Plnt (211) 234:145 154 OI 1.17/s425-11-146-6 ORIGINAL ARTICLE Shifts in xylem vessel dimeter nd emolisms in grfted pple trees of differing rootstock growth potentil in response to drought Tryn L. Buerle Michel Centinri Willim L. Buerle Received: 1 Mrch 211 / Accepted: 13 June 211 / Pulished online: 28 June 211 Ó Springer-Verlg 211 Astrct We investigted responses of plnt growth rte, hydrulic resistnce, nd xylem cvittion in scion-rootstock-comintions of Mlus domestic L. cv. Honeycrisp scions grfted onto high-shoot vigor (HSV) rootstock, (semi-dwrfing Mlling111), or onto low-shoot vigor (LSV) rootstock, (dwrfing Budgovsky 9), in response to sustrte moisture limittion. Adjustments in xylem vessel dimeter nd frequency were relted to hydrulic resistnce mesurements for high- versus low- vigor pple trees. We oserved greter tolernce to wter deficit in the high-shoot compred to the low-shoot vigor plnts under wter deficit s evidenced y incresed growth in severl plnt orgns, nd greter scion ntomicl response to limited wter vilility with c. 25% incresed vessel frequency nd c. 28% nrrower current seson xylem ring width. Wheres wter limittion resulted in greter grft union hydrulic resistnce of high-shoot vigor trees, the opposite ws true when wter ws not limiting. The grft union of the low-shoot vigor rootstock exhiited higher hydrulic resistnce under wellwtered conditions. Scions of high-shoot vigor rootstocks hd fewer emolisms t low plnt wter sttus compred to scions of low-shoot vigor rootstocks, presumly s result of lrge differences in xylem vessel dimeter. Our results Electronic supplementry mteril The online version of this rticle (doi:1.17/s425-11-146-6) contins supplementry mteril, which is ville to uthorized users. T. L. Buerle (&) M. Centinri eprtment of Horticulture, Cornell University, 134A Plnt Science Building, Ithc, NY 14853, USA e-mil: uerle@cornell.edu W. L. Buerle eprtment of Horticulture & Lndscpe Architecture, Colordo Stte University, 213 Sheprdson Building, 1173 Cmpus elivery, Fort Collins, CO 8523, USA demonstrted tht ntomicl differences were relted to shifts in hydrulic conductivity nd cvittion events, direct result of grfting, under limited soil wter. Keywords Hydrulic resistnce Wter stress Xylem ntomy Mlus domestic Rootstock Cvittion Introduction The wter reltions of grfted trees is well-studied topic tht hs produced numerous theories on the mechnisms tht result in low- versus high-vigor trees. To dte, studies on wter movement through grfted plnts hve focused on prtitioning the hydrulic pth s mens to etter understnd which plnt orgns ply the lrgest role in restricting wter flow (Bermn nd ejong 1997; Cohen nd Nor 22; Nrdini et l. 26; Cohen et l. 27). Empiricl evidence hs highlighted oth negligile (Nrdini et l. 26; Cohen et l. 27) nd decresed (Atkinson et l. 23; Bsile et l. 23; Gscó et l. 27; Gonçlves et l. 27; Trifilò et l. 27) chnges in hydrulic resistnce s result of grfting. ecresed plnt vigor through incresed hydrulic resistnce (restricted wter flow) cused y the grft union nd xylem conduit structure is widely ccepted (Gonçlves et l. 27; Tomesi et l. 21). Investigtion into vigor controlling root systems nd their effects on shoot physiology, prticulrly the mechnisms tht drive plnt growth potentil nd the role of wter trnsport, is ongoing. Nevertheless, the grft nd xylem conduit cpcity to control tree size is still unresolved. Most studies fil to exmine if root systems which promote whole plnt vigor respond differently to shifts in wter vilility (Buerle et l. 28). uring periods of wter limittion, trde-offs t the cellulr level cn impct
