Aluminium alleviates manganese toxicity to rice by decreasing root symplastic Mn uptake and reducing availability to shoots of Mn stored in roots

Transcription

1 Annls of Botny 116: , 215 oi:1.193/o/mv9, vill onlin t Aluminium llvits mngns toxiity to ri y rsing root symplsti Mn uptk n ruing vilility to shoots of Mn stor in roots Wi Wng 1,2, Xu Qing Zho 1, Zhn Min Hu 1,2, Ji ng Sho 1,2, Jing Ch 1,2, Rong Fu Chn 1, Xio Ying Dong 1 n Rn Fng Shn 1, 1 Stt y Lortory of Soil n Sustinl Agriultur, Institut of Soil Sin, Chins Amy of Sins, Nnjing 218, Chin n 2 Univrsity of Chins Amy of Sins, Bijing 149, Chin For orrsponn. E-mil: rfshn@isss..n Riv: 18 Mrh 215 Rturn for rvision: 17 April 215 Apt: 5 My 215 ulish ltronilly: 22 Jun 215 Bkgroun n Aims Mngns (Mn) n luminium (Al) phytotoxiitis our minly in i soils. In som plnt spis, Al llvits Mn toxiity, ut th mhnisms unrlying this fft r osur. Mthos Ri (ryz stiv) slings (11 ol) wr grown in nutrint solution ontining iffrnt onntrtions of Mn 2þ n Al 3þ in short-trm (24 h) n long-trm (3 wks) trtmnts. Msurmnts wr tkn of root symplsti sp, root Mn plqus, ll mmrn ltril surf potntil n Mn tivity, root morphology n plnt growth. y Rsults In th 3-wk trtmnt, ition of Al rsult in inrs root n shoot ry wight for plnts unr toxi lvls of Mn. This ws ssoit with rs Mn onntrtion in th shoots n inrs Mn onntrtion in th roots. In th 24-h trtmnt, ition of Al rsult in rs Mn umultion in th root symplsts n in th shoots. This ws ttriut to highr ll mmrn surf ltril potntil n lowr Mn 2þ tivity t th ll mmrn surf. Th inrs Mn umultion in roots from th 3-wk trtmnt ws ttriut to th formtion of Mn plqus, whih wr proly rlt to th Al-inu inrs in root rnhym. Conlusions Th rsults show tht Al llvit Mn toxiity in ri, n this oul ttriut to rs shoot Mn umultion rsulting from n Al-inu rs in root symplsti Mn uptk. Th rs in root symplsti Mn uptk rsult from n Al-inu hng in ll mmrn potntil. In ition, Al inrs Mn plqus in th roots n hng th ining proprtis of th ll wll, rsulting in umultion of nonvill Mn in roots. y wors: Aluminium, mngns toxiity, ri, ryz stiv, Gouy Chpmn Strn mol, mngns plqus, plsm mmrn surf ltril potntil, root rnhym, root symplst, i soils. INTRDUCTIN Mngns (Mn) is n ssntil lmnt for plnts n is involv in rgulting svrl mtoli prosss, suh s photosynthsis n ntioxint tivity (Mrshnr, 1995). Nvrthlss, n xss of Mn is toxi for most plnts (Milllo t l., 21). Mngns toxiity is on of th mjor ftors rstriting plnt growth on poorly rin n ii soils with high onntrtions of rily vill Mn 2þ (Mrshnr, 1995). In plnts, Mn tns to umult prominntly in shoots rthr thn roots (Lion, 21; g n llr, 25; g t l., 26). Thrfor, in ition to rs growth rt, symptoms of Mn toxiity, suh s hlorosis n nroti rown spots, r ommonly visil on lvs (Moroni t l., 23; Ross t l., 27; Mor t l., 29; Führs t l., 21; Li t l., 211). Th thrshol of Mn toxiity n tolrn vris mong iffrnt plnt spis n mong ultivrs n gnotyps within spis (Foy t l., 1988; Horst, 1988). In gnrl, ri (ryz stiv) plnts r onsir to tolrnt to Mn toxiity (Lion, 21). Compr with othr grsss, ri plnts n umult 5 1 tims mor Mn in th lvs n show milr symptoms of toxiity (Foy t l., 1978). Although Mn toxiity rrly ours in floo or py ri, this is not th s for upln ri grown on roi soils. Ai upln soils my ontin toxi lvls of oth Mn n luminium (Al) (Nlson, 1983), n s onsqun Al n Mn toxiitis my limit th yils of upln ri (onnmprum, 1975). Aluminium toxiity is gnrlly onsir to th primry limiting ftor for plnt growth in i soils (ohin t l., 24). Low onntrtions (<1 mm) of Al n inhiit root longtion n us struturl n funtionl mg to roots (ohin, 1995). Mny plnts promintly umult Al in th roots, whil som plnts, suh s ukwht (Fgopyrum sulntum) (M t l., 1998), t (Cmlli spp.) (Chnry, 1955; Zng t l., 213) n plnts in th Mlstomt (.g. Mlstom mlthrium) (Wtn t l., 25), umult high onntrtions of Al in ithr roots or lvs. High onntrtions of Al n Mn n o-our in soils, rstriting rop proution in i n high-mn soils. In prvious stuis, Austrlin soils wr lssifi into svn tgoris ovring th rng of Al n Mn omintions (Dolling t l., 21; Yng t l., 29). Most griulturl soils wr Downlo from y gust on 9 Jnury 218 VC Th Author 215. ulish y xfor Univrsity rss on hlf of th Annls of Botny Compny. All rights rsrv. For rmissions, pls mil: journls.prmissions@oup.om

