Carter R. Miller A, Ivan Ochoa A, Kai L. Nielsen A, Douglas Beck B and Jonathan P. Lynch A,C. Functional Plant Biology, 2003, 30,

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1 CSIRO PUBLISHING Funtionl Plnt Biology, 23, 3, Geneti vrition for dventitious rooting in response to low phosphorus vilility: potentil utility for phosphorus quisition from strtified soils Crter R. Miller A, Ivn Oho A, Ki L. Nielsen A, Dougls Bek B nd Jonthn P. Lynh A,C A Deprtment of Hortiulture, The Pennsylvni Stte University, University Prk, PA 1682, USA. B Centro Internionl de Agriultur Tropil, AA 6713, Cli, Colomi. C Corresponding uthor; emil: JPL4@psu.edu Astrt. We hypothesized tht dventitious roots my improve rop dpttion to low-phosphorus soils y enhning topsoil forging. In tropil field study, phosphorus stress stimulted dventitious rooting in two phosphorus-effiient genotypes of ommon en (Phseolus vulgris L.) ut not in two phosphorus-ineffiient genotypes. Although phosphorus vilility hd no onsistent effets on the length or iomss of whole root systems, it hd differentil effets on dventitious, sl, nd tproots within root systems in genotype-dependent mnner, resulting in inresed llotion to dventitious roots in effiient genotypes. Adventitious roots hd greter length per unit iomss thn other root types, espeilly under phosphorus stress. Adventitious roots hd less onstrution ost thn sl roots, despite hving similr tissue nitrogen ontent. Phosphorus stress redued lterl root density, nd dventitious roots hd less lterl root density thn sl roots. Lterl roots formed further from the root tip in dventitious roots ompred with sl roots, espeilly under phosphorus stress. Field results were onfirmed in ontrolled environments in solid nd liquid medi. Stimultion of dventitious rooting y phosphorus stress tended to e greter in wild genotypes thn in ultivted genotypes. We propose tht dventitious rooting is useful dpttion to low phosphorus vilility, euse dventitious roots explore topsoil horizons more effiiently thn other root types. Keywords: dventitious roots, sl roots, effiieny, genotype-vrition, Phseolus vulgris (ommon en), phosphorus, root rhiteture, root prtitioning, topsoil forging. Introdution Low phosphorus vilility is primry limittion to rop growth in mny ntive soils. Fertiliztion is n inomplete solution to this prolem euse of the poor vilility nd ffordility of fertility mendments in mny groeosystems, nd the fixtion of pplied phosphorus in mny tropil nd sutropil soils (Snhez 1976). Intensive phosphorus fertiliztion is lso prolemti in developed ountries, euse of resultnt wter pollution (Frnis 199). The development of rop genotypes with enhned ility to yield with low phosphorus vilility ( phosphorus effiieny ) is therefore desirle (Lynh 1998). The ommon en (P. vulgris) is n importnt soure of nutrients in the tropis nd sutropis, where its prodution is severely onstrined y low phosphorus vilility. Geneti vrition for phosphorus effiieny is present in en germplsm (Whiteker et l. 1976; Thung 199; Youngdhl 199; Lynh nd Beee 1995; Beee et l. 1997). Phosphorus effiieny in en ppers to e due to enhned ility to quire phosphorus from the soil, rther thn enhned internl utiliztion of phosphorus (Lynh nd Beee 1995; Beee et l. 1997). In generl, inresed phosphorus quisition effiieny in en is not relted to hemil modifition of the rhizosphere (Yn et l. 1995,, 1996), ut does pper to e relted to root rhiteture nd morphology, whih re highly vrile mong en genotypes (Lynh nd vn Beem 1993). Root ron osts onsume signifint frtion of ville photosynthte in low-phosphorus en plnts (Nielsen et l. 1998) so rhiteturl trits tht deploy roots to more fertile regions of the soil nd minimize interroot ompetition (Fitter 1991; Nielsen et l. 1994) my inrese phosphorus quisition effiieny. A omprison of four en genotypes ontrsting in phosphorus effiieny showed tht the root systems of two effiient genotypes were Arevitions used: CIAT, Centro Internionl de Agriultur Tropil; DAP, dys fter plnting; FLRD, distne from root tip to first lterl root; LCC, liner onstrution ost; LRD, lterl root density; PAR, photosynthetilly tive rdition; RCBD, rndomized omplete lok design; SCC, speifi onstrution ost; SRL, speifi root length; VAM, vesiulr rusulr myorrhize. CSIRO /FP /3/9973

2 974 Funtionl Plnt Biology C. R. Miller et l. le to mintin higher rtes of growth per unit respirtion thn root systems of two ineffiient genotypes (Nielsen et l. 21). Therefore, morphologil nd rhiteturl trits tht redue the metoli ost of soil explortion pper to e importnt spets of effiient phosphorus quisition in this speies. Our present fous is the sptil vrition in phosphorus vilility with soil depth. In most nturl soils, phosphorus ontent nd phosphorus vilility re greter in surfe or ner-surfe horizons thn in the susoil (Chu nd Chng 1966; Enwezor nd Moore 1966; Anderson 198; Keter nd Ahn 1986; Pothuluri et l. 1986). One reson for this is the ontinul deposition of phosphorus on the soil surfe in deyed leves nd other plnt residues. Another is tht hemil, physil, nd iologil onditions in surfe horizons re generlly more onduive to phosphorus moiliztion thn re onditions in the susoil. In griulturl soils, fertiliztion nd ultivtion inrese phosphorus vilility in the topsoil, with only very slow movement of phosphorus into the susoil in most ses. As result, phosphorus vilility usully delines sustntilly with soil depth. Root rhiteturl trits tht enhne the explortion nd exploittion of surfe horizons my therefore enhne phosphorus quisition. In ommon en, low phosphorus vilility inreses the shllowness of sl roots, espeilly in phosphoruseffiient genotypes (Bonser et l. 1996). Bsl root shllowness is orrelted with yield