lso existed within Dougls-fir stems. Third, we compred core respirtory potentil on four different indices: dry mss, volume, moles cron nd moles nitrogen. Finlly, we discussed the likely physiologicl mechnisms responsile for vritions in respirtion within trees, cross sites nd etween sesons. Mterils nd Methods Species nd site chrcteristics We collected smples from mture (6 112 yr-old) Douglsfir trees, Pseudotsug menziesii (Mir.), from site just est of the Cost Rnge in southern Oregon, ner Riddle (N2 7 - W123 22, elevtion 21 m) nd in McDonld-Dunn Reserch Forest in the Willimette Vlley, ner Corvllis, OR, USA (N 38 -W123 17, elevtion 3 m). Unless indicted otherwise, smples were collected either efore the growing seson for this region, in erly Mrch of 1998 nd 1999, or fterwrds in Octoer of 1999, 2, nd 21. The rtionle ehind selecting these smpling dtes ws to cpture mintennce respirtion, nd thus void the complictions of growth respirtion in estimting core respirtion. Tree smpling All trees smpled were free of roken tops, stem deformities, or visile disese. Twenty to 3 trees were selected rndomly from ech site to e used for one or more of the following experiments. Stem dimeter 1 m from the ground rnged from to 7 cm (Corvllis) or 7 cm (Riddle). Tree ge t rest height ws 6 19 yr (Corvllis, from cores) nd yr (Riddle, from cross-sectionl disks). Respirtion mesurements Respirtory potentil ws estimted from 12 mm increment cores extrcted from either stnding stems (1 m from ground), or felled trees (1 m from ground nd nodes 3 nd 1 from treetop). When extrcting multiple cores from specific stem height, we took them evenly from out the stem s circumference. All cores were nlyzed within 1 wk of smpling. Twenty-four hours efore mesurement, cores were cut into four segments: inner rk (phloem nd cmium) nd outer, middle nd inner spwood. Spwood ws defined s the woody tissue extending from the first growth ring interior to the inner rk to the lst growth ring interior to the trnsition zone (one or two lighter colored rings t the spwood/hertwood oundry). Outer, middle, nd inner spwood smples were otined y dividing spwood into three equl rdil lengths. For this study, respirtion of hertwood ws not mesured ecuse preliminry dt reveled lmost no CO 2 evolution from hertwood smples (<.1 nmoles CO 2 g (d. wt) 1 s 1, M. L. Pruyn, unpulished dt). Numer of rings per segment ws recorded, so tht men ge could e determined for ech segment. These segments were weighed, wrpped tightly in plstic, then stored t 2 C overnight to llow metolic ctivity in core segments to stilize (Goodwin & Goddrd, 19; Hri et l., 1991; Levy et l., 1999). Immeditely efore mesurement, core segments were reweighed nd plced in vils, which were then seled with gs-tight ruer sept. To determine rte of CO 2 production, cron dioxide concentrtion within vils ws mesured with Hewlett-Pckrd (7 A) gs chromtogrph (GC) (Hewlett-Pckrd, Avondle, PA, USA) immeditely fter closing the vils nd gin fter n incution period. Core segments were incuted t 2 C (unless indicted otherwise) New Phytologist (22) 1:

3 Reserch 361 etween GC mesurements. Incution period ws held constnt t either 6 or 2 h, with the longer incution necessry to ccommodte the processing time of lrge numers of smples. Detils of GC nlysis nd clcultion of respirtory potentil (nmoles CO 2 g (d. wt) 1 s 1 ) re in Pruyn et l. (22). From this point onwrd, we refer to the reported vlues s respirtory potentil, rther thn respirtion rte ecuse the conditions of our mesurements on these excised smples re proly different from those within the tree. Immeditely following the GC nlysis, core segments were weighed third time. The three successive wet msses verified tht wter loss ws low (etween 1 nd 3%) etween smpling nd the end of the mesurement period. Fresh volume of core segments ws estimted s displced wter y sumerged smples (D239, ASTM, 21). Dry msses were determined fter oven drying t 6 C for 8 h. The OSU Centrl Anlyticl Lortory determined totl core cron nd nitrogen content using LECO CNS-2 Micro Anlyzer (LECO, Corp., St. Joseph, MN, USA). Potentil rtifcts of technique To ddress the question of why in vitro respirtion rtes from the current technique differ from those reported in vivo, we conducted six tests to exmine potentil rtifcts from extrction, hndling nd storge efore nlysis. Effects of time in cold-storge t C To determine whether rte of core segment CO 2 production ws ffected y storge time efore respirtion mesurement, we extrcted four 12 mm dimeter cores t rest height from six trees from the Corvllis site in lte August of 2. For ech tree, ech core ws ssigned to one of four tretments: 1, 2, or 9 d of storge t C efore GC mesurement. The night efore GC nlysis, cores were segmented ccording to rdil position s descried ove, with the exception tht only inner rk, outer nd inner spwood tissues were smpled to cpture the extremes of ctivity. Core segments were then nlyzed for CO 2 production over 6-h incution period. We tested pulished results tht storge time of < 1 d would hve no effect on core segment respirtory potentil (Goodwin & Goddrd, 19; Shin & McKy, 1973). The presence of trend etween core segment CO 2 production rte nd storge time would indicte tht the response ws not stle nd thus not likely indictive of respirtion. Decresed CO 2 production rte with incresed storge time my suggest decresing wound response, decresing diffusion of stored CO 2 from within the core s it equilirtes to tmospheric concentrtions, or incresing prenchym cell deth; wheres incresed CO 2 production with storge time suggests the incresed contriution of microil respirtion. Microil presence nd respirtion rte To determine the extent to which microil respirtion contriuted to the oserved CO 2 production of core segments, we compred the respirtory potentil of smples 2 d nd 1 month fter coring. One core ech ws extrcted from three of the six trees in the storge time experiment, segmented ccording to rdil position nd mesured for core respirtory potentil s descried ove. Core segments were then plced seprtely into sterile Petri dishes contining mlt extrct gr (1.