Plasma Membrane H&#X002b;&#X2010;Atpase Activity and Graft Success of Breadfruit

Accepted Article This by is protectedarticle reserved. Allrights copyright. doi: 10.1111/plb.12879 lead to differences between this version and the throughbeen the copyediting, pagination typesetting, which process, may and proofreading hasThis article been accepted publicationfor andundergone peerfull but review not has 2 1 handling Editor: Prof.G Thiel Paper :Research type Article DR. YUCHAN ZHOU ID:0000-0001-8218-7603)(Orcid Yuchan Zhou Yuchan interspecific rootstocks marang of (

Plasma membrane H membrane Plasma Abstract Abstract * Australia Australia Faculty 4556, ofScience, of Education Sippy University Coast, and QLD Engineering, the Sunshine Downs, QueenslandLucia,Innovation,4072, andFood Alliance St of Queensland, Agriculture University for QLD Correspondence: • •

relationship of plasma membrane (PM) H (PM) membrane ofrelationship plasma odoratissimus Here we first reported the success of breadfruit Artocarpus graftingofbreadfruitun-relatedonto approach However, dwarfing rootstocks. identifying to predominantlyvegetative of propagation the species, graftingontointerspecific seedlings is an Significanthas driven damage anwind Due developing to interest dwarfing its rootstocks. in the ( Breadfruit 1, 2* and Steven J. R. Underhill

Yuchan Zhou species has not been investigated. Artocarpus altilis ) and pedalai ( pedalai ) and + -ATPase activity and success graft of breadfruit (

, e-mail: [email protected] A. odoratissimus A. sericicarpus A. ) isprimarily grown as staple a tree crop for security food the in Oceania. 1, 2 1,

+ -ATPase activity tograft success. ). To ). address the graftsurvival,low we investigated the ) and pedalai ( Version of Record. Please Version as this cite of Record. article grafting onto interspecific rootstocks, marang( ontointerspecificrootstocks, grafting

A. sericicarpus ) Artocarpus altilis ) onto ) onto A. Accepted Article Keywords Breadfruit ( Breadfruit Keywords This by is protectedarticle reserved. Allrights copyright. and the ofgraftedperformance breadfruit cultivars. In addition, vegetativelyrootstocks propagated maybe not There 2008). limited properties rootstock method Roberts-Nkrumah, is (Solomon informationregarding and suckers, root cuttings,orlayering. Breadfruit air grafting has practised, been as amainbut not propagation recalcitrant seeds (Ragone, 1997; Ragone, 2006). Clonal pr seedlessthevarietiesvarietiespropagationpreferred for breadfruit their seeded and requireddueof the is to for other fruit tree dwarfing species; however, rootstock has by achieved been Zhou Dwarfism 2014). 2012; etal., br dwarf indeveloping anincreasing has interest driven tree from m,15-30 breadfruit is prone to damage.wind Significant tree dueloss to intense tropical windstorm The speciesprimarily isgrown staplea ascrop food for securitythe Oceania. inHowever, being evergreen an of cultivars, diversitygreatmorphologicalwith in characteristics andagronomic Ragone, (Ragone, 1997; 2006). species comprises fertile and diploids sterile (2n=2x = 56) andsterile triploids (2n =3x 84), and = hundreds has [ Breadfruit pedalai ( pedalai 1.

• • Introduction Introduction

A. sericicarpus scion’s own roots decreased the leaf PM H PM leaf the decreased own roots scion’s after grafting =0.9203,after r(7) (marang, The function of PM H PM of function The graftoutcome. The recoveryof the enzyme activity was onlyin thesuccessful found grafts. We provided theevidences first for apositive correlationbetween PM H discussed. discussed. survival. The graft graftsof unions successful had higher PM H failed grafts. Rootstocks with low PM H PM low with Rootstocks grafts. failed success andthe PM H 30%for marang pedalai and rootstocks respectively.There was apositive correlationbetweengraft lowergraft survival than with those high enzyme ac Artocarpus altilis Artocarpus altilis Artocarpus altilis ); rootstock; scion scion rootstock; ); (Parkinson) Fosberg)] is a traditional fruit trees throughout the tropics. The tropics. the traditionalaFosberg)](Parkinson)throughout is trees fruit + -ATPase activities measured from the rootstockstem microsomes twomonths + -ATPase in graft union development and graftsuccess improvement is ); grafting; plasmas membrane H membrane plasmas grafting; ); P = 0.0004; pedalai (r(7) = 0. 8820, =0. pedalai (r(7) = 0.0004; + + -ATPase -ATPase activity in leaf microsomes before grafting had -ATPase activity of grafted-ATPaseplants activityof regardless of the final eadfruit varietieseadfruit (Daley et al., 2012; Roberts-Nkrumah, for for breadfruit has not been investigated. Vegetative the widespread the use of widespread in many rootstocks dwarfing tivity, withgraft successofvs 10% and60% 0%vs opagation of breadfruit isgenerally through root + -ATPase -ATPase activity compared to thoseof + -ATPase; marang ( + -ATPase activitygraft and P = 0.0017). Removal of Removal =0.0017). A. odoratissimus ); Accepted Article This by is protectedarticle reserved. Allrights copyright. and scions for prediction and improvement of graft survival for breadfruit species. rootstocks of screening fast for strategies develop to opportunity provide may knowledge The survival. graft ourknowledge, there is little informationon apossible between correlation of level H PM phloem parenchyma, stomatalguard cellsand (Pavlovkin al.,et Paiva 2002; etal., 2004; Arend et To 2008). al., Palmgren, 2001). Increase inPM H bundle differentiation,phloem loading and stress (Fromm adaptation et1989; al.,Michelet andBoutry, 1995; Thisprocessphysiologicalis essential for responses,including cambialvascular celldivision expansion, and cells. growing of membrane plasma the across ions and sugars as such nutrients of uptake the drives that force centralATPase playsa role innutrient The transport 2001). (Palmgren, generates the enzyme proton-motive 2015). However, candidates involved inthese events ar differentiation as thegraft main during events union formation et (Yin al., 2012;Melnyk andMeyerowitz, Accumulating have evidences identified re wound the capacity conditionand favourable for divisioncambium differentiationcell (Melnyk,and 2017). includegraftingtechniques,andenvironmental These scion/rootstockbeen have factors. linked age factors to Graft success oftropical fruit trees may be improved when a breadfruit grafting protocol with high graft succ overby 60% grafting. approach Clearly, to achieve on seedy granting (2005) found Kuniyuki and hand,theother Nandwani significant on gr effect had tongue)nor age rootstock graft,grafting.They neither whip and afterrootstock6 grafting (side wedge, top techniques weeks found onsurvival of scion 18~28% reported these (2008) an andRoberts-Nkrumah rootstocks. Solomon mariannensis domesticated from Species 2008). Roberts-Nkrumah, Solomon 2007; and Medagoda Chandrarathna, 2005;and (NandwaniKuniyuki, and Breadfruit interspecific grafting has been reported only on mariannensis The genus. Nkrumah,2012). Dwarfingbreadfruit rootstocksof maycome otherseededfrom species within the ideal resistancedue wind for to their shallow root sy A. camansi Artocarpus (Zerega et al., 2004). Even though, poor survival has been reported on breadfruit graftingonto breadfruit on been reported has poor survival Even et al., (Zerega 2004). though, (dugdug), A. camansi, A. and is a genus ofgenus approximately shrubs,including isa and trees 60 A. mariannensis A. A. odoratissimus A. and theoccurrenceof introgressive hybridizationbetween + -ATPase has been predominantly infound active cambial zone, xylem and areclosely breadfruit related to ( (marang) and (marang) and ess for interspecific rootstocks is a prerequisite. large-scale graftingscreening dwarfing and for properties, aft success (Solomon and Roberts-Nkrumah, 2008). 2008). aft (Solomon andRoberts-Nkrumah, On success stem (Solomon and Roberts-Nkrumah, 2008; Roberts- 2008; Roberts-Nkrumah, and (Solomon stem sponse, hormone signalling, callus formation and callus hormonesignalling, sponse, formation and e less e less membrane In plants,clear. plasma (PM) H a number of factors are considered. These generally A. sericicarpus A. A. camansi A. mariannensis A. (pedalai) (Zerega et al., 2010). (pedalai)2010). et al., (Zerega and and A. altilis A. A. mariannensis A. A. camansi ) with breadfruit being with breadfruitbeing ) could be improved to be could improved + -ATPase activity and A. altilis (breadnut), (breadnut), rootstocks rootstocks A. camansiA. Artocarpus and A. A. A. + -