performne in low-phosphorus tropil soils mong set of unrelted genotypes (Bonser et l. 1996) nd mong reominnt inred lines (Lio et l. 21). Geometri modeling showed tht shllow sl root systems suffer less inter-root ompetition for phosphorus thn deeper root systems (Ge et l. 2), nd tht shllower root systems were more ompetitive thn deep root systems for topsoil phosphorus (Ruio et l. 21), whih ws susequently onfirmed in the field (Ruio et l. 23). These results support the hypothesis tht effiient topsoil forging, in this se regulted y sl root grvitropism, enhnes phosphorus effiieny (Lynh nd Brown 21). The ommon en is typil of mny nnul diots in hving root system omposed of tproot, sl roots emerging from the sipetl end of the tproot, nd dventitious roots emerging from the suterrnen portion of the hypootyl. Beuse of their origin in the hypootyl, s well s their initil horizontl growth hit, dventitious roots re often the most shllow portion of en root system. Preliminry oservtions indited tht phosphoruseffiient en genotypes might hve vigorous dventitious rooting (Lynh et l. 1995). In this study we tested the hypothesis tht dventitious rooting ontriutes to phosphorus effiieny y ontriuting to effiient topsoil forging. In nlogy with prior results with sl root grvitropism (Bonser et l. 1996), we found tht phosphorus vilility regultes dventitious rooting in genotypedependent mnner, nd tht dventitious roots hve severl fetures tht enle them to explore the topsoil more effiiently thn other root types. Mterils nd methods Field study Plnt mteril Common en (Phseolus vulgris L.) genotypes G2333, G19839, G417, nd DOR364 from CIAT (Cli, Colomi) were used. These genotypes hve een studied y our group nd t CIAT for some yers, nd s result their reltive phosphorus effiieny is known [e.g. (CIAT 1999), or ppers y Lynh nd ollegues ited in this rtile]. The phosphorus-effiient nd reltively high yielding lndre G2333 is of Mexin origin (Mesomerin gene pool) nd indeterminte liming growth hit (type 4, ording to Singh 1982). The Peruvin lndre G19839 (Anden gene pool), of indeterminte prostrte growth hit (type 3), hs lrge seeds nd hs high phosphorus quisition effiieny. The Brzilin ultivr Crio (CIAT ession G417, Mesomerin gene pool), hs intermedite prostrte hit (type 3), is responsive to phosphorus fertiliztion, nd is hrterized s hving intermedite phosphorus effiieny. The phosphorus-ineffiient reeding line DOR364 is of Mesomerin origin nd hs n indeterminte ush hit (type 2), eret stems nd smll seeds. Field site The experimentl site ws loted in Sn Isidro el Generl, Perez Zeledon, Cost Ri (9 23 N, W, elevtion 78 m ove se level). Photosyntheti photon flux densities t middy mesured with quntum sensor (LI-189, LI-COR, In., Linoln, NE) rehed mximum of 22 µmol photons photosynthetilly tive rdition (PAR) m 2 s 1, nd rnged on verge etween 1 15 µmol photons PAR m 2 s 1. Field preprtion nd plnting ws rried out in lte Septemer Severl yers of fertilizer mngement were used to generte djent plots hving low or moderte phosphorus vilility. Prior to plnting, the medium phosphorus tretments were mended with rok phosphte, P 2 O 5, t 15 kg P h 1. Seeds were sown in rised eds (25 m wide y 15 m high) in oth min plots (P level) in similr design nd plot size (Singh 1995). Both fields were rrnged in rndomized omplete lok design (RCBD) with four loks. Dy of plnting ws loking ftor to llow stggered hrvests t equivlent plnt ge, nd there were four rndomly ssigned tretments (genotypes) per lok. Eh experimentl unit (plot) onsisted of six 6-m long rows of eh genotype. Plnts were sown t 6 m depth, 5 m sping etween plnts, nd 6 m sping etween rows. Rows one nd six nd the 1 m t oth ends of the plot were used s orders nd were not hrvested. Bloks were ordered y plntings of three rows of fifth genotype not inluded in the nlysis. Hrvests egn t 22 d fter plnting (DAP), nd eh lok, in the rndomized omplete lok design ws smpled one per week for four weeks, t 22, 29, 36, nd 43 DAP. Soil nlysis The soil ws Typi Pleudult with the epipedon to 2 m depth. Orgni mtter ontent ws etween 4 6%. Rok frgments omposed less thn 3% of soil volume. Multiple soil smples were tken from eh plot during the fourth week of hrvesting, 43 DAP. Horizontl slies of soil, 1 m thik nd 1 2 g dry weight, were tken every 5 m soil depth eginning t 5 m, until 25 m soil depth nd then irdried. Soil lods were homogenized y pssing the smple through sieve. Phosphorus vilility ws determined y Bry-2 (Olsen nd Sommers 1982) nd y the iron-oxide-impregnted filter strip method

3 Adventitious root prodution in response to low phosphorus vilility Funtionl Plnt Biology 975 (vn der Zee et l. 1987; Kuo 1996). Men ville phosphorus determintions were otined t eh 5 m soil depth for eh genotype nd for oth phosphorus levels from the men of the four loks ± stndrd error. Tissue nlysis Two plnt shoots (two smples) from plnts used for sl root trings (see elow) were hrvested weekly in eh plot (22, 29, 36, nd 43 DAP). The numers of oth dventitious nd sl roots were ounted from extrted hypootyls. The numer of leflets ws ounted, nd 15 rndomly hosen lef punhes were tken for determintion of lef re, speifi lef re, nd phosphorus ontent. Smples were dried t 45 6 C for 48 h for dry weight determintion. Stems nd leves were weighed seprtely. Dry tissues were shed t 6 C for 18 h prior to olorimetri determintion of phosphorus ontent (Murphy nd Riley 1962). Myorrhizl oloniztion At four nd six weeks fter plnting myorrhizl oloniztion ws evluted. Smples of pproximtely 25 root segments (1 3 m length) were olleted nd stored in 4% lohol. Root segments were oiled in 1% KOH t 121 C nd then rinsed with wter followed y 5% HCl. Smples were then stined for 16 h with.5% trypn