% mlt extrct, 1% gr), seled with Pr-film M lortory film (Americn Cn Compny, Greenwich, CT, USA ) nd then stored t 2 C. After 2 nd wk, the type of microe (fungl or cteril) visile to the nked eye on ech core ws recorded. We predicted tht microil growth would e visile on core surfces. After the fourth week, we gin mesured core respirtory potentil. To verify tht CO 2 produced y these core segments ws exclusively from microes nd not spwood prenchym, we exposed the cores to vitl stin. Immeditely following the GC nlysis, we sumerged them in 1% queous triphenyl tetrzoilium chloride solution (TTC), which is reduced to deep-red color in the presence of living cells (Feist et l., 1971; Ryn, 199). Red-stined tissues were considered live nd nonstined tissues ded. Using differentil scnning clorimetry to mesure core respirtory potentil To verify tht the CO 2 relesed y core segments ws product of respirtion, we compred our GC mesurements with core segment metolic het rte, tht is the rte t which het is produced y respirtion (Criddle et l., 1991), using differentil scnning clorimeter (DSC, Hrt Scientific Model 777) (Clorimetry Sciences Corp. (CSC), Americn Fork, UT, USA) in the isotherml mode (2 C). Becuse the DSC mpules were only 1 cm 3, we reduced the size of ech smple to. cm dimeter.8 cm length. Two replictes for ech rdil position (inner rk, outer spwood, nd inner spwood) from 12 trees from the Corvllis site were used for the experiment ecuse of high vriility of preliminry results (M. L. Pruyn, unpulished). Extrcted cores were stored t C, until the night efore nlysis, when they were segmented s descried ove nd stored t 2 C. The DSC nlysis ws implemented ccording to Criddle et l. (1991) nd Anekond et l. (199), with the exception tht ech phse of the rection required 7 insted of min ecuse respirtion in stem tissues is considerly less thn in leves. We nlyzed one tree per dy, nd ech replicte set (inner rk, outer spwood, nd inner spwood) seprtely, which enled us to keep cores intct until the night efore nlysis, therey reducing risk of desicction. Effects of temperture on respirtion rte To determine if response of core segment respirtory potentil to temperture ws consistent with literture vlues from intct Dougls-fir trees, four 12 mm dimeter cores were extrcted from rest height from ech of five trees t ech site (Corvllis nd Riddle) in erly Mrch 2 for totl of 2 cores per site. For New Phytologist (22) 1:

4 362 Reserch ech tree, ech core ws ssigned to one of four tempertures (, 1, 1, 2 C). Core response to temperture ws mesured, nd the Q 1 s (coefficient for chnges in respirtion with respect to temperture) for the 1 C nd 1 2 C temperture rnges were clculted s descried in Pruyn et l. (22). Sesonl vrition of within-stem CO 2 nd O 2 concentrtion We exmined the sesonl pttern of within-stem O 2 nd CO 2 concentrtions in vivo to gin insight s to how different these conditions were from the environment of cores in vitro. To determine the CO 2 nd O 2 concentrtion within stems, four holes of different depths were drilled into 12 trees t rest height (1 m from the ground) in erly Mrch of 2 from the Corvllis site. This experiment ws modeled fter Eklund (199). Depths were s follows: outer rk (1 2 cm), inner rk (1. 3 cm), middle spwood (3 7 cm) nd spwood/hertwood ( 13 cm). Depths were ssigned to lternting directionl fces y tree to distriute the depths evenly mong the four spects (N, S, E, W). There were few exceptions to this design due to overshooting the drilling depths, which introduced confounding vrile of direction on depth. To determine the four drilling depths for ech tree, we extrcted one mm increment core from ech of the NE nd SE spects. Before drilling into ech tree fce, we extrcted -mm core to the ssigned depth. This smple core ws compred with the neighoring digonl core to verify tht the depth ws correct. For exmple, this comprison would indicte if the rk thickness hd chnged, or whether we hd overshot the spwood/hertwood oundry. We mrked the confirmed depth on 12-mm uger drill-it nd then drilled into the mm incrementcore hole. Stinless steel tues were inserted ll the wy into the holes so tht only 1 cm 2 of wood (tngentil fce) ws exposed to the ir in the tue nd the other end of the tue emerged from the tree. The re round the tue t the point of insertion ws seled with silicone to void lekge, nd gs-tight ruer stopper ws inserted into the end of the tue. For trees in which resin initilly thretened to lock some of these stinless steel tues, we instlled dringe pprtuses, which consisted of Y-connecting glss tue inserted into the septum on the tree. Ech exposed end of the Y-tue ws connected to either 2-ml glss vil for gs collection, or to 1-ml vil vi septum for resin collection (pointed towrds the ground to fcilitte flow of resin). The gs collection vil ws seled with silicone septum nd open-top screw cp nd connected to the Y-tue vi doule-ended needle tht penetrted the sel of ech tue. All sept connecting Y-tues to steel tues or glss vils were tightened with plstic cinchstrps. Additionlly, ll exposed sept surfces were seled with Prfilm M nd covered with duct tpe to impede environmentl degrdtion of the ruer nd thus minimize gs lekge. All septum-seled, steel tues without dringe pprtuses