Effects of Aluminum, Iron and/or Low ph on Rice Seedlings Grown in Solution Culture

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1 INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY ISSN Print: ; ISSN Online: /2015/ DOI: /IJAB/ Full Length Artile Effets of Aluminum, Iron nd/or Low ph on Rie Seedlings Grown in Solution Culture Frhn Jmludin Ali, Jusop Shmshuddin *, Che Ishk Fuzih, Mohd Hnif Ahmd Husni nd Qurn Ali Pnhwr Deprtment of Lnd Mngement, Fulty of Agriulture, Universiti Putr Mlysi, Serdng, Selngor, Mlysi * For orrespondene: shmshud@upm.edu.my Astrt Wter in the pddy field overed y id sulfte soils hving very low ph ontins high mount of Al nd Fe tht ffets rie growth. A lortory study ws onduted to qulify rie grown under the dverse onditions n withstnd the stresses. Two rie vrieties, MR 219 nd MR 253, were grown hydroponilly t vrious ph (3, 4, 5, 6, 7), Al (0, 20, 40, 60, 80, 100 µm) nd Fe (0, 20, 40, 60, 80, 100 µm) onentrtions. After 14 dys, rie root length nd surfe re were determined using root snner. Therefter, orgni ids relesed y the roots of rie were determined y high performne liquid hromtogrphy. Results showed tht the root length deresed with inresing Al nd/or Fe onentrtion. On the ontrry, the root length inresed linerly s the ph of the solution inresed. This phenomenon ws proly in prt relted to the exudtion of oxli, itri nd mli ids y the rie roots. It ws oserved tht the mount of orgni ids relesed ws inresed with inresing Al nd/or Fe onentrtion in the solution ulture. Hene, it is elieved tht these orgni ids were responsile for helting some of the Al nd/or Fe in the solution, rendering them unville for their uptke y rie. In this wy, rie plnts n withstnd some degree of Al 3+ nd/or Fe 2+ toxiity Friends Siene Pulishers Keywords: Aid sulfte soil; Cheltion; Aluminum toxiity; Iron toxiity; Orgni id; Rie seedling Arevition: HPLC = High performne liquid hromtogrphy; GML Ground mgnesium limestone; MARDI = Mlysin Agriulturl Reserh Development Institute; SAS = Sttistil nlysis softwre; SEM = Snning eletron mirosope Introdution Rie (Oryz stiv) is stple food for over hlf of the world's popultion. More thn one illion people depend on rie ultivtion for their survivl. Relizing the importne of rie nd its eonomi role, the prodution of rie should e inresed suffiiently euse growth in rie prodution hs een slower thn the popultion growth. With little room for re expnsion, improving the fertility of mrginl soils (suh s id sulfte soils) is one of the wys to inrese rie prodution worldwide. Aid sulfte soils ourring long the ostl plins of Peninsulr Mlysi (Enio et l., 2011) n e used for rie ultivtion if properly meliorted (Shmshuddin, 2006). Under norml ondition, rie growing on these soils performs poorly due Al 3+, Fe 2+ or H + stress (Shmshuddin et l., 2013, 2014). It is reported tht low ph nd high Al nd/or Fe onentrtions re the result of pyrite (FeS 2 ) oxidtion when the soils re utilized for rie ultivtion (Shmshuddin et l., 2004). At ph <4.8, dissolved Al 3+ in the soil solution n reh the ritil level of 30 µm, whih n dmge rie plnts. Al toxiity is often relted to phosphorus defiieny euse soil with high Al onentrtion will derese the vilility of P due to Al-Fe-phosphte intertion (Wrd et l., 2008). Tht is why the most reognized symptom of Al toxiity is P-relted inhiition of root growth. In id sulfte soils worldwide, the onentrtion of Fe 2+ in the wter of rie fields inreses when they re flooded for rie ultivtion (Muhrizl et l., 2006; Shmshuddin et l., 2014). Toxiity ours if extrtle Fe in the soils exeeds 300 mg kg -1 (Doermnn nd Firhurst, 2000). Aording to Silveir et l. (2007), Fe toxiity is onsidered s one of the most signifint onstrints in rie prodution, long with Al toxiity. However, without enough Fe in rie plnts, hlorophyll nnot e suffiiently produed (Follett nd