2 238 Wng t l. Aluminium llvits mngns toxiity y ruing its vilility signt s ss 2, with low onntrtion of vill Al n high Mn onntrtion. Hvy rinfll n hng th onntrtions of vill Al n Mn, n onsquntly, hng ss 2 soils to ss 3, with mort Al n vry high Mn onntrtions (Yng t l., 29). Although high onntrtions of Mn n Al n o-our in i soil, th tolrns of plnts/ultivrs to Al n Mn o not nssrily oini (Nlson, 1983). This riss qustions s to how plnts pt to oth Mn n Al toxiity n how Mn intrts with Al in plnts. rvious rports hv init tht Al n ru Mn umultion in rly (Horum vulgr), triplx (Atriplx hstt) (Rs n Sirk, 1961), orn (Z mys) (rk, 1977), wht (Tritium stivum) (Blir n Tylor, 1997), owp (Vign unguiult) (Tylor t l., 1998) n soyn (Glyin mx)(yng t l., 29). Allvition of th ffts of Al on Mn toxiity hs n rport for triplx (Rs n Sirk, 1961) n soyn (Yng t l., 29), n os-pnnt fft of Al on Mn toxiity hs n monstrt in wht n owp (Blir n Tylor, 1997; Tylor t l., 1998). n xplntion for this llvition is tht Al my hv n ntgonisti fft on Mn uptk y plnt roots. Howvr, th xt mhnism is still poorly unrstoo. Th rsults of opittk t l. (211), s on th Gouy Chpmn Strn (GCS) mol, rvl tht th ffts of tions on Mn nutrition wr rlt to th ltril potntil (w o ) of th root ll mmrn (CM) surf. In thir stuy, whn owp ws ultur in solutions ontining iffrnt Mn onntrtions, th root tissu Mn onntrtion ws losly rlt to th tivity of Mn 2þ t th outr surf of th CM. This rsult provi nw lus out th intrtion twn Al n Mn. Th GCS mol omins ltrostti thory (Gouy Chpmn thory) with n ion-ining mol (Strn mol) to lult w o. This oul usful n powrful tool to xplin th Mn Al intrtion in plnts. To ttr unrstn th ffts of Al on Mn toxiity, w xplor how Al fft Mn toxiity using Al- n Mn-tolrnt ri plnts. ur rsults iffr from thos rport prviously; in prtiulr, th ffts of Al on root Mn umultion in ri ompltly ontrst with thos in othr plnt spis. MATERIALS AND METHDS lnt mtril n growth onitions W us Wuyunjing7, n Al-tolrnt ultivr of ri (ryz stiv vr. jponi) (Zho t l., 29) in ths xprimnts. Th ri ss wr surf-striliz in 1 % (v:v) soium hypohlorit solution for 3 min, wsh thoroughly with ioniz wtr, n thn sok in ioniz wtr ovrnight. Th ss wr grmint in n inutor t 3 Cfor24h, n thn trnsfrr to nylon nt floting on 65-L plsti pot ontin 5mM C 2 (ph 45). Th solution ws rnw vry 2. Aftr 3 ys of growth, th slings wr trnsfrr to light onitions n grown in 5mM C 2 (ph 45) for furthr 2. Thn, uniform slings (5 ol) wr trnsfrr to 15 L of hlf-strngth imur B nutrint solution (ph 45) n grown for 6. Th hlf-strngth imur B nutrint solution ontin 18 mm (NH 4 ) 2 S 4, 27 mm MgS 4 7H 2, 9 mm N 3, 18 mm C (N 3 ) 2 4H 2, 9 mm H 2 4,67 mm Mn 2 4H 2, 94 mm H 3 B 3,1 mm (NH 4 ) 6 Mo H 2, 15 mm ZnS 4 7H 2, 16 mm CuS 4 5H 2 n1mm EDTA-. Th solution ws rnw vry 2 without rtion. Finlly, 11--ol slings wr us in xprimnts with iffrnt Al n Mn trtmnts. Th slings wr grown in growth room unr 14:1-h light:- rk photoprio with : C, y:night tmprturs. Th rltiv humiity ws ontroll t %nth light lvl ws ontroll t 375 mmol photon m 2 s 1. For ll Al Mn intrtion xprimnts, four slings wr grown in h 15 L of nutrint solution. To intify suitl Al onntrtion for xprimnts on th Al Mn intrtion, 11- -ol slings wr grown in hlf-strngth imur B nutrint solutions ontining, 2, 5 or 1 mm Al for 3 wks. Bs on th rsults of this prliminry xprimnt, 2 mm Al ws hosn for furthr xprimnts. For th Al Mn intrtion xprimnts, 11--ol slings wr grown in hlf-strngth imur B nutrint solution ontining 67, 2, 5 or 1 mm Mn in th prsn or sn of 2 mm Al for 24 h (short trm) or 3 wks (long trm). Ths solutions wr rnw vry 2 without rtion. At th n of th long-trm xprimnts, w msur ri growth, Mn onntrtion, root Mn plqus, root rnhym, root imtr n root volum. At th n of th short-trm xprimnts, w trmin Mn onntrtions in th root symplst, whol roots n whol shoots. Th Al n Mn wr suppli s Al 3 n Mn 2,rsptivly. Th initil ph of h solution ws just to 45 using 1 M NH or H. All xprimnts wr onut with thr rplits, n h xprimnt ws prform t lst twi. Extrtion of root symplsti sp Th roots wr wsh with ioniz wtr n gntly lott ry, n thn ut into pis. Th pis wr pl in n i-ool filtr unit. Th root symplsti sp ws ollt y ntrifugtion oring to lug t l. (21) n Xi t l. (21). Brifly, roots tht wr frshly ut from th shoot s of four slings wr pl in filtr unit with por siz of 45 mm (Ultrfr-MC, Millipor, Billri, MA, USA). Th nutrint solution hring to th surf of th roots ws rmov y ntrifugtion t 6 g for 2 min. Apoplsti solutions wr rmov y ntrifugtion t 3 g for 15 min t 4 C. Th roots wr thn frozn t 8 C ovrnight. Th root symplsti sp solution ws otin y thwing th smpls t room tmprtur n thn ntrifuging thm t 21 6g for 1 min. Th rsius (i.. whol root) wr wsh thr tims with ioniz wtr n thn ovn-ri t 75 Ctoonstnt wight. Th Mn onntrtions in root symplsti sp n whol roots wr trmin y inutivly oupl plsm mss sptromtry (IC-MS) using n Agilnt 77x instrumnt (Agilnt, Snt r, CA, USA), s sri low. Extrtion n osrvtion of root Mn plqus Roots wr wsh twi with ioniz wtr to rmov ny xtrnous pripitt, n thn ut from h plnt t th shoot s. Root Mn plqus on th root surfs wr xtrt using th ithionit itrt iront (DCB) thniqu (Tylor n Crowr, 1983; Crowr n Coltmn, 1993), Downlo from y gust on 9 Jnury 218