lue in equl mounts of glyerol, lti id nd wter, nd destined in equl mounts of glyerol, lti id nd wter (Phillips nd Hymn, 197). Myorrhizl oloniztion ws estimted on perent sis using grid interset method (Tennnt 1975). Root system nlyses At 43 DAP multiple plnts, inluding intt root systems, were exvted. From these, four representtive plnts were hosen for dditionl nlysis of root systems. Two plnts were divided into root nd shoot. Roots from these plnts were stored in 4% lohol prior to determintion of root length, dimeter nd surfe re. Rhizoi were seprted nd root systems were divided into three groups determined y type dventitious roots rising from long the length of the hypootyl nd their lterls, sl roots rising from the se of the hypootyl nd lterls, nd tp roots, inluding lterls (Zoel 1996). Root smples were soked in queous solution ontining.1 g L 1 neutrl red dye (Sigm Chemil Co., St Louis, MO) for one hour. Roots were then snned using flt ed snner (HP SnJet II, Hewlett Pkrd, Plo Alto, CA) with refletive lid, s desried y Boum et l. (2). Averge dimeter, root length, nd surfe re (SA) were determined for eh root system seprtely with imge nlysis softwre (Delt-T SCAN, Delt-T Devies Ltd, Cmridge, UK). Dry weight of snned roots ws mesured fter drying t 65 C for 48 h. Speifi root length (SRL) ws lulted s length per unit of root dry weight. Speifi onstrution osts (SCC), gluose equivlent/tissue dry weight (g g 1 ), of dventitious nd sl root tissue on dry-weight sis were otined y elementl nlysis. Reltive frtions of omponent elements were used to determine the metoli expense of tissue in ommon urreny of gluose (MDermitt nd Loomis 1981). Ground tissue ws nlysed for elementl omposition with n elementl nlyser (CHNO Elementl Anlyser, Model EA 118, Fisons Instruments, Beverly, MA), nd speifi onstrution osts of root tissues were determined y nlysis of reltive frtions of omponent elements. Susmples were used for olorimetri determintion of phosphorus ontent fter shing (Murphy nd Riley 1962). Smples of representtive dventitious nd sl roots were olleted weekly from eh plot. Roots were wshed, nd one root of eh type ws pled flt on lined pper. Lterl roots were then tesed wy from the min root, nd lterl root rnhing ws designted y ross hshes for the length of the root for up to 26 m mesured sipetlly from the root tip. Lterl root density m 1 root length ws determined long the length of the root eginning from the lst lterl nd ontinuing sipetlly towrd the se of the hypootyl. Intermedite ounts were mde every 2 m, nd one men vlue of lterl root density, defined s numer of lterl roots m 1 root length, ws lulted. Distne from min root tip to lst (youngest) lterl ws lso mesured. Root topologil mesurements were tken in the field. From smples of dventitious nd sl roots tht were olleted weekly from oth medium- nd low-phosphorus plnts, mesurements of lterl root density (LRD), numer of primry lterl roots emerging from the min root per m min root length, were sored. Root segments from the lst emerging lterl to the root tip were ignored in this lultion, nd this dt, distne to first lterl root, is lso disussed. Seondry rnhing from primry lterls ws negligile even t 43 DAP. Dt nlysis The experiment ws nlysed s ompletely rndomized lok design omined over lotions (phosphorus tretment), with nd without susmpling depending on the vrile tested, in order to ddress the phosphorus tretment genotype intertion. Comining experiments ssumes homogeneity of vrines nd this ssumption ws tested using Brtlett s test (Beee et l. 1997). The experimentl unit (plot) onsisted of plntings of six 6-m rows of one genotype, replited four times for eh of four genotypes t two phosphorus levels, or eight plots per genotype. For dt nlysis, the replitions were onsidered s rndom effets nd phosphorus tretment nd genotypes s fixed effets (MIntosh 1983; Tern nd Singh 22). The sttistil softwre pkges SAS, version 8.2 (SAS 1985) nd Systt, version (SYSTAT 1992) were used for dt nlyses. Controlled-environment studies Plnt mteril For studies in snd ulture, solution ulture, nd igr rolls, genotypes BAT 477, BAT 881, DOR364, G19833, G19839, G417, G21212, G2333, nd G3513 were otined from the CIAT germplsm nk (Cli, Colomi). These genotypes ontrst in performne in low-phosphorus tropil soils. For the igr roll study with wild genotypes, eight essions (listed in Tle 7) of wild P. vulgris were rndomly hosen from the USDA germplsm nk. Snd ulture nd solution ulture In the solution ulture study five genotypes (G2333, G19839, DOR364, G19833, G417) were grown in nutrient solution to oserve dventitious rooting in the sene of soil mehnil impedne. The growth system onsisted of 15-L plsti ontiners filled with 12 L nutrient solution to whih ws dded 1.5% (w/v) solid-phse-uffered lumin-p (Lynh et l. 199) providing two different phosphorus desorption rtes, low P (1 µm) nd high P (1 µm). The omposition of the nutrient solution ws (in µm): 15 KNO 3, 12 C(NO 3 ), 4 NH 4 NO 3, 5 MgSO 4, 3 K 2 SO 4, 3 (NH 4 ) 2 SO 4, 25 MgCl 2, 5 Fe-N-EDTA, 1.5 MnSO 4, 1.5 ZnSO 4,.5 CuSO 4,.15 (NH 4 ) 6 Mo 7 24,.5 N 2 B 4 O 7. Seeds were surfe-sterilized with 7 mm NOCl for 2 min nd germinted in rolls of rown germintion pper soked with.5 mm CSO 4 for 6 d. The seedlings were then trnsplnted to the plsti ontiners with the nutrient solution. The plnts were grown under greenhouse onditions for two dditionl weeks prior to hrvest; shoot dry weight ws mesured nd ll root types were onserved seprtely in 25% ethnol. Roots were stined with.16% neutrl red dye (Sigm Chemil Co.) efore eing snned for root length with WinRHIZO Pro softwre (Regent Instrument In., Quee City, Quee, Cnd). After snning, roots were dried t 6 C nd root dry weight otined.