Westfll, 1992). Under low ph ondition, the vilility of Fe is inresed, using n exessive umultion of Fe in the leves of rie. Visul symptom of Fe toxiity on rie is ronzing (Firhurst nd Witt, 2002). Besides ronzing lef symptom, Fe toxiity n inrese pnile sterility tht redues plnt growth (Mthis nd Ash, 2005). Aording to Elis et l. (2011), Al 3+ nd Fe 2+ umulte in rie root due to ttrtion to the negtively-hrged ells wll, preventing ell division nd elongtion. Rie roots rpidly sor Al 3+ nd Fe 2+, using redution in root length nd inhiit root growth tht result in the urtilment of nutrient uptke. The end To ite this pper: F.J. Ali, J. Shmshuddin, C.I. Fuzih, M.H.A. Husni nd Q.A. Pnhwr, Effets of luminum, iron nd/or low ph on rie seedlings grown in solution ulture. Int. J. Agri. Biol., 17:

2 Rie Seedlings Affeted y Aluminum, Iron nd/or Low ph Stress / Int. J. Agri. Biol., Vol. 17, No. 4, 2015 result would e the redution of rie yield (Elis et l., 2011; Shmshuddin et l., 2014). Orgni ids relesed y rie roots under stress my ply mjor role in the Al 3+ nd Fe 2+ tolerne mehnisms (Gerke et l., 1994; Shmshuddin et l., 2013, 2014) nd the utiliztion of insolule nutrients, espeilly P (Jones nd Drrh, 1994). There re proly two mehnisms involved in id metl tolerne y the roots of rie: (1) internl tolerne mehnism in the symplsm; nd (2) exlusion mehnism in the poplsm nd t the plsm memrne (Kohin, 1995). For rie, the ltter is the more importnt mehnism thn tht of the former. In the exlusion mehnism, metls re prevented from entering or stying in the symplsm or oming into ontt with sensitive intrellulr sites. For exmple, Al tolerne is onferred y exlusion from root tip. Elis et l. (2011) found tht under stress ondition, plnt root were found to relese greter mount of orgni ids into the rhizosphere. These ids helte Al outside the plsm memrne, therey reduing its uptke. Ryn et l. (2001) suggested tht exudtion of orgni ids ould e tive mehnism in response to environmentl signls nd stresses. Orgni ids re known s Al-helting moleules. A study y Pineros nd Kohin (2009) showed tht low moleulr weight orgni ids sereted y plnt root helte Al in the soil solution, forming Al-itrte or Almlte, preventing it from entering the root ells. Mny tolernt plnts shre this generl resistne mehnism. However, the orgni ids so relesed y plnt roots vry depending on plnt speies (Kohin et l., 2005). Miysk et l. (1991) found tht the mount of orgni ids sereted y plnt root my determine its resistne to Al in the rhizosphere. This finding is in line with tht of M et l. (2001) nd Horst et l. (2010) who proved tht plnt root plys ritil role in the tolerne mehnism sed on efflux of the orgni ids. The most effetive orgni id in the lleviting toxi Al nd Fe effets ws itri, followed y oxli nd trtri ids. Mli, mloni nd sliyli ids were moderte in detoxifying Al nd Fe (Yng et l., 2013). Aording to Mlysin Agriulturl Reserh Development Institute (MARDI, 2010), rie vriety MR 219 is high yielding vriety nd therefore is widely grown in Peninsulr Mlysi. Reently, new rie vriety nmed MR 253 hs een relesed in Mlysi. This MR 253 rie vriety, limed to e id tolernt y MARDI, is le to perform etter thn tht of MR 219 in mrginl soils. So fr, no detiled study hs een onduted to determine the tul performne of MR 253 vriety on the id sulfte soils. Hene, study ws onduted to determine the effets of Al 3+, Fe 2+ nd low ph on root elongtion nd root surfe re of rie vrieties MR 219 nd MR 253 nd to explore the underlying mehnism of reduing H + stress s well s Al 3+ nd Fe 2+ toxiity detoxifition. Mterils nd Methods Soils nd their Lotions of Smpling Soils nd wter smples were tken from pddy field in the Integrted Agriulturl Development Projet (IADP), Kelntn, whih is loted in the northestern prt of Peninsulr Mlysi. The soils for the hrteriztion nd lssifition purposes were tken t 5 depths: 