Accepted Article SamplesH collected for PM This by is protectedarticle reserved. Allrights copyright. ( Breadfruit 2.1. Plant materials and treatments methods and Materials 2. plants. wepositive providedevidences relationshipa for betweenPMH graftunion,microsomalscion rootstock and fractions and their relationship graftFor success. tofirst time, the graf low the causing factors identify to order In pedalai. and marang rootstocks, interspecific onto grafting breadfruit of success the reported we study, this In union beforegrafting, stems immediately rootstock above graft the unionmonths two after grafting,scion apical 2.2 Preparation of microsomal fraction and measurement of plasma membrane H membrane plasma of measurement and fraction microsomal of Preparation 2.2 determined as four survival the rate grafting.after thebeginningmonths of completed withbeing the scionsolely dependent on the rootsystem of theThe rootstock.graft success was removal of the portionbottom (root of system) belowthe scion graftthe union. The graft union was then immediat rootstock of the portion united, top the well aligned. The of progress the graft was checked through unt two peeled surfaces were then bound tightly together wi removed, andslice a 3to5cm ofand 40% thicknesslong stem the from indepth was each stem.Theremoved to nextrootstockother, at each brought and scion were th sustained by own whiletheir roots thegraft heals incr to graft withmodification for st similar their on based scion the with randomly paired youngfrom plants 30 50 ~ cm tall and rootstocks were soil mixture previously as described and Underhill, (Zhou For2016). grafting scionsexperiment, were selected 25 daily ~28°Csupply. with natural daylight and water plants marang and,pedalai andfrom cuttings root plants ( Artocarpus sericicarpus Artocarpus Artocarpus altilis ) obtained) were from a commercial nurseryat

Artocarpus + -ATPase activityassay leaves rootstock included thegraft removed from intended , cultivar Cannonball), marang, cultivar ( Cannonball), species. In this technique, both the eventualscion and rootstock are self- t success, we examined the levelsofPM H em thickness.em Grafting wasperformed through approach ely above ely graftthe union cutwas off followed by the as seedlings, were grown under glasshouse grown under seedlings, at were condition as e point where the graft union towas occur, leaves were ease graft success. Briefly, to start the grafting, both grownPlants were in potscontaining vermiculite and seedlings of 2 to 3 months old. of2 months old. seedlings to 3 was Each rootstock th grafting tapes so that their cambium layersgraftingcambium tapes weretheirso that th ying the tapes mont two after + -ATPase activity and the survival of grafted Cairns, northern Queensland. Breadfruit Artocarpus

odoratissimus + -ATPase activity. activity. -ATPase hs. Afterthewere parts + -ATPase activity in ) and pedalai pedalai and ) Accepted Article centrifuged at 13,000 g for 15 min. The supernatant was then centrifuged for 30 min at 100,000 g to obtain obtain g atmin then to 100,000 for 30 The centrifuged supernatantg was 15 min. for 13,000 centrifuged at of cheesecloth and layers several through filtered homogenate The was (PVP)]. polyvinylpyrrolidone 0.5 M sucrose, 10 mm ascorbic acid, 1 mM phenylmet ethyleneglycol diaminebismM ethylenetetraacetic tetraacetic (2-aminoethyl) 20 acid (EDTA),(EGTA),acid mM 20 dithiothreitolmM (DTT), 2 with 7.7 pH Tris-Mes M [0.1 w/v) (1:3 mediumice-cold homogenised in mM DTT, 3mM for KCl] immediate H 2 M sucrose, 0.33 mM pH7.8, KPi, [5 medium insuspension resuspended were pellets microsomal Thepellet. This by is protectedarticle reserved. Allrights copyright. leaves afterof completion and grafting prepared at 4 prepared 4 at system of rootstock (Fig 1C). At 3 to 5 months after grafting, success was confirmed by appearance of shoot shoot of appearance by confirmed was success grafting, after 5months to 3 At 1C). (Fig of rootstock system onthe root dependentplant solelygraftedleaving the roots, scion of the completeafter removal over 4 weeks grafts were adhered well for both rootstocks. A successful Allgrafts were un-tied tocheckthe adhesion before remova 3.1. Success breadfruit of grafting onto interspecific rootstocks marang of and pedalai Results 3. determined bylinear regression analysisunder SPSS software (IBM SPSS Statistics version 24). comparison test at P<0.05. Significant differences were tested using ofanalysis variance (ANOVA) byfollowed Tukey’s multiple analyses Statistical 2.3. accordingto Bradford (Bradford, 1976). ATP hydrolytic activity was measured at 38 38 at measured was activity hydrolytic ATP Na sensitivecalculateddifference ATPase were formbetween the absence activitiesmM in the ofpresence 1 and phosphate released from wasATP determineddescribed as 1990). The (Kasamo, activitiesof vanadate- inorganic of amount The X-100]. Triton [w/w] 0.1% KNO3, mM 50 molybdate, ammonium mM 0.5 MgSO4, microsomalproteins) to areaction mix [50Tris mM al., 2014) with modification. The reaction was initiatedby of additionof microsomal suspension µg (~15 3 VO 4 . The activity Pi/min/mwas presented as µmol o C as previously described (Zhou et al., 2014). et 2014). (Zhou C Br al., described as previously