3 Wng t l. Aluminium llvits mngns toxiity y ruing its vilility 239 whih ws moifi s follows: ll root sgmnts from h plnt wr gitt in solution ontining 4 ml of 3 M N 2 C 6 H 5 7 3H 2 n5mlof1m NHC 3 for 3 h. At th strt of th xprimnt, 3 g of N 2 S 2 4 ws to th solution. Th xtrtnt ws thn nt into 1-mL volumtri flsk n th roots wr wsh twi with ioniz wtr, whih ws lso nt into th 1-mL volumtri flsk. Th rsulting solution ws m up to 1 ml with ioniz wtr. W prpr DCB ontrol solutions without roots s sri ov, ut sustituting n quimolr onntrtion of N 2 S 3 for N 2 S 2 4 (with rspt to N). Aftr roots h n xtrt, thy wr ovn-ri t 75 C to onstnt wight. Th ry msss of roots n shoots wr msur n ror. Th Mn onntrtions of Mn plqus wr trmin y inutivly oupl plsm tomi mission sptrosopy (IC-AES) (ptim 8, rkin Elmr) ftr pproprit ilution. Th Mn onntrtions of roots ftr xtrtion wr trmin s sri low. To tt Mn plqus y mirosopy, roots wr hrvst ftr trtmnts n ut into 5-mm pis, n thn quikfrozn n frz-ri. Aftr oting with gol, root sgmnts wr osrv unr snning ltron mirosop (SEM- 3N, Hithi, Tokyo, Jpn) n nlys y nrgyisprsiv X-ry sptromtry (EDX) (EDX-25, Hori, yoto, Jpn). srvtion of rnhym n msurmnt of root imtr n volum Nwly form vntitious roots, 3 mm long, wr ollt ftr th long-trm xprimnt. Frsh smpls wr ut into 5-mm pis using rzor l to osrv rnhym y SEM using th mtho of Dvi n lg (2). Thn, th root smpls wr viw n photogrph unr n SEM (3N, Hithi). To msur root imtr n volum, whol roots wr wsh with ioniz wtr n thn snn using root snnr (rftion V7 hoto, Siko Epson, Ngno, Jpn). Root imtr n volum wr lult using th WinRHIZ nlysis systm (Rgnt, Qu, Cn). Computtion of CM surf ltril potntil n Mn tivity t CM surf Vlus for th CM surf ltril potntil (w o ) n Mn 2þ tivity t th CM surf ({Mn 2þ } o ) wr lult using omputr progrm for th GCS mol (inri t l., 1998; opittk t l., 214). This omputr progrm ws kin gift from rofssor T. B. inri n Dr ng Wng. Brifly, th GCS mol onsists of th Gouy Chpmn portion n th Strn portion. Th Gouy Chpmn portion of th mol rfrs to ltrostti thory prtining to hrg surfs in ontt with solutions. As rgrs th Strn portion, th CM ws onsir to ontin ngtivly hrg (R ) n nutrl ( )sits to whih ions (I Z ) my in. A onvrging lultion twn th Gouy Chpmn n Strn portions llows th lultion of w o (inri n Wng, 21; Ttulin, 1999). Th w o vlus lult from th GCS mol llow th lultion of ion I Z t th CM surf ({I Z } o ) y th Nrnst qution ({I Z } o ¼ {I Z } xp[ ZFw o /(RT)]), whr F, R n T r th Fry onstnt, th gs onstnt n tmprtur, rsptivly (Wng t l., 211). For tils of th lultion of w o, rfr to inri (1994) n inri t l.(1998). Ri hrvst n Mn nlysis At th n of th long-trm xprimnt, th roots wr rins rifly with ioniz wtr n lott ry. Th roots n shoots wr thn hrvst sprtly. Th longst root n th longst shoot on h sling wr msur with rulr. Th roots n shoots wr ovn-ri t 75 C to onstnt wight n thn wigh n groun. Thn, groun smpls (1 5 mg) wr igst with 5 ml of 5:1 (v:v) onntrt nitri i:prhlori i. Aftr omplt igstion, th smpls wr ilut to 5 ml using ioniz wtr n th Mn onntrtions wr trmin y IC-AES (ptim 8, rkin Elmr). At th n of th short-trm xprimnt, roots wr hrvst n thir symplsti sp ws ollt, n thn th roots wr igst in 5 ml of ultr-tr onntrt nitri i. Aftr pproprit ilution, th Mn onntrtions in th smpls wr trmin y IC-MS using n Agilnt 77x instrumnt. Sttistil nlysis All t wr sujt to on-wy nlysis of vrin ftr tsting for normlity n homognity. Multipl omprisons mong trtmnts wr prform using Tuky s multipl rng tst t th 5 proility lvl. All ths nlyss wr prform using SAS 9.1 softwr (SAS Institut In., Cry, NC, USA). Ri growth RESULTS W onut prliminry Al toxiity xprimnt to intify suitl Al onntrtion for rsrh on th Mn Al intrtion. As shown in Supplmntry Dt Tl S1, thr ws no signifint iffrn in th root ry wights mong ll Al onntrtions, ut Al signifintly inhiit root longtion t trtmnt onntrtions of 5 n 1 mm Al ut not with th trtmnt onntrtion of 2 mm Al. Th shoot ry wight n shoot hight wr lowr in plnts trt with 1 mm Al thn in ontrol plnts ( mm Al), n shoot ry wight ws signifintly rs y 5 mm Al. Howvr, 2 mm Al h littl fft on shoot hight n shoot ry wight. Thrfor, 2 mm Al (188 mm {A1 3þ } o ) ws us in th following Mn Al intrtion xprimnts. In th sn of Al, 5 n 1 mm Mn signifintly rs root lngth rltiv to tht of plnts grown with 67 mm Mn (Fig. 1A). Howvr, thr ws no signifint iffrn in root lngth mong ll Mn onntrtions in th prsn of Al (Fig. 1A). Shoot hight n ry wights of roots n shoots signifintly rs with inrsing Mn onntrtions in th sn of Al (Fig. 1B D). Howvr, ition of Al mrkly llvit this inhiition, us ll of ths prmtrs wr inrs y Al ition t 5 n 1 mm Mn (Fig. 1B D). Downlo from y gust on 9 Jnury 218