0 15, 15 30, 30 45, 45 60, nd m using n uger. Soil nd Wter Anlyses Soil ph (in wter) ws determined y ph meter. Exhngele C, Mg, K nd tion exhnge pity (CEC) nd exhngele tions were determined using mmonium ette uffered t ph 7 (Benton, 2001). Exhngele Al ws extrted y 1 M KCl (Brnhisel nd Bertsh, 1982) nd the Al in the extrt ws determined y indutively oupled plsm optil emission spetrometry (ICP-OES). Totl C nd N were determined y CNS TruM Anlyzer. Aville P ws determined y the method of Bry nd Kurtz (1945) with the extrted P determined y uto nlyzer (AA). Extrtle Fe in the soils ws determined y doule id method. It ws extrted using 0.05 M HCl in M H 2 SO 4. A 5 g smple of the soil ws mixed with 25 ml of the extrting solution nd shken for 15 minutes. The solution ws then filtered through Whtmn filter pper numer 42 efore determining the Fe it ontined y ICP-OES. The wter tken from the pddy field ws filtered through Whtmn filter pper numer 42 efore determining its ph. The onentrtion of Al nd Fe in the wter ws determined y ICP-OES. Plnt Mteril Rie (Oryz stiv L.) vrieties MR 219 nd MR 253 used for this study were tken from Deprtment of Agriulture, Psir Ms, Kelntn, Mlysi. Lotion, Mterils nd Experimentl This study ws rried out t the Fulty of Agriulture, Universiti Putr Mlysi, Serdng, Mlysi. It ws onduted in lortory under hydroponi ondition t the mient temperture. Seeds of rie (vrieties MR 219 nd MR 253) were soked in hormone-sed hemil (Zpp ) for 24 h nd then left in the drk ple for nother 24 h. This ws short-term experiment onduted in two phses; the method used ws modified from tht of Elis et l. (2011). In the first phse experiment, three pre-soked seeds were trnsferred into test tues ontining 0.5 mm CCl 2 solution t vrious onentrtions of Al (0, 20, 40, 60, 80 nd 100 µm) using AlCl 3, Fe (0, 20, 40, 60, 80 nd 100 µm) using FeCl 3 nd mixtures of Fe nd Al (0, 20, 40, 60, 80 nd 100 µm). In the seond phse experiment, three pre- 703

3 Ali et l. / Int. J. Agri. Biol., Vol. 17, No. 4, 2015 soked seeds were trnsferred into test tues ontining wter tken from the pddy field. For this experiment, the ph of the wter ws djusted to vrious levels (3, 4, 5, 6 nd 7) using either 0.01 M HCl or 0.01 M NOH. For oth phses of the study, the seeds of rie were llowed to grow in the tues for 14 dys. No fertilizers were dded to promote the growth of the rie seedlings. Determintion of Root Morphology At dy 14, root length (m) nd root surfe re (m 2 ) of rie for oth phses of the study were determined using root snner, model Win RHIZO Visul Oservtion of Rie Root Rie root ws oserved under snning eletron mirosope (SEM). The roots of the rie seedlings were ut into piees, pre-fixed with 4% glutrldehyde overnight nd wshed with 0.1 M sodium odylte uffer 3 times for 30 minutes. Osmium tetroxide uffer (1%) ws used for post fixtion. After series of dehydrtion in etone (35, 50, 75, 95 nd 100%), the smples were dried in ritil point dryer nd mounted on luminum stus, sputter-oted in gold nd viewed under SEM (JEOL JSM-6400 tthed with OXFORD INCA ENERGY 200 EDX). Determintion of Orgni Aids The orgni ids in the ulture solution for oth phses of the study were determined using high performne liquid hromtogrphy (HPLC), Jso Borwin softwre. Aout 20 µl of the smples from eh tretment were injeted into the HPLC with UV detetor set t 210 ɳm, using Rezex ROA-orgni id H + (8%) olumn ( mm) from Phenomenex Co.; N H 2 SO 4 ws the moile phse with flow rte of 0.17 ml min 1. Peks for the orgni ids deteted were identified y ompring with the retention times otined for pure orgni ids injeted s stndrds. From the pek res, the quntity of orgni ids in the smples were lulted nd expressed in µm. Determintion of Sensitivity to Tolerne Two rie vrieties were studied to differentite their sensitivity to Al, Fe nd low ph stress. It ws done vi 2 methods: (1) using the slope of the grphs of the reltive root length or root surfe re ginst Al or Fe onentrtion; nd (2) using the ritil vlues for rie growth. The steepness of the slope of the grphs nd ritil vlues (sed on the 90% reltive root length or root surfe re of rie seedlings) were used s mesure of Al or Fe tolerne. Sttistil Anlysis The experiments were lid out in Completely Rndomized Design (CRD) with four replitions. The dt olleted from this study were nlyzed y ANOVA for nlysis of vrines nd Tukey test for men omprison using SAS version 9.2 (SAS Institute, In., Cry, N.C., USA). All digrms in this pper were drwn using Exel Progrm in the Mirosoft. Results Initil Chemil Properties of Soil nd Wter Tle 1 shows the hemil hrteristis of the id sulfte soil under study. The ph of the topsoil ws Soil ph deresed onsistently with depth, the lowest eing 3.13 t the m depth. The exhngele K, C nd Mg in the topsoil were elow the suffiieny level for rie growth, while the exhngele Al in the sme horizon ws very high (> 5 mol kg -1 ). The ville P ws lso low (< 5 mg kg -1 ) in the topsoil. Tle 2 shows tht wter ph ws 2.98, while the onentrtion of Al nd Fe were 1060 nd 192 µm, respetively. First Phse Experiment Effet of Al on the root length nd root surfe re: The reltive root length of MR 219 nd MR 253 rie vrieties were negtively nd highly orrelted with Al onentrtion nd the equtions representing the reltionship re given y Y = x (R 2 = 0.97) nd Y = x (R 2 = 0.94), respetively (Fig. 1). The reltive root surfe re of rie seedlings for oth vrieties showed the sme trend. They re negtively nd highly orrelted with Al onentrtion nd the equtions representing the reltionships re given y Y = x (R 2 = 0.95) nd Y = x (R 2 = 0.97), respetively (Fig. 2). Fig. 2 shows tht the slope of the grph for rie vriety MR 253 ws steeper ompred to tht of MR 219, while the ritil Al onentrtion for vriety MR 219 nd MR 253 were 8.91 nd µm, respetively. Effet of Fe on the root length nd root surfe re: The reltive root length of MR 219 nd MR 253 ws negtively orrelted with Fe onentrtion nd the equtions representing the reltionship re given y Y = x (R 2 = 0.98) nd Y = x (R 2 = 0.98), respetively (Fig. 1). The reltive root surfe re for oth vrieties showed similr trend. The equtions representing the reltionships re given y Y = x (R 2 = 0.93) nd Y = x (R 2 = 0.98), respetively (Fig. 2). The slope of the grph for MR 219 ws less steep ompred to tht of MR 253. The ritil Fe onentrtion for the growth of vrieties MR 219 nd MR 253 were nd 8.76 µm, respetively. On the ontrry, MR 253 ws more tolernt to Fe ompred to tht of MR 219 if it is sed on the root length (Fig. 1). 704

4 Rie Seedlings Affeted y Aluminum, Iron nd/or Low ph Stress / Int. J. Agri. Biol., Vol. 17, No. 4, 2015 Tle 1: The hemil properties of the id sulfte soil used in the study Depth ph EC Exhngle tions (mol kg -1 ) Ext. Fe * CEC Totl N Avil. P Totl C (m) ms/m C Mg K Al (mg kg -1 ) (mol kg -1 ) % (mg kg -1 ) % * CEC = tion exhnge pity Tle 2: The hemil properties of the wter tken in pddy field t id sulfte soil re ph EC Bsi tions (µm) Fe Al P ms/m C Mg K (µm) (µm) (mg L -1 ) Fig. 1: Reltionship etween reltive root length (RRL) nd ) Al onentrtion, ) Fe onentrtion, ) Al in omintion with Fe onentrtion nd d) phofmr 219 nd MR 253 Effet of Al in omintion with Fe on the root length nd root surfe re: The reltive root length of MR 219 nd MR 253 ws negtively orrelted with Al in omintion with Fe nd the equtions representing the reltionship re given y Y = x (R 2 = 0.98) nd Y = x (R 2 = 0.98), respetively (Fig. 1). The reltive root surfe re for oth rie vrieties showed the sme trend. The equtions representing the reltionships re given y Y = x (R 2 = 0.96) nd Y = x (R 2 = 0.98), respetively (Fig. 2). The slope of the grph for MR 219 ws less steep ompred to the slope of MR 253. This mens