146 Plnt (211) 234:145 154 plnt function (Lovisolo nd Schuert 1998). For exmple, xylem ntomy nd hydrulic conductnce re tightly linked, with reduction in vessel dimeter common response to soil wter deficit (e.g. Lovisolo nd Schuert 1998; Beikircher nd Myr 29). Smll conduit dimeter djustments shift specific hydrulic conductnce (K s ) exponentilly s descried y the Hgen Poiseuille lw where theoreticl conductnce is the sum of conduit dimeters to the fourth power (e.g. Tyree nd Ewers 1991). Resulting ntomicl shifts towrd nrrower xylem cn restrict wter flow (Tyree nd Ewers 1991) ut simultneously reduce conduit vulnerility to cvittion (Gonçlves et l. 27). Ultimtely, xylem dysfunction s result of cvittion produces n interruption in the wter column nd n increse in hydrulic resistnce (Tyree nd Ewers 1991). Although cvittion nd xylem vulnerility hve een well studied in oth woody nd herceous plnts, little informtion is ville on ntomicl plsticity nd the ccompnying shift in hydrulic conductivity of plnts composed of scions grfted onto geneticlly different vigor controlling root systems (Trifilò et l. 27) under wter limited conditions. Furthermore, we know of no studies tht directly investigte the interction etween vigor controlling root systems nd plnt susceptiility to cvittion under decresed soil moisture conditions. The im of this study ws to exmine ntomicl plsticity in response to limited wter vilility nd to determine if there is link etween hydrulic responses to xylem vessel ntomy nd direct mesurements of xylem cvittion in oth rootstock nd scion portions of the plnt. We hypothesized tht low-vigor plnts re less susceptile to drought either through the production of numerous xylem vessels (Trifilò et l. 27), or through the reduction in vessel dimeter. Moreover, we predicted tht these chrcteristics would confer decresed cvittion vulnerility during period of soil wter limittion. Mterils nd methods Plnt mteril nd environmentl conditions One-yer-old pple cuttings (Mlus domestic L. cv. Honeycrisp) were whip nd tongue grfted onto two rootstocks of contrsting vigor. Semi-dwrfing Mlling 111 (M.111), usully produces tree 8% the height of non-grfted specimen, herefter referred to s high-shoot vigor rootstock (HSV), nd dwrfing Budgovsky 9 (B9), usully produces tree tht is 3% the height of nongrfted specimen, herefter referred to s low-shoot vigor rootstock (LSV). Control rootstocks of M.111 nd B9 (n = 1) were not grfted while tretment rootstocks (n = 1) were grfted. Trees were plnted in 1 L plstic pots contining sustrte mixture of 1:2:1 (perlite: pet:vermiculite) nd grown t Cornell University s Agriculture Experiment Sttion (42.42 N 76.51 W). All of the plnts were well-irrigted until 24 June 29 (y of yer 175) fter which hlf of the plnts were not wtered further. Both control plnts nd within ech scion rootstock comintion hlf of the plnts were rndomly ssigned to drought tretment, (n = 5) nd hlf to well-wtered tretment (n = 5) lid out in completely rndomized design. The letter x denotes un-grfted plnts. Sustrte moisture of ech pot, expressed s volumetric wter content (VWC), ws mesured i-weekly with ML2x proes (elt T evices, Cmridge, Englnd). Redings were tken y horizontlly inserting two proes in opposite sides of the continer through drilled lterl holes t depth of 2/3 of the sustrte height (14 cm). Holes were plugged etween mesurements. The two redings tken for ech continer were verged. Plnt wter potentil nd lef gs exchnge mesurements Four times during the dry down period, one lef per tree ws removed t predwn nd its wter potentil (W p ) ws determined using Scholnder type pressure chmer (Soil Moisture Inc., Snt Brr, CA, USA) concurrent with sustrte VWC mesurements. The reltionship etween the W p nd sustrte wter content redings ws used s surrogte for sustrte wter potentil (Buerle et l. 26). Lef net photosynthesis (A net ) nd stomtl conductnce (g s ) were mesured on recently expnded mture leves using portle gs-exchnge system (Li-64; Li-Cor Inc., Lincoln, NE, USA) equipped with light nd temperture controlled cuvette. Mesurements were tken weekly etween 1: nd 12: h on one lef per tree (n = 5). Cron dioxide (CO 2 ) concentrtion nd lef temperture within the cuvette were controlled t 4 lmol s -1 nd 25 C, respectively. Instntneous wter use efficiency (WUE) ws clculted s the rtio of A net to g s nd expressed s percentge of the control (wellwtered, un-grfted rootstocks). Plnt growth nd iomss Shoot growth rtes were mesured weekly for ll plnts strting 1 week efore tretment implementtion. All plnts were hrvested t the end of the experiment, 61 dys post wter withholding, nd dry iomss ws recorded. A tolernce to wter deficit index (TOL wd_index %) ws clculted for ech grfted rootstock (Fichot et l. 29). The