were connected directly to gs-collection vils vi doule-ended needles. The connections to the gs-collection vils were initited on April 1st nd mintined for 3 wk. The seled glss vils were then removed nd the inside gs composition determined using the GC. Immeditely fter removing the seled glss tue, replcement ws connected to the steel tue for the next 3-wk period. Vils were collected during the morning (8 1 h), once every 3 wk from April through Octoer Dringe pprtuses were replced s resin-collection vils filled-up, s were ny ruer sept tht showed excessive degrdtion. To compre the mesured fluxes of stem O 2 nd CO 2 to sesonl climte trends, dily men mximum nd minimum temperture nd precipittion for the site were otined from the Oregon Stte University (OSU) Integrted Pest Mngement Wether Dt we site ( Responses in core respirtory potentil to vritions in mient CO 2 /O 2 concentrtions To understnd the effect of CO 2 /O 2 concentrtion on core segment respirtory potentil, we extrcted four 12 mm dimeter cores from five trees t the Corvllis site in Octoer Ech core ws ssigned to one of four tretments: mient in the lortory (control,.% CO 2 /21% O 2 ), O H (2% CO 2 /% O 2 ), O L (1% CO 2 /2% O 2 ), nd nitrogen-flushed (% CO 2 /% CO 2 ). Cores were cut into the three rdil segments nd stored t 2 C overnight. The morning of the experiment, segments were plced in test tues nd septum-seled. Test tues were flushed with nitrogen using two-wy syringe system. Control segments were not flushed. For the O H nd O L tretments, volume of gs equl to tht which would e dded ws removed, then the pproprite volume of ech gs ws dded to ech test tue. Approprite volumes were clculted y n ppliction of the Idel Gs Lw (concentrtion 1 volume 1 = concentrtion 2 volume 2 ). Gs concentrtions (CO 2 nd O 2 ) in the test tues were nlyzed using the GC initilly, s well s t, 1, nd 32 h. Appliction of the method stem rdil nd verticl trends in core respirtory potentil We selected nd felled three trees from the Riddle site. After felling, we swed 2 cm tll stem disks from stems t the 1th (men ± SE,.3 ±.2 m from ground), nd just ove (to void rnch whorls within the crown) the 3th (3 ± 1 m from ground) nd 1th (39 ± 1 m from ground) nodes (yers) from the treetop. Totl stem height verged t ± 2 m nd se of the live crown (first stem position ove ground level with three live rnches) t 28 ± 2 m. A second, short disk (< cm tll) ws tken from ech verticl position for clcultions of inner rk, spwood nd hertwood thickness nd for determining the ge of the tissues. The tll disks for respirtion mesurements were wrpped in mmthick lck plstic gs with moist pper toweling inside to reduce desicction efore experimenttion. Tll disks were New Phytologist (22) 1:

5 Reserch 363 stored t C. The short disks were kiln dried, nd then rdil distnces nd ring counts from pith to the distl edge of ech tissue (outer nd inner rk, spwood nd hertwood) were recorded. Within 1 wk fter hrvesting, five 8 mm dimeter increment cores were extrcted from ech height position (tll disk), wrpped seprtely in plstic gs nd immeditely returned to cold storge. The evening efore GC nlysis, cores were cut into four rdil positions (inner rk, outer spwood, middle spwood, nd inner spwood), rewrpped in plstic, nd stored overnight t 1 C. The following morning the smples were plced in vils, septum-seled, nd nlyzed for CO 2 production s descried ove. The incution period ws 22 h t 1 C. Sttisticl nlysis All dt were nlyzed in Sttisticl Anlysis Systems softwre, relese 8. (SAS Institute Inc. 1998). The Shpiro-Wilk W-test ws used to determine whether the response vriles were distriuted normlly. A trnsformtion (squre-root or nturl log) ws performed when necessry to meet ssumptions of normlity nd constnt vrince. Lest squres mens (LSMEANS), generted from the vrious SAS procedures descried elow, re reported ± pooled SE, or confidence intervls for trnsformed vriles. Within specific tle or figure, if confidence intervls were required for one vrile, they were presented for ll. A one-wy ANOVA in PROC GLM ws used to mke comprisons mong cold-storge time tretments. Specific pir-wise comprisons mong tretments were conducted for ech rdil position seprtely using Fisher s Protected Lest Significnt Difference (FPLSD) procedure (Fisher, 1966). Pired t-tests were used to compre respirtory potentil of control cores efore nd fter 3 dys on gr medium. Comprisons mong rdil positions for the DSC nlysis of core respirtory potentil were mde using PROC MIXED, with rndomized lock design nd strip-plot (split-lock) tretments (Little & Hills, 1978; Milliken & Johnson, 198). Trees were locks nd the effect of rdil position ws tested. Pir-wise comprisons mong tissue rdil positions were conducted using FPLSD procedure. Comprisons mong Q 1 vlues t different temperture rnges were mde using strip-plot nlysis in PROC MIXED. Trees were locks, nd the effects of tissue rdil position, temperture rnge, site nd ll possile interctions were tested. Pir-wise comprisons mong tissue rdil positions nd temperture rnges were conducted using FPLSD. Repeted mesures nlysis in PROC MIXED ws used to test the effects of smpling dte, rdil position nd their interction on respirtory potentil (Little et l., 1996). We initilly included spect (N, S, E, W) nd the interction of spect nd smpling dte in the model, ut dropped these effects when they proved to e nonsignificnt. Trees were treted s locks, nd the tree y tretment interction ws lso locked using the SUBJECT option in the repeted sttement with TYPE = UN covrince structure. A repeted mesures nlysis in PROC MIXED ws lso used to test the effects of incution time, tmospheric (CO 2 /O 2 ) tretment, nd their