tht MR 253 ws more sensitive to Al in omintion with Fe ompred to tht of MR 219. The ritil Al in omintion with Fe for vriety MR 219 nd MR 253 were nd 8.88 µm, respetively. Fig. 2: Reltionship etween reltive surfe res (RRSA) nd ) Al onentrtion, ) Fe onentrtion, ) Al in omintion with Fe onentrtion nd d) ph of MR 219 nd MR 253 Seond Phse Experiment The reltive root length of MR 219 nd MR 253 ws positively orrelted with ph nd the equtions representing the reltionship re given y Y = x (R 2 = 0.94) nd Y = x (R 2 = 0.86), respetively (Fig. 1d). The reltive root surfe re of rie seedlings for oth vrieties showed similr trend. The equtions representing the reltionships re given y Y = x (R 2 = 0.71) nd Y = x (R 2 = 0.72), respetively (Fig. 2d). In Fig. 2d, it is shown tht the ritil ph vlues for rie vrieties MR 219 nd MR 253 growth re 6.1 nd 6.2, respetively. This mens tht MR 219 nd MR 253 were more or less omprle in terms of their tolerne to low ph stress. 705

5 Ali et l. / Int. J. Agri. Biol., Vol. 17, No. 4, 2015 A B d e e d C D E F Fig. 3: Root surfe of rie seedlings viewed using SEM fter 14 dys tretment. A) MR 219 t 0 µm of Fe + Al showing smooth surfe. B) MR 219 root surfe t 100 µm of Fe + Al; note shrinking surfe due to Al nd Fe stress. C) MR 253 t 0 µm of Fe + Al showing smooth surfe. D) MR 253 root surfe t 100 µm of Fe +Al; note torn surfe tissue Al nd Fe stress. E) Root surfe t ph 7 id sulfte soil solution showing; note root hirs developed very well. F) Root surfe under low ph SEM oservtions on the ellulr strutures of the roots showed tht oth rie vrieties were ffeted y the high onentrtion of Al in omintion with Fe. Fig. 3 shows tht root ellulr strutures of MR 219 shrnk due to the presene of Al plus Fe t 100 µm, while those of MR 253 were ruptured. The roots of the rie seedlings were lso ffeted y the low ph s shown in Fig. 3F. For rie growing under high ph ondition, its root hirs were well developed; however, when the ph ws redued, the root hirs were either sent or their development ws signifintly urtiled. Seretion of Orgni Aids y Rie Root under Al Stress Fig. 4 nd 5 show orgni ids exuded y MR 253 ws higher ompred to those of MR 219. Mlte exuded y MR 253 ws proly tivted y the exposure to Al nd e d ws signifintly higher ompred to tht of MR 219. However, there is no signifint differene in itrte nd oxlte exudtion etween oth vrieties t low Al onentrtion. Fig. 4 shows tht the root of MR 219 sereted high mount of orgni ids t high Al onentrtion. The exudtion of orgni ids y MR 253 showed the sme trend s tht of MR 219 (Fig. 5). Orgni Aids Seretion under Fe Stress d Fig. 4: Exudtion of orgni ids y rie root MR 219 under ) Al onentrtion ) Fe onentrtion ) Al in omintion with Fe onentrtion As depited in Fig. 4 nd 5, oth rie vrieties sereted high mount of orgni ids t high onentrtion of Fe, with MR 253 sereted more orgni ids ompred to tht of MR 219, espeilly itrte nd oxlte. For MR 219, the root sereted higher mount of mlte ompred to tht of MR 253. Orgni Aids Seretion under the Stress of Al in Comintion with Fe It is seen in Fig. 4 nd 5 tht orgni ids exuded y MR 706

6 Rie Seedlings Affeted y Aluminum, Iron nd/or Low ph Stress / Int. J. Agri. Biol., Vol. 17, No. 4, ws higher thn tht of MR 219. Mlte, itrte nd oxlte exuded y MR 253 ws tivted y the exposure to Al nd Fe nd were signifintly higher ompred to tht of MR 219. The root of MR 219 sereted more orgni ids t high Al nd Fe onentrtion ompred to those t low onentrtion (Fig. 4). As the Al nd Fe inresed, the exudtion of orgni ids ws inresed. The exudtion of orgni ids y MR 253 showed the sme trend s tht of MR 219 (Fig. 5). d d Orgni Aids Seretion under Low ph Stress The exudtion of orgni ids due to H + stress for vriety MR 219 nd MR 253 is depited in Fig. 6 nd Fig. 7, respetively. It ws oserved tht the root of MR 219 sereted higher mount