Plnt (211) 234:145 154 147 index estimtes dry weight iomss in response to wter deficit, modified from Monclus et l. (26) s Biom ðdryþ wter deficit = Biom ðdry x 1 ð1þ Þ well wtered where Biom (dry) wter deficit is the dry plnt mss of trees in the grfted drought tretment nd Biom (dry) well-wtered is the dry plnt mss of trees in the un-grfted control tretment. The index llows for direct comprison of iomss production y trees under different wter tretment regimens nd reflects the plnts sensitivity to drought. Antomicl mesurements Three plnts per tretment were rndomly selected for ntomicl sectioning. One centimeter long 1-yer-old stem segments were cut 6 cm ove the grft union (scion) nd 5 cm ove the sustrte surfce (rootstock) just efore hydrulic mesurements were conducted. The smples were immeditely fixed in chromic cid cetic cid formlin mixture (CRAF III) nd stored t 4 C until ntomicl nlyses. Smples were dehydrted in grded ethnol tertiry utnol (TBA) series, followed y infiltrtion y solute TBA s descried y Ruzin (1999). Therefter, the specimens were emedded in prffin mixture (Fisher Scientific, Fir Lwn, NJ, USA) nd crosssectioned to 15 lm using rotry microtome (Spencer 82 type, Americn Opticl Corportion, Bufflo, NY, USA). Prffin-emedded sections were stined in sfrnin [1% (w:v) in ethnol 5%] nd then in fst green [.1% (w:v) in 1:1 clove oil: solute ethnol] efore prffin removl. A series of imges were tken t 2x mgnifiction using light microscope (BX6; Olympus Imging Corp., Tokyo, Jpn) connected to the digitl cmer. Five res (.9 mm 2 ech) were rndomly selected nd imged to represent the current yer s xylem growth for ech stem cross section ( totl of 15 imges per tretment). For ech imge, vessel dimeter (V, lm), vessel frequency (VF, n mm -2 ), nd the vessel re (VA, lm 2 ) were determined using the imge-nlysis softwre Imge J (Ntionl Institutes of Helth, Bethesd, M, USA, http://rs.info.nih.gov/ji/). Vessel re vlues (lm 2 ) were converted to dimeter (lm) y ssuming the vessels were circulr. Vessel susceptiility to dmge during wter trnsport ws determined y clculting the vulnerility index (VI) s VI = V/VF, where V is the vessel dimeter (lm), nd VF is the vessel frequency (no./mm -2 ) (Crlquist 1977). Moreover, in order to ssess the effect of wter deficit on the vessel size, the dimeter of the lst produced line of vessels (n = 36 per tretment) of the current yer s xylem growth were mesured to more ccurtely represent xylem production during the wter deficit period. Hydrulic resistnce mesurements Trees were tken to n ir temperture controlled room (22 C) equl to the clirtion temperture of the high pressure flow meter (HPFM) pproximtely 3 h efore strting the mesurements. The scion ws excised 6 cm ove the grft union, shved free of cortex with rzor lde, nd ttched to the HPFM. The grft portion of the stem ws still ttched to the plnt nd kept humid within plstic g tht contined moist pper towels. Hydrulic resistnces (R, MPskg -1 ) of tree segments were directly mesured with HPFM (Tyree et l. 1993, 1995) (ynmx Inc., Houston, USA). For detiled explntion of the principles of the HPFM refer to Tyree et l. (1993, 1995). Briefly, R (inverse of conductnce) ws mesured y forcing pressurized wter (P) into portion/ segment of the plnt, while simultneously recording the rte of the wter flow (F). Hydrulic resistnce ws then clculted s the slope of the plot of P versus F: R ¼ dp=df ð2þ The scion ws flushed with wter until leves ecme wterlogged (R scion stem?leves ) (Boget-Trioulot et l. 22; Cohen et l. 27). Additionl R mesurements were mde fter ll the leves were removed to otin the hydrulic resistnce of the scion stem (R scion stem ). Susequently, stem segment including the grft nd rootstock stem ws excised from the rootstock five cm ove the soil