4 976 Funtionl Plnt Biology C. R. Miller et l. In the snd ulture study five genotypes (G2333, G19839, DOR364, G19833, G417) were grown in snd ulture to oserve dventitious rooting in solid medi without omplitions from VAM nd rhizoil symioses, s well s environmentl stresses. The system onsisted of 2-L plsti pots filled with mix of sili snd nd vermiulite (2:8 v/v) nd solid-phse-uffered lumin-p (1.5% w/v) providing onstnt vilility of low (1 µm) nd high (1 µm) P onentrtion in the soil solution. Twie dy the pots were utomtilly irrigted with the nutrient solution desried ove. Seeds were germinted s efore nd fter 6 d were plnted t depth of 6 8 m in order to stimulte dventitious root formtion in the hypootyls. After two dditionl weeks plnts were hrvested nd mesurements tken s in the nutrient solution experiment. Cigr roll method Seeds were surfe-sterilized for 1 minute in 1% NOCl nd rinsed thoroughly with deionized wter. Individul seeds were pled 6 m elow the long edge of germintion pper (Anhor hevy weight seed germintion pper, 76 l., 25.4 m 38.1 m, Anhor Pper Co., St Pul, MN), whih ws soked in nutrient solution, nd then rolled nd pled upright in 25.4 m m ID PVC open-ended ylinder. Sping etween plnts ws 12 m. A nutrient solution (ph 6.5) ws provided with the following omposition (in mm): 3.1 NO 3, 1.8 K, 1.2 C, 1.4 SO 4, 1. NH 4,.825 Mg,.5 Cl, (in µm) 5. Fe-EDTA, 2. B, 1.5 Mn, 1.5 Zn,.143 Mo, nd.5 Cu s well s. ( phosphorus) nd 1. mm (+phosphorus) KH 2 PO 4. Solution ws supplied ontinuously y pillry tion through the pper roll from smll reservoir t the ottom of the tue. Reservoir level ws mintined t 1 m y ddition of 2 4 ml of nutrient solution to the top of the germintion roll one or twie dily. Nutrient solution ws renewed three times per week. This llowed the germinting seed nd developing seedling suffiient moisture for helthy root system growth for up to d. During this time ll roots, inluding dventitious roots, developed s oserved under field onditions t similr stndrd plnting depth. Plnts were grown in growth hmer with verge photosyntheti photon flux density of 1 14 µmol photons PAR m 2 s 1 with metl hlide lmps t 14/1-h dy/night. Temperture ws onstnt t 25 ± 2 C, nd reltive humidity ws 8%. Plnts were hrvested t 14 DAP for experiments 1 nd 3 nd t 15 DAP for experiment 2. Shoots nd root systems were hrvested. Lef nd stem dry weights, s well s dry weights for dventitious, sl, nd tp root systems were mesured fter 48 h t 65 C. Dt nlysis Results for igr roll experiments were nlysed s RCBD, without susmpling. Five loks, djent replitions long light-density grdient, were used, nd 16 rndomly ssigned tretment omintions, 8 genotypes 2 phosphorus levels (+ or phosphorus), were pled within eh lok. The experimentl unit onsisted of one seedling rolled in germintion pper in one ylinder with n independent reservoir. Results for snd ulture nd solution ulture were nlysed s split-plot design where phosphorus level ws onsidered the min plot nd genotypes the suplots. The sttistil softwre pkge SYSTAT, version ws used for igr roll experiments nlyses (SYSTAT 1992), while snd nd solution ulture experiments were nlysed using the GLM proedure of SASystem (SAS Institute 1985). Mens of tretments were lulted ± stndrd error. Results Soil nlysis Soil phosphorus nlysis y Bry-2 extrtion nd the ironoxide-impregnted filter strip method were omprle. Results from the filter strip method re shown in Fig. 1. Phosphorus vilility ws gretest etween 1 15 m, lowest t 1 m nd elow, nd intermedite t the very surfe. Fertiliztion inresed ville P t 5 nd 2 m depth (Fig. 1). Tissue nlysis Detiled results for plnt phosphorus ontent nd onentrtion re provided y Miller (1998). Shoot dry weight nd lef re inresed over time for ll genotypes in oth phosphorus tretments, nd ws t lest 5% lower in lowphosphorus plnts ompred with medium-phosphorus plnts t 43 DAP. In low-phosphorus plnts shoot dry weight nd lef re of G19839 nd G2333 were greter thn oth G417 nd DOR364. For plnts grown under medium phosphorus, shoot phosphorus onentrtions inresed in ll genotypes up to 36 DAP. Low phosphorus vilility redued lef phosphorus onentrtion y out 5%. At 43 DAP there ws no signifint differene in phosphorus onentrtion mong the four genotypes t either phosphorus level. Shoot phosphorus ontent ws greter in the effiient genotypes G19839 nd G2333 thn in the ineffiient genotypes G417 nd DOR364 (Fig. 2). VAM symiosis ws highly developed in ll genotypes, inresing over time from 29 DAP (53% infeted root length) to 43 DAP (75% infeted root length). No signifint differenes mong genotypes or etween phosphorus tretments were oserved for myorrhizl oloniztion (dt not shown). Numer of dventitious nd sl roots For this nd ll other trits nlysed the hypothesis of homogeneity of vrines etween lotions (phosphorus tretment) ws not rejeted (dt not shown), so oth experiments were pooled to investigte the intertion Aville phosphorus (mg kg 1 soil) Medium P Low P Soil depth (m) Fig. 1. Phosphorus vilility with depth in the field experiment, s ssyed y the iron-oxide-impregnted filter strip method t 43 DAP. Eh vlue is the men of 16 replites ± stndrd error.