4 24 Wng t l. Aluminium llvits mngns toxiity y ruing its vilility Root lngth (m plnt 1 ) A Control Ths rsults init tht Al ition llvit th toxi ffts of Mn on ri growth. Th photogrphs of th slings in th vrious trtmnts illustrt th llviting ffts of Al on Mn toxiity (Fig. 1E, F). Ri growth ws inhiit y high Mn onntrtions n ol lvs wr nrly wilting (Fig. 1E), ut ition of Al mrkly improv growth of ri n th ol lvs t 5 n 1 mm Mn (Fig. 1F). Shoot hight (m plnt 1 ) Root ry wight (mg plnt 1 ) Shoot ry wight (mg plnt 1 ) B C D f Ri Mn umultion Aftr th long-trm Al Mn trtmnts (3 wks), oth root n shoot Mn umultion signifintly inrs with inrsing Mn onntrtions, rgrlss of whthr Al ws prsnt or not (Fig. 2A, B). At 67 mm Mn, ition of Al i not signifintly fft Mn umultion in roots n shoots (Fig. 2A, B). At 2, 5 n 1 mm Mn, Al signifintly rs Mn umultion in shoots(fig. 2B) ut inrs Mn umultion in roots (Fig. 2A). Aftr th short-trm Al Mn trtmnts (24 h), Mn onntrtion in th root symplst, whol roots n whol shoots lso grully inrs with inrsing Mn in ll trtmnts (Fig. 3A C). At 67 mm Mn, ition of Al i not signifintly fft th Mn onntrtions in th root symplst (Fig. 3A), whol roots (Fig. 3B) or whol shoots (Fig. 3C). At 2, 5 n 1 mm Mn, ition of Al signifintly ru th Mn onntrtion in th root symplst (Fig. 3A) n whol shoots (Fig. 3C), onsistnt with th rsults of th long-trm xprimnt (Fig. 2B). In roots, th Mn onntrtions wr signifintly ru y ition of Al t 1 mm Mn, ut not t 2 or 5 mm Mn (Fig. 3B). Ths rsults suggst tht Al inhiit Mn uptk into th root symplst, rsulting in lss Mn ing trnsport to shoots. E Mn on. in ultur solution (µm) F Cll mmrn surf ltril potntil n Mn tivity in th ulk solution n t th ll mmrn surf At th sm Al onntrtion, th vlu of w o grully m mor positiv with inrsing Mn onntrtion (Tl 1). Aition of Al rstilly hng th w o vlus from ngtiv to positiv (Tl 1). Th vlus of {Mn 2þ } o n Mn 2þ tivity in ulk solution ({Mn 2þ } ) inrs with inrsing Mn supply, whil ition of Al rstilly rs {Mn 2þ } o ut rly fft {Mn 2þ } (Tl 1). Mn (µm) (µm) FIG. 1. Effts of Al on root lngth (A), shoot hight (B), root ry wight (C) n shoot ry wight (D) of ri unr Mn toxiity in long-trm xprimnt. (E) Toxiity symptoms of ri unr Mn toxiity. (F) Amliorting fft of Al on ri Mn toxiity. Ri slings (11 ol) wr grown in nutrint solution with 67, 2, 5 or 1 mm Mn in th prsn or sn of 2 mm Al for 3 wks. Vlus r mn 6 s.. (n ¼ 3 rplits, h omprising four slings). Diffrnt lttrs in A D show signifint iffrns mong trtmnts ( < 5; Tuky s tst). Root Mn plqus W tt root Mn plqus using EDX. Th EDX sptr show tht Mn signls h two pks, on roun 5 kv n th othr is roun 6 kv (Fig. 4F H). Sin th first on oul not sily iffrntit from othr lmnt pks, th son pk ws usully us for Mn nlysis (Btty t l., 22). This pk ws snt from ll Mn trtmnts in th sn of Al (Fig. 4A D) n from th 67 mm Mn trtmnt in th prsn of Al (Fig. 4E). Howvr, Mn signls intnsifi whn Al ws to solutions ontining 2, 5 or 1 mm Mn (Fig. 4F H). Th Mn plqus in roots wr quntifi using th DCB xtrtion thniqu. Th Mn onntrtion in th DCB xtrt from roots n in roots ftr DCB xtrtion Downlo from y gust on 9 Jnury 218

5 Wng t l. Aluminium llvits mngns toxiity y ruing its vilility A Control 25 A 2 Control Root Mn on. (µg g 1. wt) Shoot Mn on. (µg g 1. wt) B f f Mn on. in root symplst (mg L 1 ) Mn on. in whol root (µg g 1. wt) Mn on. in whol shoot (µg g 1. wt) f B C f Mn on. in ultur solution (µm) FIG. 2. Effts of Al on Mn umultion in roots (A) n shoots (B) of ri unr Mn toxiity in long-trm xprimnt. Ri slings (11 ol) wr grown in nutrint solution with 67, 2, 5 or 1 mm Mn in th prsn or sn of 2 mm Al for 3 wks. Vlus r mn 6 s.. (n ¼ 3 rplits, h omprising four slings). Diffrnt lttrs show signifint iffrns mong trtmnts ( < 5; Tuky s tst) Mn on. in ultur solution (µm) FIG. 3. Conntrtions of Mn in root symplst (A), whol root (B) n whol shoot (C) ftr short-trm trtmnt. Ri slings (11 ol) wr grown in nutrint solution with 67, 2, 5 or 1 mm Mn in th prsn or sn of 2 mm Al for 24 h. Vlus r mn 6 s.. (n ¼ 3 rplits, h omprising four slings). Diffrnt lttrs show signifint iffrns mong trtmnts ( < 5, Tuky s tst). TABLE 1. ult surf ltril potntil t outr surf of th plsm mmrn n Mn tivity in ulk solution n t th ll mmrn surf Al (mm) Mn(mM) w o (mv) {Mn 2þ } (mm) {Mn 2þ } o (mm) w o, surf ltril potntil t outr surf of plsm mmrn; {Mn 2þ },Mntivityinulksolution;{Mn 2þ } o,mntivity t ll mmrn surf. All ph vlus wr just to 45 t th strt of xprimnts. All solutions wr 1/2 imur B nutrint solution ontining vrious onntrtions of Al n Mn. Downlo from y gust on 9 Jnury 218