interction on core respirtory potentil. This repeted mesures model ws identicl to the previous model, except tht tree ws included s rndom effect nd ech rdil position ws nlyzed seprtely. A strip-plot nlysis ws used to mke comprisons (using FPLSD) mong tissues t vrious rdil nd verticl positions within trees. We tested the effects of tissue rdil position, verticl position nd their interction. This nlysis ws lso used to test the effects of rdil position, seson (Corvllis site) or verticl position (Riddle site), nd their interction on respirtory potentil on four different indices (i.e. core dry mss, volume, cron or nitrogen). Results Effects of cold-storge time nd microil respirtion There were no significnt differences in respirtory potentil (nmoles CO 2 g (d. wt) 1 s 1 ) of inner spwood from Douglsfir trees on the Corvllis site, stored for 1, 2,, or 9 d t C efore GC nlysis (P., Tle 1). Outer spwood respirtory potentil ws lso firly constnt, regrdless of storge length, with the exception of tissue stored for d, which respired significntly higher thn tissues stored for 1, 2, or 9 d (P <., Tle 1). By contrst, inner rk showed trend of incresing ctivity with storge time tht ws significnt (P =.) fter 9 d of storge, when respirtory potentil ws % higher thn fter the first dy. After 3 d on gr medium, microil growth (fungl nd/or cteril) ws visile on ll core segments (except one inner rk smple). We concluded tht microil respirtion proly did not contriute significntly to the respirtory potentil of freshly smpled cores ecuse respirtory potentil of older cores with visile microe growth ws either > % less thn (inner rk nd outer spwood) or equl to (inner spwood) tht of fresh cores with no microes visile (Tle 1). Further, we verified tht CO 2 produced y cores with visile microe growth ws exclusively from microes ecuse the TTC only stined red where microes were visile. Differentil scnning clorimetry (DSC) Respirtory potentil mesured using DSC ws significntly higher in inner rk thn in spwood, ut not higher in outer thn inner spwood (Tle 1). Inner rk nd spwood respirtory potentils mesured with the DSC were comprle with GC-mesured respirtory potentils from core segments in the storge time experiment (Tle 1). However, DSC respirtory potentils for inner spwood were generlly higher thn when mesured with the GC. New Phytologist (22) 1:

6 36 Reserch Tle 1 Respirtory potentil t 2 C of core segments from three rdil positions, testing three different vriles or methods: storge period, microil presence fter 1-month incution, nd differentil scnning clorimetry Respirtory potentil (nmoles CO 2 g (d. wt) 1 s 1 ) y rdil position Test Inner rk LSMEAN Outer spwood LSMEAN Inner spwood LSMEAN Storge period (dys t C) (n = 6) (2.6,.).63 (.3,.7).27 (.22,.32) 2.1 (3.,.9).6 (.9,.7).27 (.21,.33) 3.9 (3.1,.6).79 (.69,.9).32 (.27,.37) 9.8 (.1,.).61 (.,.71).3 (.2,.3) Microil presence (n = 3) Men Men Men (dys fter coring, microe sttus) 2 d, no growth visile.1 (.,.2).63 (.7,.69).27 (.2,.3) 3 d, microe growth visile.69 (.62,.76).3 (.2,.39).19 (.18,.2) LSMEAN LSMEAN LSMEAN Differentil scnning clorimeter (n = 12) 3.9 (3.,.) A..7 (., 1.) B..6 (.,.8) B. All cores extrcted from rest height of (n) trees t the Corvllis site. Lest Squres Men (LSMEAN, ANOVA or Strip-plot nlysis in PROC MIXED) or Men (t-test) ±9% confidence intervls. For ech test, different lower-cse letters indicte significnt differences within columns nd different cpitl letters indicte significnt differences within rows (LSMEANS y FPLSD, Mens y t-tests, P <.). Tle 2 Men Q 1 for three rdil positions in stems t oth the Corvllis nd Riddle sites Rdil position Q 1 (Temperture Rnge C) Inner rk Outer spwood Inner spwood Corvllis Q 1 ( 1). (3.3,.7).8 (3.6, 6.).3 (.1, 6.) Q 1 (1 2) 2.2 (1., 3.) 1.9 (.7, 3.1) 2. (.9, 3.2) Riddle Q 1 ( 1). (3.3,.7) 7.6 (6., 8.8) c 7.6 (6., 8.8) c Q 1 (1 2) 2. (1.2, 3.6) 1.8 (.6, 2.9) 1.9 (.8, 3.1) All cores extrcted from rest height of (n = ) trees t ech site. Lest Squres Men (Strip-plot nlysis in PROC MIXED) ±9% confidence intervls re given for ech temperture rnge (i.e. 1 nd 1 2 C). Different letters represent significnt differences mong ll mens (FPLSD, P <.). Temperture effects For ll rdil positions in trees from oth sites, the Q 1 for the 1 C temperture rnge, verging etween. nd 7.6, ws significntly higher thn for the 1 2 C rnge, verging 2. (Tle 2). The effect of temperture on Q 1 ws thus significnt (P <.1). The lrge vriility of the Q 1 verge t the lower temperture rnge resulted from outer nd inner spwood smples from the Riddle site. Thus, these two positions were exceptions to the trend of no significnt vrition mong rdil positions for ech temperture rnge nd the effect of rdil position on Q 1 ws not significnt (P >.2). However, the interction of temperture y rdil position nd the interction of temperture y site were significnt (P =.1), likely result of high outer nd inner spwood Q 1 s t the Riddle site. Sesonl vrition of within stem CO 2 nd O 2 concentrtion The effects of spect (N, S, E, nd W) nd the interction of spect nd smpling dte were not significnt to the repeted mesures model for response vriles, O 2 nd CO 2 concentrtion (P >.1). Thus, we found no evidence in these dt of confounding vrile of spect on gs concentrtion y rdil position nd dropped these effects from the model. However, we could not completely rule out the possiility of confounding ecuse of the experimentl design, which hd n unequl numer of rdil positions ssigned to ech compss direction. Within stem O 2 nd CO 2 vried significntly y smpling dte (P <.1) nd rdil position (P <.1). The interction of smpling dte y rdil position ws significnt for stem O 2 (P =.3), New Phytologist (22) 1:

7 Reserch 36 Fig. 1 Sesonl flux of within-stem gs concentrtions of trees t the Corvllis site. Concentrtion of cron dioxide (% CO 2, closed circles) nd oxygen (% O 2, open circles) in the gs phse in equilirium with () outer rk () inner rk (c) middle spwood, nd (d) hertwood. (e) Dily men mximum temperture (lines) nd minimum temperture (old lines) nd precipittion (rs). Lest Squres Mens ±9% confidence intervls (n = 12 trees) in PROC MIXED. %O 2 %O 2 %O 2 %O 2 Temperture ( o C) () Outer rk () Inner rk (c) Middle spwood (d) Spwood/hertwood oundry (e) Men dily temperture nd precipittion Julin dy Precipittion (mm) %CO 2 %CO 2 %CO 2 %CO 2 suggesting tht the reltionship etween O 2 nd smpling dte vried mong the four rdil positions. This result ws in contrst to the nonsignificnt interction of smpling dte y rdil position for stem CO 2 (P =.2). The trend in O 2 concentrtion y smpling dte nd rdil position were essentilly inversely relted to the corresponding trend in CO 2 : when O 2 ws low, CO 2 ws high. Lowest within stem O 2 (men = 6.%) nd highest CO 2 (men = 3.%) occurred t the spwood/hertwood oundry during the growing seson on Julin dy 16 (Fig. 1 d). This hypoxic environment within stems corresponded with the onset of the growing seson, higher tempertures nd less precipittion (Fig. 1e). As the growing seson ended in lte Septemer erly Octoer (Julin dy 28), within stem O 2 grdully returned to the tmospheric levels recorded on the first smpling dte in erly My ( Julin dy 123). Within stem CO 2 followed similr pttern, yet returned to tmospheric levels more rpidly. Middle spwood nd the hertwood/spwood oundry were nerly lwys more hypoxic thn the inner nd outer-rk, yet significnt differences etween these two outer, or two inner rdil positions were rre. New Phytologist (22) 1:

8 366 Reserch % O 2 nmoles O 2 g (d. wt) 1 s 1 Inner rk 2 1 () () 1 8 Tretment Effects (,, c P <.) Time Effects (, P <.) c c % CO 2 nmoles CO 2 g (d. wt) 1 s 1 % O (c) Outer spwood (d) % CO 2 nmoles O 2 g (d. wt) 1 s s % O 2 nmoles O 2 g (d. wt) (e) Incution time (h) Inner spwood (f) + c c + c c nmoles CO 2 g (d. wt) 1 s 1 % CO 2 nmoles CO 2 g (d. wt) 1 s 1 Fig. 2 Effects of four different gseous environments on respirtory potentil t 2 C, O 2 uptke (nmoles O 2 g (d. wt) 1 s 1 ) nd CO 2 production (nmoles CO 2 g (d. wt) 1 s 1 ), of cores extrcted t rest height from trees t the Corvllis site. Positive vlues for O 2 rtes nd negtive vlues for CO 2 rtes re the result of the respective diffusion of O 2 out of, or CO 2 into the core. Ech lettered pir of grphs shows gs concentrtion (%CO 2 or percentge O 2 ) within test tues, nd respirtory potentil (O 2 uptke or CO 2 production) of tissues from ech rdil position t three incution times. Lest Squres Mens ± SE (n = trees) from repeted mesures nlysis in PROC MIXED. Aove the x-xis, different letters indicte significntly different mens mong tretments t ech incution time (tretment effects), nd elow the x-xis, different letters indicte significntly different mens mong incution times for ech tretment (time effects) (FPLSD, P <.). For ech effect, dt without letters re not significntly different from one nother. Amient, open columns; % O 2 2% CO 2, htched columns; 2% O 2 1% CO 2, lck columns; N 2 flushed, gry columns. New Phytologist (22) 1:

9 Reserch 367 Responses in core respirtory potentil to vrition in tmospheric CO 2 /O 2 concentrtion The initil gs concentrtion tretments (mient, O H % O 2, 2% CO 2 ; O L 2% O 2, 1% CO 2 ; nd N 2 flushed) chnged over the course of the experiment ecuse of core segment respirtion (oxygen uptke or cron dioxide production) nd gs diffusion (stored gs from within the cores into the hed-spce of the vils or vis vers, Fig. 2). As result, gs con-centrtions were nerly equl mong the four tretments s the experiment reched completion, which chnged the effect of tretment on core respirtory potentil over time. Respirtory potentil, cron dioxide production (nmoles CO 2 g (d. wt) 1 s 1 ) nd oxygen uptke (nmoles O 2 g (d. wt) 1 s 1 ) rtes vried significntly y incution time (P <.) nd tretment (P <.) for ll three rdil positions (Fig. 2). The interction etween time nd tretment ws significnt for O 2 uptke in inner rk nd outer spwood (P <.), ut not for inner spwood (P =.7). By contrst, the interction for CO 2 production ws significnt for ll three rdil positions (P <.1). This result indicted tht the trend in core respirtory potentil over time vried y tretment. All rdil positions of core segments under mient conditions (control) showed the highest rtes of O 2 uptke nd CO 2 production mong the four tretments tested (Fig. 2). As O 2 concentrtion in the vils decresed nd CO 2 incresed over time, O 2 uptke nd CO 2 production of the inner rk nd outer spwood controls decresed significntly (Fig. 2 d). Inner spwood O 2 uptke nd CO 2 production for the controls did not show ny significnt trends over time (Fig. 2e f ). For the O H, O L, nd N 2 flushed tretments O 2 uptke ws not evident fter or 1 h of incution in smples from ny of the rdil positions (Fig. 2,c,e). The positive vlues for O 2 rtes were likely the result of O 2 diffusion from within the core segments into vil hedspce. After 32 h, it ws evident tht ll core segments (from the O H, O L, nd N 2 flushed tretments) were consuming O 2 ecuse their O 2 uptke rtes hd decresed or ecome negtive (indicting O 2 uptke). Inhiition of core segment respirtory potentil ws evident y the trend of decresed CO 2 production in the treted core segments compred with the controls (Fig. 2,d,f ). The most notle inhiition of CO 2 production rtes occurred in the O L treted core segments. The most extreme cses of this trend were in the inner spwood nd to