surfce nd connected to the HPFM through its sl portion. Immeditely fter the hydrulic R mesurement (R rootstock stem?grft union ) ws completed, the grft ws removed while the rootstock segment ws still ttched to the HPFM nd the R of the rootstock stem (R rootstock stem ) ws determined. Lef hydrulic resistnce (R leves ) ws clculted s the difference etween R of the scion with nd without leves (R scion? leves - R scion stem ). Similrly, hydrulic R of the grft union (R grft union ) ws clculted s the difference etween the R of the rootstock stem with nd without the grft. All of these mesurements were conducted s qusi-stedy stte hydrulic mesurement with F nd P pproximtely constnt (Tyree et l. 1993, 1995). Hydrulic resistnce of the root system (R root system ) ws mesured y series of trnsient mesurements. The lower prt of the trunk with its intct root system ws shved nd connected to the HPFM. To void solute concentrtion in the root tips (reverse osmosis) the pressure pplied ws incresed t the rte of 3 6 kp s -1 while mesuring the flow every 2 s (Tyree et l. 1994, 1995). The whole plnt hydrulic resistnce (R plnt ) ws then clculted s: R plnt ¼ R scionþleves þ R rootstockþgrft union þ R root system
148 Plnt (211) 234:145 154 Xylem cvittion Stem segments from two replicte plnts per tretment were rndomly selected nd frozen in situ using specilized copper pliers (NGK Metls Corp., Sweetwter, TN, USA). Stems were clmped with the two pliers nd then smpled from the most distl section to llevite rtifcts of flse emolisms due to freezing xylem tension resulting in cvittion (Fcette et l. 21). Smples were immeditely trnsferred into storge dewer of LN 2 nd cryo-plned nd polished with glss knife under -6 C nitrogen vpor (Leic Ultrcut UCT Ultrmicrotome, Leic MR, Leic Microsystems, Vienn, Austri). Specimens were wrpped in indium foil to mintin contct etween the specimen nd the holder prior to mounting on specilly designed stu, mchined to house the pple stem nd indium foil in three mm deep depression. The stu nd smple were then ttched to cryostge in LN 2 nd immeditely trnsferred to the cooled stge (-167 C) of BAL-TEC cryo-preprtion chmer (BAL-TEC cryo unit SCE 2, Leic Microsystems Inc. Bnnockurn, IL, USA) nd inserted into Hitchi S-45 field emission scnning electron microscope (Hitchi High-Technologies Corp., Tokyo, Jpn). The smple ws sulimted t -81 C for pproximtely 1 min nd then returned to the cryo-preprtion chmer where the smple ws sputter coted with 3 nm of pltinum t -15 C. The smple ws then returned to the cooled SEM stge (-165 C) nd viewed t 7 kv. Five rndom fields of view were cptured t mgnifiction tht llowed for the lrgest viewing re while still enling detection of xylem emoli (33 lm). Cvitted vessels were esily distinguished from wter filled vessels in cryo-sem microgrphs. As result of low electron emissivity of liquid in the vessels the liquid ppered grey with numerous stritions, the result of xylem sp crystlliztion. Vessels without wter ppered lck. Vessels were counted s emolized if gs spce ws locted in the vessel (McCully et l. 1998). moisture dt were normlized y log trnsformtion. Percent cvittion differences were exmined y compring cvittion clss distriution ( 25), (25 5), (5 75), nd (75 1) percentges of rootstocks with scions of grfted nd un-grfted trees. Cvittion clss differences were nlyzed using PROC FREQ (SAS Institute Inc., Cry, NC, USA). Results Averge VWC for trees in the well-wtered tretment ws mintined t pproximtely 22%, wheres VWC of the drought tretment trees decresed 7% per week in the first 2 weeks nd therefter pproximtely 2.4% per week until stiliztion t 5% during week four of the experiment. The reltionship etween VWC nd W p reveled differences in rootstock response to decresing sustrte moisture with W p higher for the LSV rootstock compred with HSV rootstock t similr VWC s when sustrte moisture ws C22% VWC (P \.1) (Fig. 1). The opposite trend ws found for lower VWC of 14% or less, where the HSV rootstock hd higher W p, i.e. less negtive, compred to the LSV plnts t similr