6 242 Wng t l. Aluminium llvits mngns toxiity y ruing its vilility A B C D Cu N Zn Cu Zn Zn Cu Cu Zn Cu Zn Zn Zn Zn Zn Cu Cu Cu Cu kv kv kv kv Y-xis full sl 561 ounts Y-xis full sl 576 ounts Y-xis full sl 72 ounts Y-xis full sl 928 ounts E F G H Mn Mn kv kv kv kv Y-xis full sl 135 ounts Y-xis full sl 1972 ounts Y-xis full sl 2312 ounts Y-xis full sl 172 ounts FIG. 4. Enrgy-isprsiv X-ry nlysis (EDX) of pripitts form in roots of ri in hyroponi ultur supplmnt with Mn n Al or Mn only in longtrm xprimnt. Th y-xis shows th ounts, whih rfr to th numr of X-rys riv n pross y th ttor. Th ounts priniplly rprsnt th intnsity of th lmnts in th surf of root ll. Ri slings (11 ol) wr grown in nutrint solution with 67 (A, E), 2 (B, F), 5 (C, G) or 1 (D, H) mm Mn in th sn (A D) or prsn (E H) of 2 mm Al for 3 wks. Mn Mn Mn N Mn grully inrs with inrsing Mn supply (Fig. 5A, B). Aition of Al signifintly inrs th DCB-xtrt Mn onntrtions in roots (Fig. 5A), onsistnt with th EDX rsults (Fig. 4). Root Mn onntrtions ftr DCB xtrtion wr highr in th prsn of Al thn in its sn. Ths rsults init tht Mn plqus in roots wr nhn y Al ition unr Mn toxiity. Root rnhym, imtr n volum W osrv roots using SEM to intify hngs in root rnhym. Thr wr fwr gs sps in roots in th sn of Al (Fig. 6A C) thn in th prsn of Al (Fig. 6D F). Morovr, Mn toxiity signifintly rs th vrg imtr n volum of roots, whil ition of Al inrs ths prmtrs (Fig. 7). Ths rsults init tht Al filitt th formtion of mor root rnhym n thikr, lrgr roots unr Mn toxiity. DISCUSSIN In prliminry xprimnts, w intifi 2 mm Al s n pproprit onntrtion to trmin th ffts of Al on Mn toxiity in ri, us of its wk toxiity to ri growth. Shn t l. (26) rport tht th Al onntrtion rng in solutions of i soils from southrn Chin ws 1 2 mm. Ths soils lso ontin high onntrtions of totl Mn (763 mgkg 1 soil) n xhngl Mn (196 mg kg 1 soil) (Chins Ntionl Soil Survy ffi, 1998). Givn tht th mount of xhngl Mn vris pning on th totl Mn ontnt n wtrlogging, it ws rsonl to us 2 mm Al to intrt with 67, 2, 5 n 1 mm Mn in ths Al Mn intrtion xprimnts. ur rsults show tht Al llvit Mn toxiity to ri slings, onsistnt with prvious rports for rly n triplx (Rs n Sirk, 1961), orn (rk, 1977), wht (Blir n Tylor, 1997), owp (Tylor t l., 1998) n soyn (Yng t l., 29). ur t show tht Al ition rs Mn umultion in th root symplst, whol roots n shoots in 24-h trtmnt n in shoots in 3-wk trtmnt, whih rsult in llvition of Mn toxiity. Ri grw muh ttr t 5 n 1 mm Mn supply in th prsn of Al thn plnts grown t 2 mm Mn supply in th sn of Al in th longtrm trtmnt (Fig. 1D). Howvr, shoot Mn umultion in th formr ws highr thn in th lttr (Fig. 2B). This might involv in th ffts of Al on Mn intrnl toxition, suh s Mn istriution twn poplst n symplst or ol n nw lvs. Ths prosss oul rgult y syls6 n snram3 unr iffrnt Mn nvironmnts (Sski t l., 211; Ymji t l., 213). This is topi ning furthr invstigtion. In prvious stuis it hs n postult tht ntgonisti ffts of Al on Mn, or non-spifi omptition twn Al n Mn, my rsponsil for ru Mn umultion in plnts. Howvr, ths is hv not n tst using til n p nlyss. In this stuy w us th GCS mol, whih is s on lssil ltrostti thory (Gouy Chpmn thory) (inri, 1994; inri t l., 1998), to xplin how Al ru Mn umultion in ri plnts. W osrv tht Al rs Mn umultion in th root symplst n th whol root in short-trm Al trtmnt, rsulting in lss Mn ing trnsport to th shoots. Downlo from y gust on 9 Jnury 218

7 Wng t l. Aluminium llvits mngns toxiity y ruing its vilility 243 Mn on. in DCB xtrt (µg g 1. wt) A Control 5 1 Mn on. ftr DCB xtrt (µg g 1. wt) B Mn supply (µm) Mn supply (µm) FIG. 5. Mngns onntrtions in ithionit itrt iront xtrt (A) n root (B) ftr long-trm trtmnt with Mn n Al or Mn only. Ri slings (11 ol) wr grown in nutrint solution with 67, 2, 5 or 1 mm Mn in th prsn or sn of 2 mm Al for 3 wks. Vlus r mn 6 s.. (n ¼ 3 rplits, h omprising four slings). Diffrnt lttrs show signifint iffrns mong trtmnts ( < 5, Tuky s tst). A B C SE 28-Apr-14 WD22 4mm 8 kv x13 3um SE 28-Apr-14 WD22 3mm 8 kv x1 5um SE 28-Apr-14 WD22 7mm 8 kv x16 3um D E F SE 28-Apr-14 WD24 mm 8 kv x1 5um SE 28-Apr-14 WD22 8mm 8 kv x1 5um SE 28-Apr-14 WD22 2mm 8 kv x1 