lesser extent inner rk, where negtive CO 2 rtes were recorded, indicting the likely diffusion of CO 2 from the vil hedspce into the core segment. Stem rdil nd verticl trends in core respirtory potentil When considering only stem spwood respirtory potentil, the effects of stem rdil position (P =.3) nd the interction of rdil y verticl position (P =.) were significnt, wheres the effect of stem verticl position ws not significnt (P =.1). At ll three verticl positions, outer spwood respirtory potentil ws > 6% higher thn middle or inner, nd there ws no significnt difference etween middle nd inner spwood (Fig. 3). The significnt interction term indicted tht the reltionship etween respirtory potentil nd rdil position (ring numer) vried y stem verticl positions. When oth inner rk nd spwood respirtory potentil were compred, effects of stem rdil position, verticl position nd their interction were ll significnt (P <.3, Fig. 3). At node 1 from the treetop, inner rk respirtory potentil ws > 3 times higher thn ll spwood positions (Fig. 3). However, t node 1 inner rk respirtory potentil ws equl to tht of ll spwood positions, except inner spwood. Outer nd middle spwood positions t node 1 from the treetop were t lest % higher thn their corresponding rdil positions t node 1 (Fig. 3). Respirtory potentil using moles cron or moles nitrogen s sis At oth the Corvllis nd Riddle sites, core segment respirtory potentil per moles cron (µmoles CO 2 moles C 1 s 1 t 2 C) followed trends similr to the mss nd volume sed indices (Tle 3). The effect of rdil position ws significnt t oth sites for ll three indices (P <.1). For the cron, mss nd volume indices, effects of seson nd the interction of rdil position y seson were not significnt (P >.2), wheres effects of verticl position nd the interction of rdil y verticl position were (P <.1). For these indices respirtory potentil t treetops ws t lest 1. times greter thn t the ses. The within-stem trends for respirtory potentil per moles nitrogen (µmoles CO 2 moles N 1 s 1 t 2 C) were not s ovious s when indices of cron, mss or volume were used (Tle 3). For exmple, lthough the effect of rdil position on respirtory potentil per unit nitrogen ws significnt (Riddle site, P =.1), the inner rk vlue ws only 1.2 times greter thn tht of spwood compred with > times greter for ny of the other indices. Moreover, there were no significnt differences mong stem rdil nd verticl positions for respirtory potentil per moles nitrogen t the Corvllis site. Discussion The methods tests indicted tht respirtion rtes in core segments were stle nd not likely from wounding or microil respirtion (Tle 1). Previous studies hve lso shown stle respirtion rtes in stored smples of tree stem tissue, for storge lengths < 1 d, in two hrdwoods (Goodwin & Goddrd, 19) nd in Pinus rdit (Shin & Mcky, 1973). In the current study, inner rk respirtory potentil ws less stle thn the spwood tissues nd thus New Phytologist (22) 1:

10 368 Reserch.8 () Respirtory potentil (nmoles CO 2 g (d. wt) 1 s 1 ) cd cd c cd d d () Numer of rings from rk Fig. 3 Reltionship etween tissue ge nd respirtory potentil t 1 C (nmoles CO 2 g (d. wt) 1 s 1 ) of cores extrcted from two to three verticl positions within () spwood nd () inner rk nd spwood of trees from the Riddle site. Different shpes represent ech stem rdil position, nd different shdes represent ech verticl position. Lest Squres Mens ±9% confidence intervls from strip-plot nlysis in PROC MIXED (n = three trees). In ech pnel, different letters indicte significnt differences (FPLSD, P <.). For clrity, not ll significnt differences were indicted in (), nmely: node 1, inner rk vs inner spwood; outer spwood, node 1 vs node 1; nd middle spwood, node 1 vs node 1. Inner rk, circles; outer spwood, tringles; middle spwood, squres; inner spwood, dimonds. Blck, node 1; gry, node 3; white, node 1. should e nlyzed within the first few dys of smpling. In previous study, we concluded tht the CO 2 production of extrcted ponderos pine cores ws from the respirtion of living prenchym cells nd not from degssing of stored CO 2 y demonstrting tht there ws no respirtion from cores exposed to toxic fumignt compred with controls (Pruyn et l., 22). Results from differentil scnning clorimetry in the current study further supported this conclusion. In the current study, the men Q 1 of 2. for ll tissues t oth sites for the 1 2 C rnge is consistent with literture vlues for intct Dougls-fir stems (L. A. Cernusk, N. McDowell, N. Blster, & J. D. Mrshll, unpulished dt). The higher spwood Q 1 s in the current study for the 1 C temperture rnge re unusul compred with Lrcher (1983), who reported tht plnt Q 1 s pproch 2. t 2 C, ut hve een recorded for decomposing root tissues (Chen et l., 2). These dt suggest tht t lower tempertures ( 1 C), Dougls-fir spwood my hve een more rective to temperture increses thn would e expected from literture vlues. Further reserch is necessry to scertin whether Q 1 lower thn 2. should e used when normlizing respirtion mesurements of Dougls-fir spwood to tempertures in the 1 C rnge. The reltive uniformity of spwood Q 1 within given temperture rnge (excluding the 1 C rnge t the Riddle site) ws notle ecuse spwood ge rnged from 2 to 3 yr. Thus, respirtory enzymes in live stem wood of these Dougls-fir trees responded to temperture similrly, regrdless of tissue ge. The O 2 nd CO 2 concentrtions within Dougls-fir stems in vivo differed from the mient concentrtions of the stndrd in vitro set-up used in the current study, which likely impcted the respirtion rtes of extrcted cores. Low O 2 nd high CO 2 within stems ws lso recorded in Pinus stroes nd Pice ies during the growing seson (Chse, 193; Eklund, 199, respectively). Men O 2 from the Dougls-fir trees in the current study did not rech levels s low, nor did men CO 2 rech levels s high s in these previous studies. However, individul Dougls -fir trees did rech such levels of O 2 (2%) nd CO 2 (9%), ut trees were not synchronized y smpling dte, resulting in the lower mens reported here. The inverse reltionship etween O 2 nd CO 2 suggests tht respirtion of the living cells in the spwood regultes their concentrtions New Phytologist (22) 1:

11 Reserch 369 Tle 3 Respirtory potentil on cron, nitrogen, mss, or volume sis of cores extrcted from 1 to 2 stem verticl positions from trees t the Corvllis (Mrch nd Octoer) nd Riddle (Mrch) sites Cron nd Nitrogen mesurements y loction, seson, nd stem position Rdil position Inner rk Outer spwood Inner spwood Corvllis, OR Mrch nd Octoer (1 m from ground) Respirtory potentil per moles Cron t 2 C (µmoles CO 2 moles C 1 s 1 ) Mrch.13 (.1,.17).2 (.18,.33).16 (.12,.22) c Octoer.9 (.7,.13).2 (.18,.33).12 (.9,.16) c Respirtory potentil per moles Nitrogen 2 C (µmoles CO 2 moles N 1 s 1 ) Mrch 3 (2, 9) c 29 (1, 3) c 22 (8, 36) c Octoer 9 (3, 63) 1 (27, ) 33 (19, 8) c Respirtory potentil per grms dry mss t 2 C (1 3 µmoles CO 2 g (d. wt) 1 s 1 ) Mrch. (.1, 7.) 1.3 (.77, 1.39).68 (.,.92) cd Octoer 3.9 (2.9,.2).97 (.72, 1.31) c.8 (.3,.6) Respirtory potentil per core volume d t 2 C (1 3 µmoles CO 2 cm 3 s 1 ) Mrch 2.7 (2., 3.6). (.,.7).33 (.2,.) c Octoer 1.8 (1.3, 2.). (.,.7).26 (.19,.3) c Riddle, OR Mrch (Node 1 nd Node 1 from treetop) Respirtory potentil per moles Cron t 1 C (µmoles CO 2 moles C 1 s 1 ) Node 1. (.,.6).1 (.3,.2).6 (.,.1) Node 1.1 (.3,.2).6 (.,.1).3 (.,.1) Respirtory potentil per moles Nitrogen t 1 C (µmoles CO 2 moles N 1 s 1 ) Node (.2, 11.7) 12.8 (2.9, 21.8) 9.1 (.6, 13.8) Node (.9, 16.) 1.2 (., 2.). (2., 2.2) Respirtory potentil per grms dry mss t 1 C (1 3 µmoles CO 2 g (d. wt) 1 s 1 ) Node (2., 2.). (.2,.6).2 (.1,.) c Node 1. (.2,.6).2 (.1,.) c.1 (.,.3) c Respirtory potentil per core volume t 1 C (1 3 µmoles CO 2 cm 3 s 1 ) Node 1 1. (.9, 1.1).23 (.1,.31) c.13 (.,.22) d Node 1.28 (.2,.37).16 (.7,.2) cd.8 (.,.17) d Note: mss nd volume indices re in units of 1 3 µmoles CO 2, wheres molr indices re in µmoles CO 2. For ech mesurement, Lest Squres Mens (Strip-plot nlysis in PROC MIXED) ±9% confidence intervls for Corvllis (n = ) nd Riddle (n = 3) trees. For ech group of two rows, different letters indicte significnt differences mong mens for ll rdil positions (FPLSD, P <.). (Eklund, 199). Further, the sesonl hypoxi within tree stems hs een ttriuted to the formtion of hertwood extrctives in Acci mernsii (Crrodus, 1971) nd ltewood formtion in Pice ies (Eklund, 199). Oxygen supply to spwood is hypothesized to e function of oth rdil influx into the trunk through intercellulr gs spces nd trnsport of dissolved oxygen vi trnspirtion in the xylem (Gnsert et l., 21). Considerle soil wter deficiency during dry periods my reduce trnspirtion nd prevent the entry (O 2 ) nd exit (CO 2 ) of dissolved gses vi the trnspirtion strem. The resulting CO 2 uild-up is ugmented y high respirtion rtes, chrcteristic of the growing seson nd ssocited with cmil divisions nd photosynthte moiliztion nd storge (Chse, 193). Hypoxi within stems my crete negtive prtil pressures, fvoring the rdil influx of oxygen through rk lenticels to intercellulr spces of the cortex nd phloem. The neighoring cmium nd xylem re reltively impermele to gs ecuse they lck continuum of intercellulr spces (Hook et l., 1972), which explins the CO 2 increse nd O 2 decrese from outer rk to the spwood/hertwood oundry in the current study, Chse (193), nd McDougl & Working (1933). Evidence tht New Phytologist (22) 1:

12 37 Reserch O 2 is lso supplied to spwood from the soil nd then the trnspirtion strem ws demonstrted in Pice ies stems, where oxygen levels decresed with stem height during the growing seson (Eklund, 2). Further evidence of the trnspirtion strem s n O 2 source within stems is reported in Betul pedul, where sp flow ws found to e mjor determinnt for the diurnl flux of dissolved O 2 concentrtions (Gnsert et l., 21). When vil CO 2 concentrtions were incresed in the current study to mimic within-stem conditions in vivo, core respirtory potentil ws decresed compred with controls. This result is consistent with reports of direct, immedite nd reversile effect of reduced respirtion in isolted tissues (Amthor, 1991) or intct plnts (Griffin et l., 1996) under conditions of elevted CO 2. The effect of respirtory potentil inhiition ws not s evident from ptterns of O 2 uptke in tretments of low O 2 ecuse of O 2 diffusion from within core segments into the vil hedspce. This diffusion of dissolved O 2 from within cores into the vil hedspce ws likely the result of positive prtil pressure inside cores, creted y the low O 2 tretments. The living cells within core segments were thus le to ccess nd use gseous nd dissolved O 2 in respirtion nd produce CO 2, even when vil O 2 concentrtion ws low or ner zero. This ility of prenchym cells to respire under low gseous O 2 vilility suggests mechnism for how cells respire deep within stem spwood. Accumultion of intrcellulr CO 2 from eroic nd/or neroic respirtion (depending on the oxygen microenvironment of ech cell) my crete grdient for O 2 diffusion into cells from nery intercellulr ir spces, or the trnspirtion strem within conducting elements of the xylem. Other mechnisms, such s the lterntive pthwy (cynide-resistnt) or neroic respirtion my enle respirtion to continue despite high mient CO 2, simultneously mintining grdient for O 2 influx while providing energy for stem tissue nolism (Hook