VWC (P =.25). Plnt growth nd wter use efficiency The LSV plnts grew t similr rte to tht of the HSV plnts under well-wtered conditions (LSV, 1.49 ±.45 cm dy -1 vs. HSV, 1.47 ±.46 cm dy -1 ), during the first 5 weeks of t nlysis A two-wy nlysis of vrince (ANOVA) ws used to test the effect of vigor nd wter sttus tretments nd their interction on shoot length, hydrulic resistnce nd xylem ntomy properties of un-grfted nd grfted trees. Tukey pirwise comprison test ws used to compre mens mong tretments (P \.5). The nlyses were performed using the MIXE procedure of SAS sttisticl pckge (SAS Institute Inc., Cry, NC, USA). The slope nd intercept of pirs of liner regressions were compred using Sttgrphics Plus version 5.1 (Sttisticl Grphics, Englewood Cliffs, NJ, USA). Wter potentil nd soil Fig. 1 Reltionship etween sustrte volumetric wter content (VWC) (%) nd pre-dwn plnt wter potentil (MP) for root system ssocited with high-shoot vigor (HSV) (closed circle) nd root system ssocited with low-shoot vigor (LSV) (open circle) grfted onto Honeycrisp scion. Lines indicte the non-liner logrithmic reltionship (HSV, continuous line, nd LSV, discontinuous line (P \.1)
Plnt (211) 234:145 154 149 the experiment with full experiment verge of LSV,.86 ±.8 cm dy -1 versus HSV, 1.1 ±.15 cm dy -1, dt not shown. Comprison of HSV-W, HSV- nd LSV-W nd LSV- to well-wtered un-grfted plnts showed greter reduction in shoot length in the LSV-W nd LSV- plnts (Fig. 2). When HSV- nd LSV- plnts were compred to the grfted well-wtered controls no significnt differences were found (Fig. 2). Tolernce to wter deficit index indicted lower tolernce to drought in ll orgns of the LSV plnts, (i.e. drought sensitive) when compred to the un-grfted control (Fig. 3). Eliminting the effects of grfting on TOL wd_index % reveled higher TOL index in the scion nd root portions of HSV- plnts (i.e. drought tolernt) (Fig. 3). These results rise from the smller overll reduction in shoot length of HSV trees (c. 35% reduction in HSV- vs. c. 6% reduction in LSV-) compred to the well-wtered, un-grfted control nd the lrge differences etween mss of un-grfted controls (e.g. LSVx-W 45 ± 2.3 g; HSVx-W 15 ± 4.4 g). Moreover, HSV grfted trees hd higher WUE compred to LSV trees when expressed s percent of the well-wtered grfted control (Fig. 4). TOL wd_index (%) TOL wd_index (%) 24 HSV - LSV - 2 16 12 8 4 8 6 4 2 Leves Scion Rootstock Roots Fig. 3 Tolernce to wter deficit index [TOL wd_index (%)] s clculted for the drought stress grfted low-shoot vigor rootstock (LSV-; grey rs) nd high-shoot vigor rootstock (HSV-, lck rs) (±1 SE). t re expressed s percentge (%) of well wtered un-grfted control () nd well-wtered grfted control (). Points of significnce re denoted y n sterisk (P \.5) Wood nd vessel ntomy Exmintion of the lst produced line of vessels from the current yer s xylem (time of highest wter deficit) reveled significnt interctions etween oth vigor nd wter sttus for scions (P =.27) nd rootstocks (P \.1). Scion xylem vessel dimeter (lm) nd current % of control 12 1 8 6 4 2 * * Instntneous WUE (% of control) 32 3 28 26 24 22 2 18 16 14 12 1 8 6 HSV- LSV- 177 183 191 196 198 23 212 217 224 231 y of the yer % of control 12 HSV HSV LSV LSV W W 1 8 6 4 2 HSV LSV Fig. 2 Finl shoot length under well-wtered nd drought conditions (men ± 1 SE). t represent finl shoot length s percent of the control, un-grfted well-wtered plnts, (%) for high-shoot vigor rootstock (HSV) nd low-shoot vigor rootstock (LSV), under wellwtered (HSV-W, LSV-W) nd drought (HSV-, LSV-) conditions () nd grfted well-wtered plnts () Fig. 4 Instntneous wter use efficiency (WUE) expressed s percentge of the control (well-wtered, grfted rootstocks) (%) for the grfted low-shoot vigor rootstock (LSV) (closed circle) nd highshoot vigor rootstock (HSV) (open circle) grfted onto identicl scions (Honeycrisp) under drought conditions over the course of the study (±1 SE) seson xylem ring width (mm) were generlly similr within wter sttus tretments (Tle 1). Under drought conditions, the scions of trees on the HSV rootstock hd 25% incresed xylem vessel frequency (P =.29), despite 28% nrrower xylem ring, ut not significnt (P =.67) (Fig. 5) compred to the LSV rootstock (Fig. 5d) (Tle 1). Vigor nd drought resulted in significntly decresed vulnerility index in the scions of