of orgni ids t low ph ompred to those t high ph. As the ph inresed, the exudtion of orgni ids ws redued. The trend for the exudtion of orgni ids y MR 253 ws similr to tht of MR 219 (Fig. 7). d d d d d Disussion The soil under study ws true id sulfte soil. It ws lssified s Typi Sulfquept (Soil Survey Stff, 2010), shown y its low soil ph (< 3.5) nd the presene of yellowish jrosite within the 0 50 m depth (Shmshuddin, 2006). Under this ondition, the onentrtion of Al nd Fe in the wter ws expeted to e ove the ritil level for rie growth (Shmshuddin et l., 2013, 2014). The wter ph ws lso expeted to e elow the ritil level of 6 (Elis et l. 2011). The onentrtion of Al in wter is dependent on its ph. As the ph flls elow 5.0, the onentrtion of Al inreses. In the urrent study, the ph of the wter ws 3.98; hene, Al onentrtion ws expeted to e high, exeeding the ritil level for rie growth (Shmshuddin et l., 2013, 2014). Al onentrtion in the wter of 1060 µm ws fr ove the ritil level of 15 µm (Elis et l., 2011). Dt in Tle 1 show tht the exhngele C nd Mg were elow the ritil level. The required level of C is 2 mol kg -1 soil (Plhres, 2000), while tht of Mg is 1 mol kg -1 soil (Doermnn nd Firhurst, 2000). C nd Mg defiienies n e llevited y pplying ground mgnesium limestone (GML) t the pproprite rte (Shmshuddin, 2006, 2014). Due to high Al onentrtion, the growth of rie root ws inhiited. Mny studies hve een onduted to explin the uses of Al inhiiting the growth of rop roots. For instne, Lio et l. (2006) found tht non-tolernt Al plnts were dly ffeted y exposure to Al. Al disturs the growth of ells in the elongtion zone of roots (Bin et l., 2013). Furthermore, Brker nd Pilem (2007) explined tht Al n redue root ell division; hene, it uses disruption of root p proesses, inhiiting root elongtion. We elieved tht Al n ffet rie roots in similr fshion. Elis et l. (2011) explined tht solule Al 3+ umulted d d d in the rie root tissue prevented ell division nd eventully its elongtion. It is likely tht rie vriety MR 253 ws more tolernt to Al ompred to tht of MR 219 (sed on root surfe re). However, dt from root length study showed otherwise. It proly mens tht these two rie vrieties hve no differene in terms of their tolerne to Al toxiity. We elieved tht more Al 3+ were umulted in the root pex (elongtion zone, root p nd meristem) ompred to tht of the mture root tissue. Initilly, the root pex rpidly ound Al 3+ in the poplst with the rte eoming lower therefter (Bin et l., 2013). The rpid inding of Al ws proly due to the presene of peti mtrix in the poplst ell wlls. This peti mtrix ontined roxyli groups tht were negtively-hrged nd hve high ffinity for Al 3+ (Chng et l., 1999). The inding etween the peti mtrix nd Al used loosening of enzyme in the ell wlls, resulting in the loss of its funtion. Hene, it deresed ell wll extensiility physilly nd physiologilly. Thus, the root length ws Fig. 5: Exudtion of orgni ids y rie root MR 253 under ) Al onentrtion ) Fe onentrtion ) Al in omintion with Fe onentrtion 707

7 Ali et l. / Int. J. Agri. Biol., Vol. 17, No. 4, 2015 Fig. 6: Exudtion of orgni ids y rie root MR 219 under ph stress Fig. 7: Exudtion of orgni ids y rie root MR 253 under ph stress redued nd the root growth ws urtiled due to less nutrients uptke (Wehr et l., 2004). Plsm memrne n ts s rrier of Al euse ions like polyvlent Al 3+ re insolule in the lipid ilyers. However, Klug et l. (2011) found tht up to one hlf of the totl of Al in the root pex ws loted in the symplsm. When Al rossed the memrne, mny potentil hrmful intertions n our. Al ound to the lipid ilyer inhiited trnsport, displed ions from ritil sites nd disrupted intrellulr metolism (Delhize nd Ryn, 1995). The ritil level for Fe vries depending on the vegettive stge of rie growth. Aording to Doermnn nd Firhurst (2000), highly tolernt rie vriety n