et l., 1972; Amthor, 1991). The trends of incresing respirtory potentil from pith to rk nd tree se to treetop tht were identified within Dougls-fir stems t the Riddle site were similr to those in ponderos pine (Pruyn et l., 22). Respirtory potentil (normlized to 2 C) ws similr in oth species, with the exception of inner rk, which ws 3 times higher in ponderos pine thn in Dougls-fir. Also, respirtory potentil from rest height of the Riddle trees ws nerly equl to vlues reported for O 2 uptke in stem tissue smples from Frxinus nigr nd Acer rurum (Goodwin & Goddrd, 19), ut twice tht of Pinus rdit (Shin & Mcky, 1973). Possile explntions for the ltter discrepncy were differences in smple dimensions (. cm rdil thickness vs our 2 cm long cores) nd mesurement techniques (volumetric respirometer vs our gs chromtogrph) etween studies. Further reserch is needed to scertin whether similr within-stem trends exist in other species, nd to incorporte diurnl or sesonl effects. Potentilly, the heightened respirtory ctivity of outer spwood ws relted to its role in supporting growth nd secondry wll formtion in the cmil zone (Goodwin & Goddrd, 19), nd/or other physiologicl ctivities ssocited with xylem mintennce (Lev-Ydun & Aloni, 199). Enhnced ctivity my lso e linked to role in crohydrte storge. Studies hve shown incresed mounts of solule crohydrtes nd strch in the outer spwood nd inner rk compred with the inner spwood nd hertwood of Pinus sylvestris (Srnpää & Höll, 1989). The reduced ctivity of middle nd inner spwood my e explined y ge-relted chnges nd/or dormncy of metolic ctivity in spwood prenchym cells. These theories re supported y findings tht ry cell nucler morphology chnged from outer to inner spwood in vrious conifer species, thus indicting decresed ry vigor (Frey-Wyssling & Bossrd, 199; Yng, 1993; Grtner et l., 2). Also, hertwood formtion or wound repir hs een ssocited with enzymtic or chemicl chnges in rys of middle or inner spwood, suggesting spwood prenchym cells my e geneticlly regulted to remin dormnt until rectivtion y signls from the cmil nd/or picl meristem (Shin & Mcky, 1973; Bmer, 1976). The notly higher respirtory potentil within the inner rk nd outer spwood t nodes 1 nd 3 compred with the sme positions t node 1 my lso e explined y their loction, which ws within the crown, where physiologicl ctivities, such s growth, sustrte supply, metolism nd trnsport re high. Our results showed rtes of CO 2 production tht were nerly equl for the vrious rdil positions when computed on nitrogen sis vs on core cron content, dry mss, or volume sis, which is likely ecuse tissue nitrogen content is n index of enzyme mounts in the tissue. Vlues for respirtory potentil on nitrogen sis for outer nd inner spwood in the current study were 1. 2 times higher thn oxygen consumption on nitrogen sis of outer nd inner spwood extrcts from two hrdwoods (Goodwin & Goddrd, 19). Although cron nd nitrogen my e more representtive of living cell mteril within stem tissue, it cnnot e n solute sis for mesurement ecuse some of the extrcted cron nd nitrogen ws likely otined from ded cells (Goodwin & Goddrd, 19). Thus, other indices in ddition to tissue nitrogen content should e explored for correltions to respirtory trends, such s totl nonstructurl crohydrtes (sugr, strch, nd lipids), or percent live prenchym cell volume. Cron dioxide production of extrcted wood cores ws generlly 3 1 times higher thn IRGA mesurements of Dougls-fir stem respirtion (L. A. Cernusk et l., unpulished) nd thus did not represent norml production within intct tree stems. The current results suggest tht wounding, microil respirtion nd lekge of stored CO 2 re not likely explntions for this discrepncy. We suggest three possile resons why extrcted cores respired t higher rtes: inner rk nd spwood Q 1 (for temperture rnges 1 C) my e New Phytologist (22) 1:

13 Reserch 371 higher thn hs een indicted previously y IRGA mesurements, which hs implictions for normlizing respirtion to 1 C; within stem concentrtions of high CO 2 nd low O 2 my inhiit respirtion in situ; nd some respired CO 2 my exit the stem vi the trnspirtion strem nd not rdil efflux, so tht not ll respired CO 2 is cptured y the IRGA systems. Grdients of core respirtory potentil within nd mong trees could e used s n index for scling to the whole-tree level, in conjunction with IRGA mesurements to provide reference for in situ respirtion rtes. However, the IRGA rtes will hve to ccount for respired CO 2 tht hs left the stem undetected vi the trnspirtion strem. Core sed estimtes for respirtion could lso e pplied to such reserch res s storge physiology, metolism, wood development, ecosystem nd individul tree modeling of cron pools, nd wood function in different life forms nd growth hits. Acknowledgements We thnk the following people for their expertise in the use nd ppliction of the vrious techniques used in this study: Dr Roert Griffiths nd Dr Willim Hicks regrding gs chromtogrphy; Cmille Freitg nd Connie Love regrding fungl/cteril identifiction; Dr Thimmpp Anekond regrding differentil scnning clorimetery; nd Dr Lis Gnio, Dr Mnuel Huso, nd the OSU Sttistics Deprtment regrding the repeted mesures nlyses. We thnk Jen- Christophe Domec for useful discussions regrding experimenttion nd physiology. Also, we thnk Roseurg Forest Products in Riddle, OR, nd the OSU Reserch Forests for providing the wood smples for this study. This reserch is funded y the USDA CSREES nd nd NSF DEB We lso pprecite the reviews on erlier versions of the mnuscript y two nonymous reviewers nd Dr Anekond. References Amthor JS Respirtion in future, higher-co 2 world. 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