5um FIG. 6. Effts of Al n Mn supply on rnhym formtion in long-trm xprimnt. Ri slings (11 ol) wr grown in nutrint solution ontining 67 (A, D), 5 (B, E) or 1 (C, F) mm Mn without (A C) or with (D F) 2 mm Al for 3 wks. Aftr th 3-wk trtmnt, nwly form vntitious roots wr ut trnsvrsly 1 mm hin th root tip. Gnrlly, Al is onsir to toxi for plnt growth, spilly root growth. Howvr, Al hs n shown to llvit H þ (inri, t l., 1992; inri, 1993) ncu 2þ toxiity (Wng t l., 28; opittk t l., 211). In our prvious rports w show tht Al nhn th growth of Lspz iolor (Chn t l., 21) n ri (Zho t l., 213)inth prsn of mmonium, whih n phytotoxi t high onntrtions (Britto n ronzukr, 22). It hs n suggst tht th llvition of Cu 2þ n H þ toxiity rsult from th Al-inu rs in th ngtivity of w o,whih orrspon to ru Cu 2þ or H þ tivity t th CM surf (inri, 1993; opittk t l., 211; Wng t l., 212). In th prsnt stuy w lult tht Al rstilly inrs th positivity of w o, rsulting in lowr {Mn 2þ } o (Tl 1). Downlo from y gust on 9 Jnury 218

8 244 Wng t l. Aluminium llvits mngns toxiity y ruing its vilility Root vrg imtr (mm) Root volum (m 3 ) A B Mn on. in ultur solution (µm) This my hv ontriut to th rs Mn onntrtions in th root symplst in th short-trm Al trtmnt (Fig. 3A) n onsquntly lss Mn ing trnsport to th shoots (Fig. 3C). Th roots r th min sit of Al toxiity, whil th symptoms of Mn toxiity r minly osrv in th shoots (stunt growth, n hlorosis n nroti lsions in th lvs) (ohin t l., 24). Thus, th toxiity of Mn to plnts gnrlly rsults from xss Mn umultion in th lvs (Foy t l., 1978, 1988; Wissmir t l., 1992; Migok n lous, 27). In ri, th roots r muh lss snsitiv to Mn toxiity thn r th shoots (Nlson, 1983; Lion, 21). Givn th ly in th trnslotion of Mn from th roots to th Control FIG. 7. Root vrg imtr (A) n volum (B) ftr long-trm trtmnt with Mn plus Al or Mn only. Ri slings (11 ol) wr grown in nutrint solution ontining 67, 2, 5 or 1 mm Mn without or with 2 mm Al for 3 wks. Vlus r mn 6 s.. (n ¼ 12 rplits). Astrisks ov olumns init sttistilly signifint iffrns twn ontrol n Al trtmnt ( < 5; inpnnt smpls t-tst). shoots, Al my hv rprss root Mn uptk in th short-trm xprimnt. This woul prvnt xss Mn umultion in shoots, thry llviting Mn toxiity. W osrv tht Al inrs Mn umultion in roots ftr th 3-wk Al n Mn trtmnts (Fig. 2A). This rsult ontrsts with thos rport for othr plnt spis (Rs n Sirk, 1961; rk, 1977; Blir n Tylor, 1997; Tylor t l., 1998; Yng t l., 29). It is iffiult to xplin this s on th rs Mn umultion in th root symplst n whol roots in th short-trm Al Mn trtmnts (Fig. 3). Thr ws n inrs in Mn plqus in th Al trtmnts (Figs 4 n 5A), onsistnt with th Al-inu inrs in Mn umultion in roots. Bus Mn plqus rry vril hrg, thy hv grtr pity for soring tion n. In lin with this, w lso tt, n N in Mn plqus (Btty t l., 22; Chn t l., 26). Th vlopmnt of root strutur (rnhym, n root imtr n volum) ws lso improv y Al unr Mn toxiity (Figs 6 n 7). Th improv root strutur my hv ply rol in th inrs Mn umultion in roots in th long-trm Al trtmnt. A similr fft ws rport for silion (Horiguhi n Morit, 1987). Tht is, silion promot th vlopmnt of root n root rnhym n improv th oxiizing powr of th root, llowing mor Mn to oxiiz on th root surf (Horiguhi n Morit, 1987; Luiz t l., 21). ur rsults lso show tht root Mn onntrtions ftr DCB xtrtion wr highr in Al Mn trtmnts thn in Mn-only trtmnts (Fig. 5B), suggsting tht th inrs Mn umultion in roots oul ttriut not only to th inrs in Mn plqus, ut lso to inrs Mn ining in th roots. Svrl prvious stuis hv rport tht Al inrs ll wll ptin n hmillulos (Chng t l., 1999; Tuhi n Mtsumoto, 21; Tuhi t l., 24; Yng t l., 28), whih r primry Al-ining omponnts (ohin t l., 25; Horst t l., 21). W lso osrv tht Al inrs th mounts of ths ll wll omponnts in ri (Wng t l., 215), suggsting tht Al-inu inrs in ptin n hmillulos might rsult in inrs Mn ining in th roots. Thrfor, inrss in oth Mn plqus n Mn ining oul l to mor unvill Mn ing umult in th roots. Togthr with th Al-inu rs in root symplsti Mn uptk, ths hngs rsult in lss Mn ing trnsport to shoots, thry llvitingmntoxiityinri. In onlusion, our rsults show tht Al llvit Mn toxiity in ri. This ws ttriut to rs shoot Mn umultion rsulting from n Al-inu rs in root symplsti Mn uptk. Th rs in root symplsti Mn uptk rsult from n Al-inu hng in ll mmrn potntil. Also, Al inrs Mn plqus in th roots n hng th ining proprtis of th ll wll, rsulting in umultion of unvill Mn in roots. To our knowlg, this is th first rport tht Al n inrs Mn umultion in roots. SULEMENTARY DATA Supplmntry t r vill t org n onsist of Tl S1: fftsofluminiumtiffrnt onntrtions on ri growth in th 3-wk (long-trm) xprimnt. Downlo from y gust on 9 Jnury 218