The relationship betweengraft success and stem PM H

+ graft union sections fromboth failedThe fraction an.graft unionsections microsomal were -ATPase activity assay. o C for 30 min following a previously described procedure (Zhou et et (Zhou procedure described previously following Ca 30 min for -MES, pH 6.5, 3 mM Tris-ATP, 50 pH6.5, mM 3 mMKCl, 3 Tris-ATP, -MES, mM hylsulfonyl fluoride (PMSF) and 2% (w/v) insoluble 2% insoluble and (w/v) (PMSF) hylsulfonyl fluoride grafting event was initially identified by survival for l of l thescion’s self-roots. At thisstage, 100% of the g Protein. Protein concentration was determined iefly, tissues (leaves or stems at ~50 g) were were g) at~50 or stems (leaves tissues iefly, + -ATPase activitywas

Accepted Article activities respectively. Eachspecies group had at least 7 rootstocks (n H orderInhighto examinewhetheractivity H+-ATPase PM four months after of beginning the grafting (two months activity, with Pi/min/mgProtein. Rootstocks of each species were grouped according to their levels of PM H PM of levels their to according grouped were species each of Rootstocks Pi/min/mgProtein. Pi/min/mgProtein, whereas thoseofpedalaito 0.40 were µmol in the rangeof 0.070.37 µmol to ATPase activityassay. These showed the PMactivities H+-ATPase ofmarang were leavesin the0.05 range of preparation microsomal and for sampled were 1A) section) (Fig. (potentialgraftsection bound the intended 3.3. PM H PM 3.3. This by is protectedarticle reserved. Allrights copyright. and marang higherfor 89% 104% and with grafts for both rootstocks, ofunsuccessful significantly than those higher healthy (successful) grafts. These showed PM H+-ATPase activitiesgraft from unionsof successful grafts were microsomal sampled were ofgraft for grafts theseunion The regulationnutrient2001). andturgor uptake (Palmgren, for responsible pump a primaryATPase,proton H+- PM of activity the examine to us prompted symptoms stress These roots. own scion’s their of removal (Melnyk, 2017). Themajority of thegrafts failed showed leaf wilting and drooping within 2weeks after passivenot woundingnecessarily response, associated withvascular functional graft and connection success 1C), graftsuggest though These even survival finalwas low. healing the the and adhesion may from come a unions. All graftsthe adhered similarwith between strengthheld grafting partners in the graftjunction (fig. Toinvestigate the causeof low graft survival, we exam unions graft from activity H+-ATPase PM of Comparison 3.2. attempted). 32 of out (3 pedalai on breadfruit grafted for 9 % was of rate asurvival and attempted), 28 of of25 asurvivalrate removal), scionroot months after a characteristicsof active growth pa growth from the terminalbud ofgrafted the scions along with emergence of new utmost (Fig.leaves 1E, Fig. 2), + - ATPase activity in rootstocks before grafting. At the beginning of grafting, rootstock leaves peeled from from peeled leaves rootstock grafting, of beginning the At grafting. before rootstocks in activity ATPase -

pedalai rootstocks respectively pedalai 3). rootstocks (Fig. + -ATPase activity leaves in rootstock -ATPase activity ≤ 0.14, 0.15 to 0.25 and 0.15 to 0.14, ttern in breadfruit (Zhou and Underhill, 2016). At the end of 4 months (2 ofthemonths (2 At end 4 Underhill, 2016). and ttern (Zhou breadfruit in ≥ 0.26 µmol Pi/min/mgProtein representing the low, medium and high high and medium low, the representing Pi/min/mgProtein µmol 0.26 before grafting and graft success success graft grafting before and ined theined physical biochemical and properties ofthe graft % wasobserved forgrafted breadfruiton marang (7 out after theafter completionof grafting).These results showed ATPase activity assay incomparison with thoseof the is required for graft surv for is required ≥ 7). 7). Thegraft survival wasdetermined ival, weexaminedival, the PM + -ATPase Accepted Article Toof PM H examine further the relationship continued to drop, with an 84% decrease at day 6 compared decrease6 dayat 84% withan continued drop, to outcome, with reduction overday 35% compared to 0.In the unsuccessful grafts, the activities enzyme activities significantly after decreased of removal the ro H PM of pattern those unsuccessful grafts months four grafting. after Th withgrafting. survivedscionscomparedTheof were activities the enzyme after months scion’sownroots two 3.5. Changes of PM-H of 3.5. Changes 0.9203; (r(7)= activities of microsomesprepared from the stems rootstock at this stage and thegraftsuccess for marang both 3.4. PM H PM 3.4. significantly differentboth rootstock for species 4). (Fig. not was activities high and medium the between groups those However, 4). (Fig. respectively rootstocks pedalai and marang vs for 36% 0% 63%and vs with 10% groups, activity high in those to survival compared rate This by is protectedarticle reserved. Allrights copyright. PM the H groups in low pedalaimarangforboth rootstocks, and regressionanalysis5) confirmed(Fig.a significant, there was positive relationshipH the between PM increased as stemthe rootstock activities ATP frommediumhigh increased low, to levelsLinear (Fig. 5). (n 9 rootstocks ac mediumandhigh Pi/min/mgProtein beinglow, 11 µmol species were alsogrouped according to their levels ofstemATPase activity, with same of the Rootstocks Pi/min/mgProtein. µmol 0.17 to 0.028 of range the in were pedalai of those whereas PM H After completionof grafting, the scion apicalgrafted leaves onmarang rootstockswere assayed for microsomal stem PM H PM stem the months of two end theat showed,These 1C). (Fig. stems of union andscion graft the destructive analysis abovewere thegraftmicrosomal sampledunion preparation for and ATPase activityorderanalysis in to avoid + -ATPase activity every two days for 6 days, with day 0 as the time immediately before removal of the the of removal before immediately time the as 0 day with days, 6 for days two every activity -ATPase + + -ATPase -ATPase Pi/min/mgProtein, activities of marang were rootstocks thein of0.031 range 0.19to µmol P = 0.00043) and pedalai rootstocks (r(7) and (r(7) pedalai=0.8820, = 0.00043) rootstocks ≥ + -ATPase activity between the twochange 9).These showed thegraftsuccessof ontobreadfruitmarang pedalai or significantly