15 Plnt (211) 234:145 154 Tle 1 Averge xylem ring width (mm), vessel frequency (VF; n mm -2 ), nd vessel dimeter (V; lm) for the scion nd rootstock portions of the grfted trees (HSV; LSV) under well-wtered conditions (W) nd drought conditions () over the current yer xylem ring Scion portion Rootstock portion Xylem ring width (mm) VF (n mm -2 ) V (lm) Xylem ring width (mm) VF (n mm -2 ) V (lm) Vigor (V) HSV.93 444.1 24.5 (23.2).87 323. 23.8 (2.9) LSV 1.2 38.1 25.9 (22.8).72 336.8 23.4 (19.9) Wter sttus (WS) W 1.46 444.1 25.7 (27.6 ) 1.26 37.3 24.1 (22.8 ).67 38.1 24.7 (18. ).32 352.5 23.1 (17.9 ) V 9 WS HSV-W 1.3 394.6 25.2 (28.4 ) 1.37 313.4 23.9 (24.3 ) HSV-.56 493.5 23.9 (18. ).37 332.6 23.8 (17.3 c) LSV-W 1.62 365.5 26.3 (26.7 ) 1.16 31.2 24.4 (21.2 ) LSV-.78 394.6 25.5 (19. ).27 372.4 22.4 (18.5 c) Two-wy ANOVA (P vlues) V.61.11.155 (.522).94.653.68 (.63) WS \.1.11.273 (\.1) \.1.165.324 (\.1) V 9 WS.712.111.756 (.27).477.44.389 (\.1) The vlues in prenthesis represent the dimeter of the outermost line of vessels. Within column different letters indicte significnt differences etween mens t P =.5 level (Tukey s test), no letters present indicte no significnt difference grfted trees (vigor, P =.12; wter tretment, P =.16) (HSV, 5.65; LSV, 6.83) nd (well-wtered, 6.8; wter stress 5.68). rought lso cused significnt reduction in xylem ring width for oth root systems compred to well-wtered plnts (P \.1) (Tle 1). Rootstock clculted vulnerility index ws not significnt for either vigor (P =.693) or wter sttus (P =.269). Hydrulic resistnce High vigor incresed hydrulic resistnce in un-grfted trees (P =.25) with greter resistnce under drought (P =.2) (Fig. 6). Compred to the grft union nd scion resistnces, rootstock stem nd root system hydrulic resistnce ws negligile regrdless of wter sttus (wter tretment, P =.82, P =.73; interction, P =.217, P =.32), (Online resource 1). Among grfted trees, there ws significnt wter sttus effect on HSV- trees (P =.5) nd significnt interction etween tree wter sttus nd rootstock (P =.1) on grft union resistnce (Fig. 6). Our study found the grft union of well-wtered LSV-W trees imposed 2% higher hydrulic resistnce to the tree when compred to the un-grfted control (1,245.6 vs. 8,524.9 MP s kg -1 ), wheres grfting HSV-W trees resulted in n 86% reduction in hydrulic resistnce (3,687. vs. 26,996.4 MP s kg -1 ). Under wter limited conditions, the grft union of the HSV- rootstock imposed 63% greter hydrulic resistnce compred to the LSV- grfted trees (26,. vs. 11,258.7 MP s kg -1,(P =.5) (Fig. 6). Cvittion Well-wtered grfted rootstocks hd similr percentge of emolisms present t the time of smpling oth ove (P =.112) nd elow (P =.331) the grft union. Compred to the un-grfted rootstock controls, under wellwtered conditions, grfted trees hd fourfold increse in emolisms in the HSV rootstock (control 4%; HSV 16%) (P =.2) while the scion remined unchnged (P =.13). Conversely, the numer of emolisms in the LSV rootstock ws reduced y hlf when grfted (control 34%; LSV 15%) (P =.1) nd y pproximtely 95% (P \.1) in the scion. While oth un-grfted rootstocks hd similr levels of emolisms under drought (HSV, 5%; LSV, 53%) (P =.785), emolism trends in grfted trees were distinct from those of well-wtered trees with the LSV rootstock developing more emolisms in oth the rootstock (43%) (P \.1) nd scion (52%) (P =.1) compred to the HSV (rootstock, 27%; scion, 29%) (Fig. 7). iscussion Severl studies hve exmined the correltion etween grfting nd vegettive vigor of trees from hydrulic