9 Wng t l. Aluminium llvits mngns toxiity y ruing its vilility 245 ACNWLEDGEMENTS This work ws support y th Ntionl y Bsi Rsrh rogrm of Chin (No. 214CB441), th Strtgi riority Rsrh rogrm of th Chins Amy of Sins (No. XDB153) n th Nturl Sin Fountion of Chin (No ). LITERATURE CITED Btty LC, Bkr AJM, Whlr BD. 22. Aluminium n phospht uptk y hrgmits ustrlis: th rol of, Mn n Al root plqus. Annls of Botny 89: Blir LM, Tylor GJ Th ntur of intrtion twn luminum n mngns on growth n mtl umultion in Tritium stivum. Environmntl n Exprimntl Botny 37: Britto DT, ronzukr HJ. 22. NH 4 þ toxiity in highr plnts: ritil rviw. Journl of lnt hysiology 159: Chng YC, Ymmoto Y, Mtsumoto H Aumultion of luminium in th ll wll ptin in ultur too (Niotin tum L.) lls trt with omintion of luminium n iron. lnt, Cll & Environmnt 22: Chn RF, Shn RF, Gu, Dong XY, Du CW, M JF. 26. Rspons of ri (ryz stiv) with root surf iron plqu unr luminium strss. Annls of Botny 98: Chn ZC, Zho XQ, Shn RF. 21. Th llviting fft of mmonium on luminum toxiity in Lspz iolor rsults in rs luminum-inu mlt srtion from roots ompr with nitrt. lnt n Soil 337: Chnry EM A prliminry stuy of luminium n th t ush. lnt n Soil 6: Chins Ntionl Soil Survy ffi Soil of Chin [in Chins]. Bijing: Chins Agriultur rss, 95 96, 95. rk RB Efft of luminum on growth n minrl lmnts of Al-tolrnt n Al-intolrnt orn. lnt n Soil 47: Crowr AA, Coltmn DW Formtion of mngns oxi plqu on ri roots in solution ultur unr vrying ph n mngns (Mn 2þ )onntrtion onitions. Journl of lnt Nutrition 16: Dvi JL, lg NB. 2. Root ll ultrstrutur in vloping rnhym tissu of thr wtln spis. Annls of Botny 86: Dolling J, Mooy, Nol A, t l. 21. Soil iity n iifition in Austrli. Ntionl Ln & Wtr Rsours Auit rojt Rport. Cnrr, Austrli. Foy CD, Chny RL, Whit MC Th physiology of mtl toxiity in plnts. Annul Rviw of lnt hysiology 29: Foy CD, Sott BJ, Fishr JA Gnti iffrns in plnt tolrn to mngns toxiity. In: R Grhm, R Hnnm, N Urn, s. Mngns in soils n plnts. Dorrht, Nthrlns: Springr luwr Ami ulishrs, Führs H, Bhrns C, Gllin S, t l. 21. hysiologil n protomi hrtriztion of mngns snsitivity n tolrn in ri (ryz stiv) in omprison with rly (Horum vulgr). Annls of Botny 15: Horiguhi T, Morit S Mhnism of mngns toxiity n tolrn of plnts VI. Efft of silion on llvition of mngns toxiity of rly. Journl of lnt Nutrition 1: Horst WJ Th physiology of mngns toxiity. In: R Grhm, R Hnnm, N Urn, s. Mngns in soils n plnts. Dorrht, Nthrlns: Springr luwr Ami ulishrs, Horst WJ, Wng YX, Etih D. 21. Th rol of th root poplst in luminium-inu inhiition of root longtion n in luminium rsistn of plnts: rviw. Annls of Botny 16: inri TB Aluminum nhnmnt of plnt growth in i rooting mi. A s of riprol llvition of toxiity y two toxi tions. hysiologi lntrum 88: inri TB Us of Gouy-Chpmn-Strn mol for mmrn-surf ltril potntil to intrprt som fturs of minrl rhizotoxiity. lnt hysiology 16: inri TB, Wng. 21. Th surf hrg nsity of plnt ll mmrns (r): n ttmpt to rsolv onfliting vlus for intrinsi Journl of Exprimntl Botny 61: inri TB, Ryn R, ohin LV Intrtiv ffts of Al 3þ,H þ, n othr tions on root longtion onsir in trms of ll-surf ltril potntil. lnt hysiology 99: inri TB, Yrmiyhu U, Rytwo G Computtion of surf ltril potntils of plnt ll mmrns. Corrsponn to pulish zt potntils from ivrs plnt sours. lnt hysiology 118: lug B, Horst WJ. 21. xlt xution into th root-tip wtr fr sp onfrs prottion from luminum toxiity n llows luminum umultion in th symplst in ukwht (Fgopyrum sulntum). Nw hytologist 187: ohin LV Cllulr mhnisms of luminum toxiity n rsistn in plnts. Annul Rviw of lnt hysiology n lnt Molulr Biology 46: ohin LV, Hokng A, iñros MA. 24. How o rop plnts tolrt i soils? Mhnisms of luminum tolrn n phosphorous ffiiny. Annul Rviw of lnt Biology 55: ohin LV, iñros MA, Hokng A. 25. Th physiology, gntis n molulr iology of plnt luminum rsistn n toxiity. lnt n Soil 274: opittk M, Blmy FC, Wng, Mnzis NW ult tivity of Mn 2þ t th outr surf of th root ll plsm mmrn govrns Mn nutrition of owp slings. Journl of Exprimntl Botny 62: opittk M, inri TB, Wng, Blmy FC, Rihmn SM, Mnzis NW Allvition of Cu n rhizotoxiitis in owp (Vign unguiult) s rlt to ion tivitis t root-ll plsm mmrn surf. Environmntl Sin & Thnology 45: opittk M, Wng, Mnzis NW, Niu R, inri TB Aw-ssil omputr progrm for lulting ltril potntils n ion tivitis t ll-mmrn surfs. lnt n Soil 375: Li, Song AL, Li ZJ, Fn FL, Ling YC Silion mliorts mngns toxiity y rgulting mngns trnsport n ntioxint rtions in ri (ryz stiv L.). lnt n Soil 354: Lion FC. 