activity in rootstock stems and graft success success graft and stems inrootstock activity + -ATPase

activity in scion leaves after completion of grafting. completion inscion of activity leaves after + -ATPase activity to graft success graft to activity -ATPase ese resultsese showedwere distinct theredifference the in ot system of the scions regardless of the final graft graft final the of regardless scions the of system ot tivities respectively. Eachspeciesgrouphad least at to those at day 0. In contrast, in the survived survived inthe In contrast, 0. day at those to groups (Fig. 6). At day 2, the PM H the PM 2, Atday 6). (Fig. groups P = 0.0017). = 0.0017). + -ATPase activitiessignificantlyhad lower , ≤ rootstock stems immediately immediately stems rootstock 0.05, 0.06 0.10to and + + -ATPase -ATPase ≥ 0. 0. Accepted Article H similar strength in graft junction 1),(Fig. the graft union ofandgrafts.We 100% found while that the failed graftsuccessrate, we examined for the phy graft union This by is protectedarticle reserved. Allrights copyright. scions, the PMH close to those at day 0 (Fig. 6). As a result,a6, H at PM scion the As day 0day(Fig.6). close to thoseat they all subgenus under the are doratissimus clade previously treated as one species, mariannensis reflect the phylogenetic relationship between breadfruit and these and previo to compared lower much are species, pedalai odoratissimus) and pedelai sericicarpus) (A. (Fig. 12).& Thegraft successof marang 25% for and for 9% ( breadfruit study, this In Discussion 4. graftedgroup than 4-fold higher of the thosewere grafts unsuccessful (Fig.6). the fundamental role of H PM inwith agreement are Our results 2001). Palmgren, 1995; Boutry, (Michelet and cells ofgrowing membrane generates the proton-motive force that drives theuptake proton pump that roles plays in key nutrient andacquisition partitioning (Palmgren, 2001). The enzyme 21 genewerethe upregulated atgraft family interfaceof ( litchi ( Hickory of union graft the day graftingafter of PM (Mo both al., Upregulation H et 2018). pecan ( differentiallygraftproteinupregulated30 in Expressiondaysgrafting was of ATPase union of the after PM phloem loading and stressad +

-ATPase activity tothoseofcompared unsuccessful grafts (Fig.3). H PM d after grafting (Chen et al., 2017). PM ATPase genes were also upregulated at the graft union of grapevine grapevine of union graft the at upregulated also were genes ATPase PM 2017). al., et (Chen grafting after d A. mariannensis A. mariannensis Carya illinoensis ) are represented inaseparated) arerepresented series section and respectivelybreadfruit species from although arethe closest relativesof breadfruit species, with the three species forming together aseparate

+ -ATPase activitygradually day recovered after 2, with activity levelsday relatively being at 6 (Nandwani and Kuniyuki, 2005; Medagoda and Chandrarathna, 2007). The results may TheMedagoda andChandrarathna, may 2007). results 2005; andKuniyuki, (Nandwani ) (Mo et al., 2017), together with upregulation of PM ATPase genes at 8d, 15d and 30 30 and 15d 8d, at genes ATPase PM of upregulation with together 2017), al., et (Mo ) Artocarpus altilis) Carya cathayensis + aptation (Fromm et al., 1989;Michelet andBoutry, 1995; 2001). Palmgren, -ATPaseplantgrowth in physiological and responses,including expansion, cell Artocarpus (Zerega et al., 2010) et (Zerega al., Artocarpus A. communis, A. communis, was successfully graftedonto interspecific rootstocks, marang (A. ) (Qiu et al., 2016; Xu et al., 2017). Members of PM ATPase of ATPase PM Members 2017). 2016;Xuetal., etal., ) (Qiu us observationon breadfruit graftingto species, graft union adhered well between grafting partners, with grafting with partners, betweengraft union well adhered sical and biochemical andsical between difference thesurvival of nutrients of nutrients such assugars theplasma ionsacross and of successful grafts showed significantly higher PM PM higher significantly showed grafts successful of whereas pedalai ( pedalai whereas Litchi chinensis

. + Inorder to identify factors causing the low -ATPase protein and genes was observed in in observed genes and was protein -ATPase Artocarpus + -ATPase activities of the successfully A. sericicarpus A. + ) for compatiblegraft at 14 -ATPase anis integral membrane species. A. camansi A. ) and marang ( A. camansi A. and

d and A. A. A. A.

Accepted Article suggest PM H suggest PM of union recently reported being differentially up-regulated e measured from the rootstock stem microsomes two months after grafting (Fig. 5). These results suggest PM H PM suggest These grafting results 5). after months (Fig. two microsomes stem rootstock measured the from (Michelet Boutry, and Palmgren, 2001). 1995; Therefore, 1999; Ehlert et al., 2011; et Spartz al., 2014). Higher of expression PM H ofmembrane; 2)stimulation cell and through apoplast expansion andVan acidification Volkenburgh, (Stahlberg plant growth developmentand through two basic 1) processes: energization of uptakenutrient plasma across selection of rootstocks with high levels of PM H of PM levels high with of rootstocks selection before grafting had significantly lower graft survival th This by is protectedarticle reserved. Allrights copyright. including genes, heterograft grafting14after day et al., (Cookson 2014). Furthermore, at leastseven of isoforms ATPase PM othernutrients essential osmoticfor regulation and cell enlargement in differentiating cambial cells (de Boer et We further investigated whether high level of PM H PM of level high whether investigated further We uptakeof most nutrients intocambial cells is an energy-dependentprocess under the control of PMH improvegraft success (Marsch-Martinez et Sanabam al.,Miles, 2013; et The2015; al., Dabirian2017). and to found was grafting before soils or medium growth into sucrose of application fact, In 2017). al., et Xu 2017; energy metabolism during graft union formation (Melnyk 2015;Meyerowitz, and Muneer et al., 2015; Mo et al., of and sucrose upregulation significant metabolic flux with in highlyactive is graft union have that indicated evidences Many 2002). al., et Arend 2001; Palmgren, 1995; Boutry, and (Michelet cells parenchyma xylem and tissueswherehighof rates solute exchangeacross plasmaguard ismembranerequired, such cells, as phloem 2012; Melnyk, 2017). Strong evidences have demonstrated the operation of proton pump theoperationof proton demonstrated have evidences Melnyk, Strong 2012; 2017). leadingvascular to is regenerationofconsidereda hallmarkgraftas success Errea, 2005; and (Pina Yinetal., cambial differentiationcell throughturgor-driven cell expa Furthermore, therewas apositive correlation ( 4). (Fig. rootstocks pedalai for 30% and marang for 60% to increase could breadfruit of success graft graft survival. This let toour findings that rootstocks with lowest PM H essential for growth of graft union cells. Furthermore, PM H PM Furthermore, cells. union graft of growth for essential or (Fig. 3-5) rootstocks may there suggest is not enough ATPase may have a role in graft union formation. As a primary ion pump, PM H roleprimarygraft PM unionhave a a As ATPase pump, may ion formation. in Arabidopsis thaliana + -ATPase maybe required for thesuccessfuldevelopmentof graft union. At1g80660 , At3g47950 from from 2406 h hto after grafting (Melnyk al., et These 2018). evidences together , At5g57350 P < 0.01)between< graft success andthe H PM + -ATPase activitygrafting, before leaves -ATPase the rootstock from , + ither asymmetricallyither or symmetrically thegraft around At4g29900, At1g17260 At4g29900, -ATPase activity before graftingcould be beneficial for an those with highest enzyme activity (Fig. 4). Through proton-motive force to sustain the uptake of nutrients