Plnt (211) 234:145 154 151 Fig. 5 Light microgrphs of pple stem cross sections, scle r.5 mm nd 1 lm, inset. Representtion of specimens under wellwtered conditions (), scion portion of HSV under wter limittion (), rootstock portion of HSV under wter (c), scion portion of LSV under wter limittion (d), nd rootstock portion of LSV under wter limittion (e). Inset imges re xylem vessels under incresed mgnifiction (29) Hydrulic resistnce (MP s kg -1 ) 5x1 4 4x1 4 3x1 4 2x1 4 1x1 4 5x1 4 4x1 4 3x1 4 2x1 4 1x1 4 V =.25 WS =.2 V x WS =.34 HSVx HSVx LSVx LSVx W W V =.274 WS =.5 V x WS =.1 HSV HSV LSV LSV W W Fig. 6 Hydrulic resistnce (MP s kg -1 ) of the scion of un-grfted trees denoted y lowercse x (), nd the grft union portion of the stem () (±1 SE) for high-shoot vigor rootstock (HSV) nd lowshoot vigor rootstock (LSV), under well-wtered (HSV-W, LSV-W) nd drought (HSV-, LSV-) conditions. Lower cse letters indicte men differences (P B.5) perspective (Nrdini et l. 26; Cohen et l. 27; Gscó et l. 27); however, we re wre of only one study tht hs evluted how such reltionships shift in response to sustrte moisture stress (Solri et l. 26). Using system tht llowed for direct mnipultion of geneticlly identicl scions grfted onto two geneticlly distinct root systems we were le to exmine the reltionship etween root system growth ttriutes nd ntomicl shifts tht re relted to hydrulic responses under different levels of wter vilility. Not surprisingly, grfted HSV plnts produced longer shoots thn LSV plnts when well-wtered nd considerly shorter shoots compred to the un-grfted control when sujected to drought. However, the reltive proportion of stem? shoot iomss differences etween tretments ws greter for the LSV rootstock. Elimintion of the grfting effect resulted in similr responses of the HSV nd LSV trees to drought. Using un-grfted trees s the control, the HSV rootstock tolernce to wter deficit reflects the relloction of iomss to the rootstock nd n incresed ility to thrive under drought. These results re prtilly due to the lrge differences in mss etween LSV nd HSV trees. Similr trends were found for the scion nd root portion of trees when compred to the un-grfted wellwtered controls. Clerly, one wy in which root systems my tolerte chnges in environmentl conditions such s exposure to sustrte moisture deficit is y diverting
152 Plnt (211) 234:145 154 Fig. 7 Representtive cryo- SEM xylem cvittion frequency imges of pple scion cross sections on two rootstocks of () high-shoot vigor (HSV) rootstock nd () low-shoot vigor (LSV) rootstock under drought conditions. Scle r 15 lm ssimiltes to increse the soring root system surfce re, llowing for greter wter forging ility (Buerle et l. 28). Moreover, the higher vlues of W p nd WUE of grfted HSV plnts compred to the LSV oserved under drought conditions confirm the HSV hs greter ility to tolerte wter deficit. Previous studies hve indicted the importnce of lrger xylem vessels for greter hydrulic conductnce nd incresed plnt growth under well-wtered conditions (e.g. Rodriguez-Gmir et l. 21). According to the rootstock medited growth regultion hypothesis, the ct of grfting cn lter xylem vessel dimeters (Trifilò et l. 27). However, proposed trde-off occurs s smll increses in cross sectionl xylem dimeter result in proportionlly lrger decreses in conduit resistnce to cvittion, jeoprdizing wter trnsport sfety (Tyree nd Zimmermn 22). Previous reserch hs demonstrted reduction in xylem vessel dimeter s the result of grfting onto lowvigor rootstocks under well-wtered conditions (Gonçlves et l. 27; Tomesi et l. 21) suggesting more restriction in wter trnsport (Tyree nd Ewers 1991). In response to soil moisture stress, however, we oserved n interction etween plnt growth rte nd xylem dimeter response to soil moisture limittion; reltive to the LSV, the HSV incresed hydrulic resistnce y xylem vessel dimeter reduction. We found greter xylem vessel dimeter djustment in the HSV scion compred to the rootstock in response to drought. This is likely the result of the rootstock hving inherently smller xylem vessel dimeters under wellwtered conditions (Tle 1). Therefore, oth the scion nd rootstock in the HSV comintion led to reduced vessel dimeter compred to the well-wtered trees, suggesting incresed hydrulic