5 Adventitious root prodution in response to low phosphorus vilility Funtionl Plnt Biology 977 Shoot P ontent (mg plnt 1 ) Medium P G2333 G19839 DOR364 G417 Low P Fig. 2. Shoot phosphorus ontent in four en genotypes 43 DAP in response to phosphorus vilility. Vlues re the mens of four replites ± stndrd error. Medium P dventitious Medium P sl etween phosphorus tretment nd genotypes. Adventitious roots were more numerous thn sl roots (Fig. 3). Numers of sl roots were reltively onstnt throughout the four-week smpling period. In ontrst to sl roots, numers of dventitious roots inresed over the four-week smpling period. At ll hrvest times n inrese in the numers of dventitious roots produed under low phosphorus ws oserved for the phosphorus-effiient G19839 (Fig. 3). A similr inrese ws oserved for phosphoruseffiient G2333 in dventitious root numers for 36, nd 43 DAP (Fig. 3). Phosphorus-ineffiient genotypes either filed to respond to low phosphorus with inresed numers of dventitious roots (G417) or only showed n inrese t 43 DAP (DOR364). Root system nlysis Root dimeter ws smllest for dventitious roots, slightly greter for sl roots, nd lrgest for tp roots (Tle 1). Low phosphorus vilility inresed tproot dimeter in genotypes G2333 nd DOR364 (Tle 1). Root-hir density ws omprle in ll genotypes t oth phosphorus levels Low P dventitious Low P sl Numer of roots G19839 G2333 G417 DOR Time (dys fter plnting) Fig. 3. Numer of dventitious nd sl roots over time in four en genotypes in response to phosphorus vilility. Vlues re the mens of four replites with two susmples ± stndrd error. Brs within hrvest dte with the sme letter re not signifintly different ording to ANOVA post ho test (P<.5).

6 978 Funtionl Plnt Biology C. R. Miller et l. Tle 1. Root dimeter (mm) for ontrsting en genotypes 43 DAP, s influened y phosphorus vilility Eh vlue is the men of four replites with two susmples ± stndrd error. For omprisons on intertions etween genotypes nd phosphorus tretment, mens followed y the sme supersript letter in eh olumn re not signifintly different ording to the SAS LSMEANS proedure (P.5). Phosphorus tretment: MP, medium phosphorus; LP, low phosphorus Genotype Phosphorus tretment Intt root Adventitious Bsl Tp G2333 MP.685 ± ±.4.65 ± ±.4 LP.78 ±.2.63 ± ± ±.5 G19839 MP.771 ± ± ± ±.4 LP.713 ±.2.64 ±.2.69 ±.3.81 ±.5 G417 MP.734 ± ± ± ±.7 LP.759 ± ±.3.69 ± ±.5 DOR364 MP.691 ± ±.3.65 ± ±.4 LP.81 ± ±..686 ± ±.11 Overll men.733 ± ± ± ±.1 (verge of 48 replites = 221 ± 5 hirs mm 2 root surfe re) with the exeption of dventitious roots of the phosphorus-effiient genotype G2333 t medium phosphorus, whih hd 281 ± 14 hirs mm 2 root surfe re (verge of four replites, two susmples per replite). Low phosphorus redued totl root system length in ll genotypes (Tle 2). The phosphorus-effiient genotypes G2333 nd G19839 hd greter nd sttistilly signifint (P.5) root length under oth medium nd low phosphorus thn the phosphorus-ineffiient genotypes. Phosphorus vilility hd differentil effets on distint root types in genotype-dependent mnner. Adventitious roots were less negtively ffeted y low phosphorus thn sl roots (see Tle 5). Root iomss responses to low phosphorus vried mong genotypes (Tle 3). Phosphorus-effiient genotypes hd greter dventitious root dry weights under oth medium nd low phosphorus thn phosphorus-ineffiient genotypes. Tle 2. Root length (m) for ontrsting en genotypes 43 DAP, s influened y phosphorus vilility Eh vlue is the men of four replites with two susmples ± stndrd error. For omprisons on intertions etween genotypes nd phosphorus tretment, mens followed y the sme supersript letter in eh olumn re not signifintly different ording to the SAS LSMEANS proedure (P.5). Phosphorus tretment: MP, medium phosphorus; LP, low phosphorus Genotype Phosphorus tretment Intt root Adventitious Bsl Tp G2333 MP ± ± ±.8 4. ±.8 LP ± ± ± ±.3 d G19839 MP 2.78 ± ± ± ±.9 LP ± ± ± ±.7 G417 MP ± ± ± ±.4 d LP 9.4 ± 1.3 d 2.59 ± ± ±.2 d DOR364 MP ± ± ± ±.5 LP ± 1.3 d 3.26 ± ± ±.4 d Tle 3. Root dry weight (g) of ontrsting en genotypes 43 DAP, s influened y phosphorus vilility Eh vlue is the men of four replites with two susmples ± stndrd error. For omprisons on intertions etween genotypes nd phosphorus tretment, mens followed y the sme supersript letter in eh olumn re not signifintly different ording to the SAS LSMEANS proedure (P.5). Phosphorus tretment: MP, medium phosphorus; LP, low phosphorus Genotype Phosphorus tretment Intt root Adventitious Bsl Tp G2333 MP.218 ±.2.51 ± ±.1.55 ±.1 LP.17 ±.1.49 ±.1.92 ±.1.29 ±.1 G19839 MP.276 ±.1.62 ±.1.14 ±.2.74 ±.2 LP.191 ±.2.37 ±.1.89 ±.1.66 ±.1 G417 MP.29 ±.2.26 ± ±.2.4 ±.1 LP.11 ±.1 d.21 ±.1.55 ±.1.34 ±.1 DOR364 MP.198 ±.1.23 ± ±.2.58 ±.1 LP.121 ±.1 d.26 ±.1.72 ±.1.26 ±.1