nsion (Palmgren,nsion 2001). Cambial cell differentiation a very activity low of PM H+-ATPase in graft union + -ATPase has long been recognised for promoting promoting for been recognised has -ATPase long + + -ATPase activitymicrosomesin leaf -ATPase has been reported for those , At2g18960 At2g18960 + -ATPase is thought to isthought affect-ATPase and and

in uptake of K + -ATPase activities At3g42640 + -ATPase were were + and and + - Accepted Article (Portillo, 2000; Zhou et al., 2014). In particular, PM H refl and nutrients stress, osmoticwater-deficiency,and increase and its role in graft union formation may provide opportunity for graft improvement through PM H PM graft through opportunity improvement for providemay union formation graft its roleincrease in and Aloni et al.,Melnyk, 2010; 2017). It the known is not correlationwhether of H PM playan important rolein the differentiationof vascular graftduringtissues union formation (Arend et al., 2002; PavlovkinetSpartz 2002; et al., 2014). al., Accumulatin 2002). PM H PM 2002). recovery of PM H of PM recovery closingbeenhas agentsgraft to shown improve success (DabirianMiles, Therefore, 2017). and the early the early stage of graft union establishment. In fact, andCoaker, 2011), an important stress response for control are consistent with the function ofPM H removalof the scion (Fig. roots 6) suggests a significant The induction of PM H of PM The induction Melnyk, 2017). selection orderin to highachieve graft success in horticulture practices (Melnyk and Meyerowitz, 2015; for criteriaof rootstock one important the differentiate divide is and capacityhigh to Notably,grafting tissues. associated withhigh PM H al., differentiation Induced 2008). ofva ( pine maritime of tissues wood-forming This by is protectedarticle reserved. Allrights copyright. al., 1985; Pavlovkin et al., 2002; Arend et al., 2004). Twoplasma membrane H through graftunion or exchange ofgenetic materials betweencannot rootstock bescion and out ruled successcurrentour study associated inis with auxin re (Stegemann and 2009;Bock, Xia 2018). etal.,Further investigation natureH the PMof intothe differentiating tissues (Arend et al., 2002; Fromm, 2013; Spartz et al., 2014). al., et Spartz 2013; Fromm, 2002; al., et (Arend tissues differentiating The results that levels of PMH manipulation. monitoringATPase and context, a very low PM H PM low very a context, was found to be associatedwith low cell extensibility aging in (Sveinsdottira organs et al., 2009). In this + -ATPase is alsokey a enzyme for H + -ATPase -ATPase activity ingrafted plants may not only reflect the return of electrochemical gradient + -ATPase by isgrowthduring welldocumented auxin cambial -ATPase 2001; (Palmgren, + + -ATPase activitymaysuggest ain rootstocks low capacity in cell of expansion -ATPase activityin the xylem and parenchyma phloem cells (Pavlovkin et al., + -ATPase activityin scion leaves of grafted plantsdecreasedimmediately after Pinus pinaster Pinus scular bundles and bundles scular functional and connectionin + -ATPase in triggering primary res + effluxresulting in cell-wall loosening and cell extension in reduction of application transpirationin through stomata- ect early disturbance ionhomeostasis in and metabolism + ) during the development ofduring secondary (Paiva development the xylem et ) -ATPase playcrucialin a role stomatal closure(Elmore g evidences have also demonstrated that auxin response that auxin g demonstrated have also response evidences sponse. sponse. In addition, the possibility ofmRNA transfer shock may occur as a result of separation. The results results The separation. of aresult as occur may shock of water loss isthat critical for graft survivalduring R ponse to abiotic stresses including educed PM H + -ATPases were upregulated in + -ATPase activity tograft Ricinus communis + -ATPase activity activity -ATPase + -ATPase were were + -