sfety (Hcke et l. 26). If the scion operted independently then we would hve expected the scion of two root systems with dissimilr growth rtes to show similr xylem vessel dimeter in response to grfting nd drought. Insted, our results suggest greter plsticity in HSV conferring rootstocks tht results in smller ove-ground portion of the plnt, lthough still lrger thn tht produced y dwrfing rootstocks. Competitively, trees tht re le to djust nd optimize their hydrulic rchitecture hve greter resistnce to cvittion (Tyree nd Zimmermn 22), n importnt ttriute in response to soil wter vriility (Fonti et l. 21). Our study found incresed resistnce in the grft union of the low-vigor rootstock under well-wtered conditions, which supports the hypothesis tht prtitioned hydrulic resistnce designtes the grft union s one cuse of dded resistnce to wter flow (Gscó et l. 27; Atkinson et l. 23) (lthough see Bsile et l. 23; Nrdini et l. 26), with the grft union of the LSV rootstock incresing resistnce y n dditionl 35% over tht of the HSV rootstock (Fig. 6). When drought event ws implemented the HSV rootstock displyed higher hydrulic resistnce thn the LSV, n unexpected result. Investigtion into mechnisms driving differences in hydrulic resistnce etween the two rootstock scion comintions under oth well-wtered nd drought conditions reveled greter trde-off in sfety versus trnsport efficiency in the HSV grft union. In the scion lrger reduction in xylem vessel dimeter s evidenced y the difference in wellwtered versus droughted xylem vessel dimeter in the youngest ring of xylem signified the hydrulic ottleneck in vigorous growers, while higher numer of cvitted xylem vessels rendered xylem vessels t lest temporrily dysfunctionl in low shoot vigor plnts. However, despite the higher percentge of emolized xylem vessels, hydrulic resistnce did not sustntilly shift in the low vigor plnts. When our results re ssessed together with tht of Hcke et l. (21), the findings re consistent with the hypothesis tht ntomicl shifts cn lter hydrulic
Plnt (211) 234:145 154 153 conductivity in order to circumvent cvittion events under limited soil moisture conditions. Cvittion events re common in nture nd cn occur nd refill on dily sis (Ewers et l. 1997; McCully et l. 1998; Holrook nd Zwieniecki 1999). There is still question surrounding wht percentge of stem xylem cn form emolisms without complete hydrulic system filure (McCully et l. 1998; Brodri nd Cochrd 29). Whether or not vessels fully or prtilly refill nd wht ffect tht my hve on xylem ftigue re eyond the scope of this pper. However, it is importnt to consider the reltive differences in emolism formtion etween plnts of vrying growth potentil ecuse prtilly impeded hydrulic flow in xylem due to cvittion cn render the plnt highly susceptile to filure if refilling does not occur. Brodri et l. (21) found the rte of sl re growth in conifers tightly coupled to the rte of xylem emolism repir, indicting tht growth rte is n integrl component of plnt growth nd survivl under drought. Similrly, we found emolism formtion nd conductivity constrints imposed y the reduction in vessel size in scions grfted onto high shoot vigor rootstocks to illustrte the role of the grft union under limited soil wter. Our results hve implictions for how we del with grfted trees nd environmentl vriility during the growing seson. We found differentil drought responses etween plnts of differing shoot vigor s result of root system growth potentil tht my identify link etween plstic xylem vessel ntomicl responses nd hydrulic xylem cvittion control on potentil growth rte in oth rootstock nd scion portions of the plnt under limited soil wter. More specificlly, our results rejected our hypothesis of reduction in drought susceptiility in low-vigor grfted plnts nd insted show tht scions of high-vigor grfted plnts reduce xylem vessel dimeter while producing more xylem vessels, thus resulting in decresed numers of emoli during drought. Acknowledgments We thnk, S. 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