7 Adventitious root prodution in response to low phosphorus vilility Funtionl Plnt Biology 979 In ontrst, sl roots did not differ mong genotypes t either phosphorus level. G19839 hd signifintly more tproot under low phosphorus thn other genotypes. Adventitious roots were generlly less negtively ffeted y low phosphorus thn sl roots (Tle 5). There ws drmti effet of root type on speifi root length (SRL; see Tle 9). Adventitious roots hd the gretest speifi root length (117.2 ± 3.8 m g 1 dry weight), followed y sl roots (89. ± 2.4 m g 1 dry weight), then tp roots Constrution ost (g gluose equiv. g 1 DW) Nitrogen ontent (%) Medium P sl dventitious Low P Fig. 4. Root speifi onstrution osts (upper pnel) nd nitrogen ontent (lower pnel) t 43 DAP for dventitious nd sl roots of ommon en 43 DAP s influened y P vilility. Eh vlue is the men ± stndrd error of four replites, eh of whih hd four susmples. Brs with the sme letter re not signifintly different ording to ANOVA post ho test (P.5). (64.4 ± 2.9 m g 1 dry weight). The ineffiient genotypes hd greter dventitious SRL thn effiient genotypes irrespetive of phosphorus level (131.2 ± 5.3 m g 1 dry weight for ineffiient genotypes vs ± 4.1 m g 1 dry weight for effiient genotypes). Constrution osts of dventitious nd sl roots Genotypes did not differ in root speifi onstrution ost (SCC), so dt for genotypes were omined to exmine differenes mong root types nd phosphorus tretments (Fig. 4). Phosphorus vilility ws positively orrelted with SCC for oth dventitious nd sl roots. Adventitious roots hd lower SCC thn sl roots t oth medium nd low phosphorus. Root nitrogen ontent ws lso positively orrelted with soil phosphorus level ut ws not signifintly different mong root types (Fig. 4). By omining speifi onstrution ost with speifi root length, we lulted liner onstrution ost (LCC), whih provides n estimte of the metoli ost of root elongtion, whih is more losely relted to phosphorus quisition thn is root mss. Adventitious roots hd 42% lower LCC thn sl roots (Tle 4). The LCC of dventitious roots ws not ffeted y phosphorus vilility, ut ws signifintly lower in ineffiient genotypes thn in effiient genotypes (Tle 4). In ontrst, the LCC of sl roots ws not ffeted y genotype, ut ws signifintly lower under low phosphorus vilility (Tle 4). Root rnhing In generl, dventitious roots hd lower lterl root density (LRD) thn sl roots, nd low-phosphorus dventitious roots hd the lowest LRD in 15 of 16 ses (Fig. 5). Tle 4. Liner onstrution ost (mg gluose equivlent m 1 ) for ontrsting en genotypes 43 DAP, s influened y P vilility Eh vlue is the men ± stndrd error of the men of four replites with two susmples. For omprisons of intertions etween genotypes nd phosphorus tretment, mens followed y the sme letter in eh olumn re not signifintly different ording to the SAS LSMEANS proedure (P.5). Phosphorus tretment: MP, medium phosphorus; LP, low phosphorus Genotype Phosphorus tretment Adventitious Bsl G2333 MP 3.67 ± ±.28 LP 3.63 ± ±.6 G19839 MP 4.64 ± ±.48 LP 3.42 ± ±.2 Men of effiient genotypes 3.84 ± ±.24 G417 MP 3.5 ± ±.68 LP 2.89 ± ±.42 DOR364 MP 2.68 ± ±.34 LP 3.15 ± ±.7 Men of ineffiient genotypes 3.5 ± ±.35 Overll men 3.44 ± ±.21

8 98 Funtionl Plnt Biology C. R. Miller et l. Phosphorus-effiient genotypes hd greter LRD thn phosphorus-ineffiient genotypes over time (dt not shown). Low phosphorus inresed the distne from the root tip to the first lterl root (FLRD) in dventitious roots, ut in sl roots low phosphorus did not inrese FLRD exept for the ineffiient genotype DOR364 (Fig. 6). Adventitious roots hd greter FLRD thn sl roots (Fig. 6). Lterl root density (roots m 1 ) Medium P dventitious Medium P sl Low P dventitious Low P sl G2333 G19839 G417 DOR 364 Genotype Fig. 5. Lterl root density of dventitious nd sl roots of ommon en s ffeted y P vilility. Vlues re the men ± stndrd error of four replites over four weeks of smpling. Within genotype, rs with the sme letter re not signifintly different ording to ANOVA post ho test (P.5). Studies in ontrolled environments Results in snd ulture lrgely onfirmed the prinipl results from the field study (Tle 5). Genotypes vried in dventitious rooting in response to low phosphorus vilility (Tles 5, 6). As in the field study, G2333 nd G19839 hd more dventitious rooting in terms of oth root length nd root iomss t low phosphorus vilility thn DOR364 nd G417. Similr results were otined in nutrient solution (dt not shown in the interests of revity). Genotype survey Phosphorus stress inresed dventitious rooting in terms of root iomss nd perentge of totl root iomss in seven of eight ultivted genotypes (Tle 7) nd seven of eight wild genotypes (Tle 8). Wild Phseolus essions generlly produed more dventitious root iomss nd less sl root iomss thn ultivted genotypes. While sl root Distne to first lterl root (m) Medium P dventitious Medium P sl Low P dventitious Low P sl G2333 G19839 G417 DOR 364 Genotype Fig. 6. Distne from root tip to the first lterl root of dventitious nd sl roots of ommon en s ffeted y P vilility. Vlues re the men ± stndrd error of four replites over four weeks of smpling. Within genotype, rs with the sme letter re not signifintly different ording to ANOVA post ho test (P.5). Tle 5. Anlysis of vrine for root dry weight (RDW) nd root length (RL) for dventitious nd sl roots in ontrsting en genotypes growing in field onditions in Cost Ri (CR) or under greenhouse onditions (GH) in rtifiil sndy soil medi evluted in high- nd low-phosphorus onditions Numers in prenthesis re degree of freedom for GH experiment (split-plot design) when different thn field experiment (omined RCBD). * Indites signifine t P.5, ** indites signifine t P.1 Men squres Field (CR) Sndy soil (GH) Adventitious Bsl Adventitious Bsl Soure df RDW RL RDW RL RDW RL RDW RL Rep (R) Phosphorus tretment (P) * ** 13.8* 2398.** ** ** ** Rep (P) P R Genotype (G) 4 (3) 34.4** ** ** ** ** ** P G 4 (3) * 679.3* 444.9* ** Error 5 (24)