Accepted Article Arend,M.,Weisenseel, M. H., Brummer,M., Osswald,W. andFromm, J.H. (2002) Seasonal changesof thequantitativemicrogram quantities andsensitive method of A proteinBradford, for rapid M. M. (1976) Arend, M., Monshausen, G.,Wind, C., Weisenseel, M. H. activity in leaves before grafting had significantly lower relationship between PM H+-ATPase activityand graftsuccess. First, rootstocks withvery low PM H+-ATPase grafted onto interspecifi successfully ofbreadfruit scions the rootstock stems two months after grafting. Third, the recovery of PM H PM of recovery the Third, grafting. after months two stems rootstock the Second, therewas positive correlationa betweengraft success the PM and H+-ATPase activity measured from This by is protectedarticle reserved. Allrights copyright. H PM of level higher that findings the on based together, Taken water loss, leadingto ahigher chanceof survival. to sustain nutrient andcellular uptakes homeostasis, but Aloni, B.,Cohen, R., Karni,L., Akta 6. References manuscript. project HORT 2014/077. The authors are indebted to the reviewers comments whose have improved the Centre Australia the by funded was work This Acknowledgments 5. dwarfing. breadfruit for success graft improve to selection rootstock for strategy screening develop was associated withlong-term the survival ofthegraf Plant Physiology, ion andendogenous plasmamembrane H(+)-ATPase Environment onof membrane the plasma H(+)-ATPase the wood rayparenchyma poplar. in Plant,& Cell utilizing the principle ofprotein-dye binding. Analytical Biochemistry, interactions. Scientia Horticulturae, 27 , 1288-1296. 129 , 1651-1663. 1651-1663. , s, H. and Edelstein, M. (2010) Hormonal signaling in rootstock-scion in rootstock-scion signaling Hormonal M. (2010) andEdelstein, H. s, 127 , 119–126. 119–126. , for International Agriculture Research (ACIAR) through InternationalResearch through Agriculture for (ACIAR) ted plants. The plants. ted provide opportunity may information to c rootstocks of marang and pedalai, we found a positive pedalai, and we found apositive ofmarang c rootstocks graft survival thanhigh those withenzyme activity. and Fromm, J. (2004)of Effect deficiencypotassium also ofcapability thesuggest restoration control to current during cambial growth in poplar plants. poplarplants. growth cambialduring in current + -ATPase activity was found in the graft union graftunion the in was found activity-ATPase + -ATPaseactivity scion from leaves 72 , 248-252. 248-252. , Accepted Article de Boer, A. H., Katou, K., Mizuno, A., Kojima, H. and Okamoto, H. (1985) The role ofThe electrogenic role Okamoto, xylem (1985) and H. Kojima, H. A., Mizuno, de K., Katou, A. H., Boer, Fromm, H., Katagiri, F. and Chua, N. (1989) An octopi An (1989) Chua, N. and F. H., Katagiri, Fromm, This by is protectedarticle reserved. Allrights copyright. Fromm, development Xylem J. intrees: from (2013) ca ofPlas The the andElmore,(2011) Role Coaker, G. J. Ehlert, c., c., j.,Plassard, s. Tardieu,Cookson, and t.f. (2011)Do pH Simonneau, inchanges leafthe apoplast by damage of characterization and Assessment (2012) B. L. Roberts-Nkrumah, and M. Gloster, O., Daley, Dabirian, S. andMiles, A. C. (2017) Increasingof Survival Splice-grafted Watermelon Seedlings Using a Chen, Z., Zhao, J. T., Hu, F.C., Qin, Y. H., Wang, X. H. and Hu, G. B. (2017) Transcriptome changesbetween Kasamo,( membranericeMechanismfrom plasma H+-ATPase the activation K.(1990) of for Antitranspiran (2017) A. C. Miles, and S. Dabirian, C. Trossat-Magnin, Magnin, N., S., Delrot, N., Z. L. Mendome, C., Hevin, C., J. Moreno, M. J., S. Cookson, expression afactor ASF-1, binds and from tobacco nuclear Cell,Plantextracts. Plant, Molecular Cell & Environment, fusicoccin.of unguiculata: zylem absorption pumps the the and Plant, Vigna from ofauxin inK+ effects Food Crop Society, ( hurricane Tomas tomajor tree crops with Sucrose Application. Hortscience, HortTechnology Scientific Reports, compatible and incompatible graft combination of Litchi chinensis by digital gene expression profile. (Eds). Cellular Aspects of Wood Formation. PlanFormation. (Eds).of Cellular Wood Aspects Environment, induced bypolyethyleneof glycol and down-regulation root hydraulic Plant, conductivity. & Cell of elongation rate contribute leaf inhibition rapid to culture cells bymolecular speciesof a phospholipid. Plant Physiology, 2481. thegraft interface comparedwhen with autograf at genes instress responses induces involved and nonself rootstocks with Ollat, (2014) Heterografting N. artocarpus camansi artocarpus 34 , 1258–1266. 1258–1266. , 4 27 , 416-427. , 416-427. 7 48 , 494-501. 494-501. , , 3954-3954. , 3954-3954. ) in St.) Lucia St. and Grenadines. and Vincent the Proceedings theof Caribbean 8 , 124-131. 124-131. , , 579–586. 579–586. , 52 , 579-583. 579-583. , special emphasisbreadfruit on ( t Application IncreasesGrafting Successof Watermelon. maMembrane H+-ATPase inPlant–Microbe Interactions. ted controls. Journal ofExperimental Botany, t Cell Monographs. 20, Springer. 20, Cell Springer. tBerlin, Monographs. Heidelberg mbial divisions to mature wood cells. in. Fromm, J. J. Fromm, cells. in.mature wood mbial to divisions ne synthase enhancerdirects elementtissue specific by water stress? Comparison of stress responses of stress Comparison byresponses stress? water 93 , 1049-1052. artocarpus altilis 1 , 977-984. Oryza sativa ) and breadnut ) and breadnut 65 , 2473– , L.) L.) Accepted Article This by is protectedarticle reserved. Allrights copyright. Melnyk, W. Plant C. grafting:(2017) insigh Marsch-Martinez, Franken,N., J., Gonzalez-Aguilera, K. Paiva, J. A. P., Garnier-Géré, P. H., Rodrigues, J. C., Alves, A., Santos, S., Graça, J., Provost,L., G. Chaumeil, micronesia. in production of breadfruit improvement and (2005) Grafting H. A. and Kuniyuki, D. Nandwani, Study Proteomic R. B. (2015) Y. andJeong, G. A., Park, Manivnnan, P., C.Muneer, H., Soundararajan, Ko, S., of differentially Analysis accumulatedR. W.Z. F. C. Su,(2017) Mo, Y., and He, proteins Peng, H., H. Z.Mo,Feng, Z., and Liu, F.W., Su, G., Peng, (2018) Michelet, B. andBoutry,M. Theplasma (1995) membrane H+-ATPase highly (A regulated enzyme with Melnyk, W. C. Meyerowitz, E.anda. M. (2015)Plantgrafting. Current Biology, Melnyk,W.,C.Gabel, A.,Hardcastle, T. J.,Robinson, Miyashima, S., S., Grosse, I.Meyerowitz, and M. E. Medagoda, I. and Chandrarathna, K. W. M. C. J. (2007) of Grafting breadfruit altilis)(artocarpus using breadnut Methods, Methods, collar-free efﬁcientgraftingAn R. ﬂat-surface method seedlings. (2013) thaliana Arabidopsis for Plant pinaster) wood-forming tissues during a growing season. New Phytologist, Phytologist, New season. growing a during tissues wood-forming pinaster) Silva-Perez,P., D., Bosc, D. Fevereiro,A., P. and C. Plomion, (2008) Plasticity ofmaritime pine (Pinus Pacific Region. ActaHorticulturae, Proceedingsof the International SymposiumonHarn DifferentLight Intensities. Plos One, Related Connections Watermelon Sc in Vascular to associatedScientiapecangraft illinoensis).union (Carya with Horticulturae, formation in Union (Caryain Pecan Development illinoinensis).Genes, Plantmultiple Physiology, physiological functions). America, of States United mechanism that activatesvascular regeneration. Proceedings of the NationalAcademy of Sciencesof the (2018)Transcriptome dynamics graftjunctions atArabidopsis reveal intertissue an recognition camansi (Artocarpus 9 , 14. 14. , ) asstock. root Acta Horticulturae, 115 , E2447–E2456. E2447–E2456. , 694 ts into tissue regeneration. Regeneration, 10 , 307-310. 307-310. , , e0120899. e0120899. , Transcriptomic Analysis Provides Insights into Grafting Transcriptomicinto Insights Grafting Analysis Provides L., deFolter, S., Angenent, G. Alvarez-Buylla,and E. 108 ions Graftedions onto Bottle-Gourd Rootstock under essing thePotentialessing of in Horticulture the Asian- 757 , 1-6. , 1-6. 9 , 149-152. 149-152. , , 71. 25 179 , R183-R188. , R183-R188. 4 , 1080-1094. 1080-1094. , , 3–14. 3–14. , 224 , 126-134. 126-134. , Accepted Article