21. Tolrn of ri to xss mngns in th rly stgs of vgttiv growth. Chrtristion of mngns umultion. Journl of lnt hysiology 158: Luiz AZJ, Rnils LFF, Júlio CLN, Gspr H, Viníius TÁ. 21. Ri grown in nutrint solution with oss of mngns n silion. Rvist Brsilir Ciêni o Solo 34: M JF, Hirt S, Mtsumoto H High luminum rsistn in ukwht. II. xli i toxifis luminum intrnlly. lnt hysiology 117: Mrshnr H Funtions of minrl nutrints: mironutrints. In: H Mrshnr,. Minrl nutrition of highr plnts, 2n n. Lonon: Ami rss, Mrshnr H Apttion of plnts to vrs hmil soil onitions. In: H Mrshnr,. Minrl nutrition of highr plnts, 2n n. Lonon: Ami rss, Migok M, lous G. 27. Th proprtis of th Mn, Ni n trnsport oprting t plsm mmrns of uumr roots. hysiologi lntrum 129: Milllo R, Rys DM, Ivnov AG, Mrí LM, Alri M. 21. Mngns s ssntil n toxi lmnt for plnts: trnsport, umultion n rsistn mhnisms. Journl of Soil Sin n lnt Nutrition 1: Mor l M, Ross A, Rir A, Rngl Z. 29. Diffrntil tolrn to Mn toxiity in prnnil rygrss gnotyps: involvmnt of ntioxitiv nzyms n root xution of roxylts. lnt n Soil 32: Moroni JS, Sott BJ, Wrttn N. 23. Diffrntil tolrn of high mngns mong rps gnotyps. lnt n Soil 253: Nlson LE Tolrns of 2 ri ultivrs to xss Al n Mn. Agronomy Journl 75: g V, llr U. 25. Sltiv trnsport of zin, mngns, nikl, olt n mium in th root systm n trnsfr to th lvs in young wht plnts. Annls of Botny 96: g V, Wisskopf L, llr U. 26. Hvy mtls in whit lupin: uptk, root-to-shoot trnsfr n ristriution within th plnt. Nw hytologist 171: onnmprum FN Growth-limiting ftors of roi soils. In: Mjor rsrh in upln ri. Los Bños, hilippins: Intrntionl Ri Rsrh Institut, Rs WJ, Sirk GH Intr-rltionship of luminium n mngns toxiitis towrs plnts. lnt n Soil 14: Downlo from y gust on 9 Jnury 218

10 246 Wng t l. Aluminium llvits mngns toxiity y ruing its vilility Ross A, Rngl Z, Mrí LM. 27. Mngns supply n ph influn growth, roxylt xution n proxis tivity of rygrss n whit lovr. Journl of lnt Nutrition 3: Sski A, Ymji N, Xi JX, M JF sysl6 is involv in th toxifition of xss mngns in ri. lnt hysiology 157: Shn RF, Chn RF, M JF. 26. Bukwht umults luminum in lvs ut not in ss. lnt n Soil 284: Tuhi A, Mtsumoto H. 21. Chngs in ll-wll proprtis of wht (Tritium stivum) roots uring luminum-inu growth inhiition. hysiologi lntrum 112: Tuhi A, ikui S, Mtsumoto H. 24. Diffrntil ffts of luminium on osmoti potntil n sugr umultion in th root lls of Al-rsistnt n Al-snsitiv wht. hysiology lntrum 12: Ttulin SA Surf ltrosttis of iologil mmrns n ion ining. In: TS Sørnsn,. Surf hmistry n ltrohmistry of mmrns. Nw York: Mrl Dkkr, Tylor GJ, Crowr AA Us of th DCB thniqu for xtrtion of hyrous iron oxis from roots of wtln plnts. Amrin Journl of Botny 7: Tylor GJ, Blrny FC, Ewrs DG Antgonisti n synrgisti intrtions twn luminum n mngns on growth of Vign unguiult t low ioni strngth. hysiology lntrum 14: Wng, Zhou DM, inri TB, t l. 28. Cll mmrn surf potntil (w ) plys ominnt rol in th phytotoxiity of oppr n rsnt. lnt hysiology 148: Wng, inri TB, Zhou D, opittk M, ijnnurg WJGM lsm mmrn surf potntil: ul ffts upon ion uptk n toxiity. lnt hysiology 155: Wng, D Shmphlr AC, opittk M, Zhou DM, ijnnurg WJGM, Lok Dvlopmnt of n ltrostti mol priting oppr toxiity to plnts. Journl of Exprimntl Botny 63: Wng W, Zho XQ, Chn RF t l. 215.Altr ll wll proprtis r rsponsil for mmonium-ru luminium umultion in ri roots. lnt, Cll & Environmnt, in prss. oi: /p Wtn T, Jnsn S, ski M. 25. Th nfiil fft of luminium n th rol of itrt in Al umultion in Mlstom mlthrium. Nw hytologist 165: Wissmir AH, Dining A, Hrgnrör A, Horst WJ, Mix-Wgnr G los formtion s prmtr for ssssing gnotypil plnt tolrn of luminium n mngns. lnt n Soil 146: Xi JX, Ymji N, si T, M JF. 21. lsm mmrn-loliz trnsportr for luminum in ri. roings of th Ntionl Amy of Sins of th Unit Stts of Amri 17: Yng JL, Li YY, Zhng YJ, t l. 28. Cll wll polyshris r spifilly involv in th xlusion of luminum from th ri root px. lnt hysiology 146: Yng ZB, You JF, Xu MY, Yng ZM. 29. Intrtion twn luminum toxiity n mngns toxiity in soyn (Glyin mx). lnt n Soil 319: Ymji N, Sski A, Xi JX, Yokosho, M JF A no-s swith for prfrntil istriution of mngns in ri. Ntur Communitions 4:2442. Zng QL, Chn RF, Zho XQ, t l Aluminum oul trnsport vi phlom in Cmlli olifr Al. Tr hysiology 33: Zho XQ, Shn RF, Sun QB. 29. Ammonium unr solution ultur llvits luminum toxiity in ri n rus luminum umultion in roots ompr with nitrt. lnt n Soil 315: Zho XQ, Guo SW, Shinmhi F, t l. 213.Aluminium tolrn in ri is ntgonisti with nitrt prfrn n synrgisti with mmonium prfrn. Annls of Botny 111: Downlo from y gust on 9 Jnury 218