9 Adventitious root prodution in response to low phosphorus vilility Funtionl Plnt Biology 981 Tle 6. Root dry weight (RDW) nd root length (RL) for dventitious nd sl roots in ontrsting en genotypes growing in snd with lumin-p 21 dys fter sowing, s influened y phosphorus vilility Eh vlue is the men of four replites ± stndrd error. For omprisons on intertions etween genotypes nd phosphorus tretment, mens followed y the sme supersript letter in eh olumn re not signifintly different ording to the SAS LSMEANS proedure (P.5). Phosphorus tretment: HP, high phosphorus; LP, low phosphorus Adventitious Bsl Genotype Phosphorus tretment RDW (g) RL (m) RDW (g) RL (m) G2333 HP.32 ± ± ± ± 4.6 LP.114 ±.2 d 2.5 ± 3. d.174 ± ± 2. def G19839 HP.368 ± ± ± ± 12.4 LP.17 ± ± 4. d.291 ± ± 6.3 d G19833 HP.356 ± ± ± ± 11.7 LP.137 ±.1 d ±.3 d.175 ± ± 4.1 def G417 HP.93 ±.1 d ± 4.4 d.2 ± ± 2.8 de LP.77 ±.2 d ± 4.4 d.19 ± ± 4.8 f DOR364 HP.23 ± ± ± ± 7.7 def LP.68 ±.1 d ± 1.9 d.138 ± ± 3.7 ef prodution inresed under phosphorus stress for ultivted genotypes, phosphorus stress generlly redued prodution of sl roots in wild essions. Disussion In this study we oserved tht in ommon en, phosphorus influenes dventitious rooting s reltive proportion of totl root iomss or totl root length, nd tht phosphoruseffiient en genotypes hve superior dventitious rooting under phosphorus stress. This is novel oservtion tht supports the hypothesis tht topsoil forging is omponent of effiient phosphorus quisition. This phenomenon is nlogous to the regultion of sl root grvitropism y phosphorus vilility, whih lso serves to inrese Tle 7. Adventitious rooting of ultivted en genotypes in response to phosphorus vilility t 14 nd 15 DAP in ontrolled environment Eh vlue is the men of 1 replites ± stndrd error.,, Signifint t P.5,.1, nd.1, respetively; ns, not signifint Adventitious (mg) Bsl (mg) Tp (mg) Adventitious (% of totl) + P BAT ± ± ± ± 1.1 BAT ± ± 8 8 ± ± 1.1 DOR ± ± 5 69 ± ± 2.7 G ± ± ± ±.6 G ± ± ± ±.4 G ± ± ± ±.4 G ±.7 58 ± 7 89 ± ±.6 G ± ± 9 93 ± ±.7 P BAT ± ± 8 41 ± ± 1.3 BAT ± ± 7 41 ± ±.9 DOR ± ± 9 22 ± ± 1.3 G ± ± ± ± 1. G ± ± 11 4 ± ±.5 G ± ± ± ±.9 G ± ± 7 32 ± ± 1.1 G ±.9 93 ± 9 67 ± ±.7 F from ANOVA P 2.21** 27.96***.94 ns 8.91* genotype 3.69** 35.3*** 1.63 ns 9.35*** genotype P 1.27 ns.58 ns 2.18* 1.4 ns

10 982 Funtionl Plnt Biology C. R. Miller et l. topsoil forging in effiient en genotypes (Bonser et l. 1996). Adventitious roots hve severl dvntges over sl roots in terms of phosphorus quisition. The first is the ololiztion of root forging nd phosphorus vilility. The origin of dventitious roots from the hypootyl omined with their horizontl growth hit ples them in the topsoil, wheres sl roots of most genotypes grow downwrd t some ngle tht eventully extends them into the susoil. In soils where phosphorus vilility delines with depth, dventitious roots should ssist phosphorus quisition y enhning plnt forging in the most phosphorus-rih soil environment. A seond potentil enefit is tht dventitious roots disperse more rpidly from the plnt thn sl roots, therey reduing inter-root ompetition for phosphorus. The vertil omponent of the growth vetor of sl roots rings them loser together, therey inresing inter-root ompetition. Modeling studies show tht shllow sl roots re more effiient thn deeper roots in soils with uniform phosphorus vilility, euse of this dispersion (Ge et l. 2). In this regrd dventitious roots re even more dispersed thn shllow sl roots euse they originte over the entire suterrnen length of the hypootyl, wheres sl roots originte in nrrow region where the tp root joins the hypootyl. A third potentil enefit, s shown in this study, is tht dventitious roots hve signifintly lower metoli onstrution ost per unit iomss thn sl roots (F for root type effet = 16.8, signifint t P<.1), despite similr N ontent (Fig. 4). This differene ws greter when metoli osts were expressed per unit of root length dventitious roots were, on verge, 42% less ostly thn sl roots per unit of root length (Tle 4). Sine phosphorus forging is primrily funtion of explortion of new soil domins undepleted of phosphorus (Silerush nd Brer 1984), nd root ron osts re signifint onstrint to en growth under phosphorus stress (Nielsen et l. 1998, 21), this ftor ould sustntilly improve phosphorus effiieny. A fourth potentil enefit, s shown here, is tht dventitious roots hve more dispersed rnhes thn sl roots. This is evident oth in lterl root density, whih is signifintly lower in dventitious roots thn in sl roots, espeilly under phosphorus stress (Fig. 5), s well s the distne from the root tip to the first lterl root, whih is lso greter in dventitious roots thn in sl roots, espeilly under phosphorus stress (Fig. 6). The dispersion of lterl rnhing on dventitious roots would inrese the volume of soil explored per unit of metoli investment in root growth. Our results re onsistent with report of Tle 8. Adventitious rooting of wild en genotypes in response to phosphorus vilility t 14 DAP in ontrolled environment Eh vlue is the men of five replites ± stndrd error. *, **, *** Signifint t P.5,.1, nd.1, respetively; ns, not signifint Adventitious (mg) Bsl (mg) Tp (mg) Adventitious (% of totl) + P PI ± ± 4 18 ± ± 2.2 PI ±.9 93 ± 6 36 ± ±.6 PI ± ± 8 34 ± ± 2.3 PI ± ± 7 4 ± ± 1.5 PI ± ± 9 35 ± ± 3.5 PI ± ± 4 28 ± ± 4. PI ±.9 49 ± 9 34 ± ± 1.3 PI ± ± 5 38 ± ± 1.9 P PI ±.8 11 ± 2 24 ± ± 1.5 PI ± ± 5 37 ± ± 2.4 PI ± ± 5 56 ± ± 2. PI ± ± 5 53 ± ± 2.3 PI ± ± 5 44 ± ± 1.5 PI ± ± 5 37 ± ± 2. PI ± ± 4 47 ± ± 2.6 PI ± ± 5 41 ± ± 2.8 F from ANOVA P 8.74* 4.21 ns 17.14** 7.4* genotype 1.77 ns 1.3*** 5.78* 9.5*** genotype P 1.5 ns 2.68*.74 ns 1.2 ns