Spartz, A. K., Ren, H., Park,M. Y., Grandt, K. N.,Lee, H., S. Murphy, A. S., Sussman,M. R., Overvoorde, J.P. Solomon, F.Solomon, L. J. and B. evaluation(2008) Roberts-Nkrumah, An of grafting successful influencingfactors of Sanabam, R., Singh, N. S., Handique, P. J. and Devi, H. L.Roberts-Nkrumah, B. Breadnutandpropagation, (2012) breadfruit amanual commercialfor propagation, Ragone, D. (2006) Ragone, D. (1997) Breadfruit, Portillo, F. (2000) Regulation of plasma membrane H membrane plasma of Regulation (2000) F. Portillo, Pina, of new A. advancesof P. (2005) inmechanism graftErrea, and A review compatibility–incompatibility. Qiu, L., Jiang, B., Fang, J., Shen, Y.,Fang, Z., Rm, S., Yi, K., Shen, C., Yan, D. and Zheng, B. (2016) Analysis This by is protectedarticle reserved. Allrights copyright. Evidence Ullrich, C. for ofactivity xylem high Läuchli,(2002) and R., Wächter, A. Pavlovkin, H., Okamoto, J., Palmgren, M. plasma membrane H+-ATP G. Plant (2001) and W. Gray, M. (2014) SAUR ofinhibition phosphatases plasmaactivates PP2C-D membrane H+- ATPases to promote to ATPases cellpromote expansion in breadfruit on chataigne rootstock. 2008 Proceedings of the Caribbean Food Crops Society, Society, Crops Food Caribbean of the Proceedings 2008 rootstock. on chataigne breadfruit PCR. Scientia Horticulturae, Blanco)vitromicroshoot production tip using in ttp://www.fao.org/3/a-i3085e.pdf Organization oftheUnited Nations. Foodof Agriculture Department Agriculture, and their culture environment anduse. Permanent AgricultureResources Holualoa, Hawaii Gatersleben, (Germany). http://www.jeffmarck.net/breadfruitdocs/Ragone_1997_IPGRI_breadfruit.pdf (IPK), Research Plant Crop and Genetics of Plant Institute Leibniz 10, crops. neglected and underutilized Scientia Horticulturae, pathway during graft process. BMC Genomics, graft BMC Genomics, during process. pathway ofhickory transcriptome(Carya in uncover cathayensis), the and dynamicsinthehormonal signaling Acta, of of parenchyma and ray cells in the interface of hoststem and Agrobacterium tumefaciens-induced tumours of Physiology Molecular Biology, Plant Plant and Ricinus communis 1469 , 31-42. , 31-42. Artocarpus altilis Artocarpus .of JournalBotany, Experimental 106 Artocarpus altilis , 1-11. , 1-11. 189 (breadfruit).in. Elevitch., C. R. Traditional (Eds). oftrees Pacific Islands: , 208-213. , 208-213. Arabidopsis (Parkinson) Fosberg - Promoting the conservation and use of of conservation and use the Promoting Fosberg - (Parkinson) 17 + -ATPase in-ATPase plants.Biochimica fungi and Biophysicaet S. (2015) Disease-freekhasi mandarin (Citrus reticulata grafting and its assessment using DAS-ELISA and RT- , 935. 935. , 52 . Plant Cell, Cell, Plant . ase: Powerhousesnutrientuptake.Review for Annual , 817–845. 817–845. , 53 , 1143-1154. , 1143-1154. 26 , 2129-2124. , pp 85-100 pp 85-100 44 , 304-312. 304-312. , Accepted Article Sveinsdottira, H., Yan, F.,Zhu, Y. Y., Peiter-Volk,T. and Schubert, S. (2009) Seed ageing-induced inhibition of Stegemann, S. and R. Bock, ofgeneticExchange (2009) This by is protectedarticle reserved. Allrights copyright. Zheng,and B. (2017) Yan,Liu, D. H., C., Shen, L., W., Guo, L., Zhao, Qiu, Tong, Y., H., Yuan, Xu, D., Xia, C., Zheng, Y., Huang, J., Zhou, X., Li, R., Zha,M., Wang, S., Huang, Z., Lan, H., Turgeon, R.,Fei, Z. and The effect E. Volkenburgh, (1999) Van Stahlberg, R. and Zhou,and Y.S. Breadfruit Underhill, (2016) altilis) (Artocarpus gibberellin 2-oxidasestemgenes elongation in ( of breadfruit Dwarfing (2014) R. J. S. Underhill, and B. M. Taylor, Y., Zhou, Zhou, Y., Pan, X., Qu, H. and Underhill, S. J. R. (2014) Low temperature altersplasma membrane lipid andZerega,T.N. (2010) Motley, Supardi, Phylogeny M. recircumscription of N. N., J. and Artocarpeae Zerega, andMotley, N., Ragone, D. T. (2004) Comple J. Yin, H., Yan, B., Sun, J., Jia, P., Zhang,Z., Yan, X., Chai, J., Ren, Z., Zheng, G. andLiu, H. (2012) Graft-union germinationgrowth post-germination and isroot to related lower plasma membraneactivityof H+- 324 and photosynthesis. Planta, H+-ATPase plasma-membrane the of - Role Argenteum. var. L. sativum of Pisum leaves in expansion ATPase in maize roots. Journal of ATPase Journal maizein Plant Physiology, roots. into Response Mechanisms to Grafting Comparative Proteomic Analysis of the Graft Unions Physiology Plant System. Heterograft benthamiana/Tomato ofZhang,Mechanisms C. Elucidation the (2018) opportunities and American challenges. and abiotic stress response. Plant andand Biochemistry, Physiology Plant response. abiotic stress Bioenergetics of and Journal Biomembranes, development. blackheart during fruit pineapple of activity ATPase and composition (Moraceae) with afocuson Artocarpus. Systematic Botany, Implications for migrations human inOceania.of American Journal Botany, Botany, Experimental of Journal accumulation. auxin development: a delicate process that involves cell–cell betweencommunication scion stock and for local , 649–651. 649–651. , 208 , 188-195. 188-195. , 46 , 59-69. , 59-69. Process. Frontiers Plantin Science, Journal of Experimental Agriculture, of light on membrane potential, apoplastic pH and cell x originsx altilis,of breadfruit (ArtocarpusMoraceae): material between cells in plant tissue grafts. Science, of Long-Distance Movement mRNA in a Nicotiana 166 63 in Hickory (Carya cathayensis) Provides Insights Insights Provides cathayensis) (Carya Hickory in , 4219–4232. 4219–4232. , , 128-135. 128-135. , 177 35 , 766-782. , 766-782. , 745-758. , 745-758. 98 , 81-88. , 81-88. 8 , 676. 91 4 , 1743-1763 , 760-766. 760-766. , Artocarpus altilis ) trees: ) trees: Accepted Article Fig. 3 Comparison of Fig. membrane 3Comparison plasma H seeFig. 