11 Adventitious root prodution in response to low phosphorus vilility Funtionl Plnt Biology 983 Tle. 9 Speifi root length (SRL, m g 1 ) for ontrsting en genotypes 43 DAP, s influened y P vilility Eh vlue is the men ± stndrd error of the men of four replites with two susmples. For omprisons on intertions etween genotypes nd phosphorus tretment, mens followed y the sme letter in eh olumn re not signifintly different ording to LSMEANS SAS proedure (P.5). Phosphorus tretment: MP, medium phosphorus; LP, low phosphorus Genotype Phosphorus tretment Intt root Adventitious Bsl Tp G2333 MP ± ± ± ± 9.6 LP ± ± ± ± 7.5 G19839 MP ± ± ± 6.8 d ± 3.3 LP ± ± ± ± 5.6 Men of effiient genotypes ± ± ± 4. G417 MP ± ± ± 6.5 d ± 9.8 LP ± ± ± ± 7.2 DOR364 MP ± ± ± 3.4 d ± 6.7 LP 1.14 ± ± ± ± 7.6 Men of ineffiient genotypes ± ± ± 3.8 deresed lterl root density in phosphorus-stressed en, n effet medited y ethylene (Borh et l. 1999). Given the importne of root hirs in phosphorusquisition effiieny (Ghooni nd Nielsen 1998; Btes nd Lynh 2), it is intriguing tht we oserved greter root hir density on the dventitious roots of phosphoruseffiient genotype. Sine this ws only oserved in one genotype t one phosphorus level, it is premture to generlize tht dventitious roots hve more root hirs, ut this ould e n dditionl enefit for phosphorus quisition. Topsoil forging y dventitious roots my hve temporl s well s sptil enefits for plnt phosphorus eonomy. Between 29 nd 43 DAP the verge length of sl roots did not inrese signifintly in three of the four genotypes (DOR364 hd moderte growth) under phosphorus stress. Bsl roots grew modertely etween 29 nd 43 DAP in three of the four genotypes under medium phosphorus (phosphorus-effiient G19839 ws the exeption). Therefore, if the sl root system is to ontinue growth nd explortion of low-phosphorus soil during lte vegettive growth, this must e omplished y lterl rnhing. While the length of sl root lterls ws not mesured throughout the experiment, this did not inrese over time. Lterl root density did inrese slightly during the experiment, ut not signifintly. Therefore, it ppers tht during vegettive development, root growth shifts from the sl (nd perhps tp) root system to dventitious roots. A numer of environmentl ftors influene dventitious rooting in the field, notly soil moisture nd ertion ner the hypootyls. For this reson we onfirmed field results in oth nutrient solution nd snd ulture in ontrolled environments. Results in ontrolled onditions supported our field results, inditing tht other ioti nd ioti ftors do not override the regultion of dventitious rooting y phosphorus vilility nd genotype. We ntiipted tht the response of dventitious roots to phosphorus vilility might e more pronouned in ultivted genotypes, whih unlike their wild nestors, hve een seleted under onditions in whih they re plnted in the soil t some depth, wheres wild seeds might germinte very ner the soil surfe, nd so my not develop lrge dventitious root systems. Contrry to these expettions, wild genotypes were generlly more responsive thn ultivted genotypes (Tles 7, 8). The tendeny of wild ens to form dventitious roots my represent n dpttion to the vining growth hit of wild ens, sine vining shoots might ontt the soil t vrious lotions. We hve shown tht dventitious roots hve severl ttriutes tht my enhne phosphorus-quisition effiieny in en, nd hve shown tht phosphorus vilility inreses dventitious rooting in phosphorus-effiient genotypes. These results suggest tht dventitious rooting my e useful trit for the geneti enhnement of rop germplsm for low-fertility soils. Aknowledgments We thnk Mr Arturo Sorio for ssisting in prepring the field plots. This reserh ws supported y USDA/NRI grnts to JPL nd to JPL nd Kthleen Brown, nd The Dnish Counil for Development Reserh grnt 9819 to KLN. Referenes Anderson G (198) Assessing orgni phosphorus in soils. In The role of phosphorus in griulture. (Eds FE Khswneh, EC Smple nd EJ Kmprth) pp (Amerin Soiety of Agronomy, Crop Siene Soiety of Ameri, Soil Siene Soiety of Ameri: Mdison, WI) Btes TR, Lynh JP (2) Plnt growth nd phosphorus umultion of wild type nd two root hir mutnts of Aridopsis thlin (Brssiee). Amerin Journl of Botny 87, Beee S, Lynh J, Glwey N, Tohme J, Oho I (1997) A geogrphil pproh to identify phosphorus-effiient genotypes mong lndres nd wild nestors of ommon en. Euphyti 95,

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