1C). Allvalues represent mean ±of SE three biological replicates. roots, own scion the of removal after 1 month to (equivalent grafting after months 3 from union graft the above Fig. 2graftedbreadfruit of scion Elongation in stems intervals after the completion of grafting. graft portions;S3, rootstock removed from the S2, stems This by is protectedarticle reserved. Allrights copyright. Sampes collected for plasma membrane H 6monthsgrafting. after onpedalai andmarang rootstocks (F) (G) plants breadfruit Grafted grafting.F-G, 3 after months rootstocks marang plants(E) breadfruit onpedalaisystem and Grafted of rootstock. the D-E, (D) root the on dependent solely being scion the with arrows), (red away cut were union graft the below scion the parterns were well united (seeinsertedpicture), the portion of top the above,of therootstock and bottom portion Alignmentof two peeled surfaces with grafting tapes. long40% and thickness) were removedgraft atthe sections wherethe occur arrows). was union to (red B. we (left) seedling a rootstock (right) and plant breadfruit scion A. interspecific onto graftingof rootstocks. approach breadfruit Representatives Fig. 1 legend Figure theof absence presence and 1 mMNa ATPase, represented as the vanadate-sensitive ATPase, was calculated from the difference between activities in H- PM of activity The 1A). (Fig. grafting before union graft intended the of leaves rootstock from prepared breadfruitonto pedalai marang and rootstocks.ATPhydrolytic activitywas assayed inthe microsomal fractions Fig. 4 membraneRelationship between plasma H significantly different ( are letter different with Values replicates. biological four of ± SE mean represent values All Protein. S4).PM H-ATPase activity, representedthevanadate-sensitive as Pi/min/mg ATPase, presented is asµmol 1C, Fig. roots, own scion’s of removal (after completion grafting after weeks two union graft the from prepared graftsontomarang rootstocks. pedalai and hydrolytic ATP activity was microsomal assayed inthe fractions P <0.05). 3 VO re brought nextother. to Leaves each slice anda of the 5 stem (3 ~ cm 4 + . Rootstocksof each species grouped accordingwere to the levelsof +- -ATPase assay: S1, rootstock leaves from the potential graftunion; potential the from leaves S1, rootstock assay: -ATPase ATPase activities from graft union of successful and unsuccessful unsuccessful and successful of union graft from activities ATPase +- ATPase activities from rootstock leaves and graft success of C. Completion of grafting two months later. After two plants. The elongation of scion stems was measured measured plants. The stemsof was elongation scion union; S4,union; scion apical leaves collected at2-day Accepted Article significantly different (P<0.05). B. Linear regr B. significantlydifferent (P<0.05). experiment.values All representmean ± of SE three se groups respectively. for mediumhigh activity and low, Fig. 5 Relationship between plasma membrane plasma Fig. between 5Relationship H ( values representmean of ±SE three separate Valuesexperiments. different with letter are significantly different leastsevenrootstocks The eachexperiment.for graftsuccess determined wasmonthsgrafting. four after All Pi/min/mgProtein respectively.groups mediumhigh activityEach group representing andcontained at thelow, breadfruit pedalai ontomarang RootstockH PM rootstocks. A. and absence and ofpresence 1 mM Na represented as thevanadate-sensitive ATPase, was calc immediately graft above the union two months after gr success. ATP hydrolytic activitywas assayed in the microsomal fractionsprepared from rootstock stems This by is protectedarticle reserved. Allrights copyright. ATPase activitylevels in themicrosomal fraction,with PM H significantly different (P<0.05). ofexperiments aspercentage expressed the activ and are unsuccessful were determinefourgrafting. months after Thevalues mean represent ±SE of three separate or successful as outcome, graft The Protein. Pi/min/mg µmol as presented are ATPase, vanadate-sensitive the and after(Day 2 toDay of 6)the removal scion rootsThe (Fig. 1C). activities of PM H-ATPase, represented as hydrolyticmicrosomal activity the preparedin fractionsassayed was of from leaves apical scions before (day0) activitiesgraft and success. Correlation coefficient: 0.9203; r(7)= 0.0017pedalai. forActivities of PM H Fig. 6 Changes in plasma membrane H membrane plasma in 6Changes Fig. after grafting. representofmeanThe samples. atleastgraft succnine P <0.05). <0.05). + -ATPase activity in the microsomal fractions, with 3 VO + + 4 ATPase are presented as µmol Pi/min/mgATPase Allpresented Protein. activity are asvalues µmol -ATPase activity of scion leaves after removal of-ATPase ATP leavesof activity scion scion roots. removal after . Rootstocks of. Rootstocks grouped eachspecies according toPM were H ession for relationship between rootstock stem PM H PM rootstock stem relationship between for ession +- ATPase activities from rootstock stems graftand success of ≤ ess is based on the percentage of survival four months onof survivalfouris based percentage the ess 0.05, 0.06 to 0.10 and and 0.10 to 0.06 0.05, ulateddifference the from between activitiesthe in Each group had at least nine rootstocks for each ity at day 0 (100%). Values withdifferent letter Values are ity0 (100%). at day parate experiments. Values with different letter are afting (Fig. 1C). The activity ofPM H-ATPase, Theactivity 1C). afting (Fig. ≤ 0.14, 0.15 0.25to and P = 0.00043 for marang; r(7) = 0.8820, =0.8820, for marang; r(7) =0.00043 + ATPase activityATPase grouping graft and ≥ 0. 11 µmol Pi/min/mgProtein Pi/min/mgProtein µmol 11 0. ≥ 0.26 µmol µmol 0.26 + ATPase P +- = Accepted Article This by is protectedarticle reserved. Allrights copyright.

Accepted Article This by is protectedarticle reserved. Allrights copyright.