withstnd Fe of up to 300 mg kg -1. In id sulfte soils, extrtle Fe vries widely depending on the environmentl onditions. Fe 2+ (the form of iron toxi to rie) onentrtion in wter inreses with flooding nd it my exeed the ritil level. For this study, the soil ws tken during dry seson nd the extrtle Fe in the topsoil ws less thn 300 mg kg -1, ut t the lower depth it exeeded this vlue. Fe onentrtion in the wter ws 192 µm, wy ove the respetive ritil level of nd 8.76 µm found for MR219 nd MR 253. Hene, rie plnted on this soil is expeted to suffer from Al 3+ nd/or Fe 2+ stress (Zhu et l., 2009; Shmshuddin et l., 2013, 2014). Root growth ws lso inhiited y the exess of Fe. The pk of Fe is 3, so its vilility in the wter ws very high. This metl n e tken up y rie roots in the form of Fe 2+. This form of Fe would e ville when the pddy field ws flooded for rie ultivtion (Muhrizl et l., 2006). Plnts hve two distint mehnisms to tke Fe, Strtegy I nd Strtegy II (Mrshner, 1995). Strtegy I involves idifition, redution nd trnsporttion t the plsm memrne of the root epiderml ells, while Strtegy II is seretion of phytosiderophores in response to Fe defiieny. For rie, it n dpt oth mehnisms euse it is growing under neroi onditions where solule iron is prevlent. Fe 2+ is tken up t the root ortex, whih enter the xylem. When Fe 2+ ws undnt within the ell, it might tlyze the tive oxygen genertion (Mrshner, 1995). The tivity of phenol oxidses inresed nd the oxidized polyphenols lter umulted, resulting in Fe toxiity (Mthis nd Ash, 2005). It is limed tht MR 253 is slightly tolernt to idity. However, our results indited no signifint differene etween MR 253 nd MR 219. We found tht t very low ph, the root hirs of oth vrieties were either sent or poorly developed, ut when the ph ws ove 6, the root hirs were in perfet order. This ws euse rie requires the ph of out 6 to grow optimlly. But, wter ph in our study re ws It mens tht under nturl onditions, id sulfte soils in Mlysi re unsuitle for rie ultivtion. The ritil wter ph for oth rie vrieties ws out 6, whih is similr to tht otined y Elis et l. (2011). This ph level is rther unusul for the res overed y id sulfte soils in Mlysi (Shmshuddin, 2006). To rise wter ph to the required level we need t lest to pply 4 t GML h -1 (Rosilwti et l., 2014). Even t this rte of lime pplition, wter ph ws elow 5, mening tht the rie ws still sujeted to Al toxiity (Shmshuddin et l., 2013, 2014). Liming t higher rte is uneonomil nd therefore not sustinle in the long run. However, due to GML pplition t 4 t h -1 rie is le to grow nd produes resonle yield due to its ility to protet itself ginst idity (Shmshuddin et l., 2014). The ph of wter is dependent on Al nd/or Fe onentrtion; the respetive pk of these metls is 5 nd 3. At ph elow their pk, the metls dissolved redily to exist in their ioni forms. In the urrent study, the ph of the wter tken from the pddy field ws 2.98, while the onentrtion of Al nd Fe were 1060 nd 192 µm, respetively. Thus, it ws possile tht the orgni ids so exuded y the rie roots t low ph ws not due to H + stress only, ut more importntly euse of the presene of high onentrtion of Al nd/or Fe. This explntion is onsistent with tht of Pineros nd Kohin (2009) who explined tht less orgni 708

8 Rie Seedlings Affeted y Aluminum, Iron nd/or Low ph Stress / Int. J. Agri. Biol., Vol. 17, No. 4, 2015 ids were sereted y the rie roots t high ph due to the immoiliztion of the phytotoxi Al 3+. If the orgni ids were indeed exuded due to low ph stress s it hppens in orn, the plusile mehnism would e s follows. We know tht the pk of mny orgni ids rnges from 4 to 5 (De Conink, 1978). In the se of orn, oxli id hydrolyzes following this eqution: H 2 C 2 O 4 + H 2 O HC 2 O H 3 O +, pk > 4 At low ph (for exmple 3), oxli id ws exuded y the rie roots. If the wter ph ws elow its pk vlue, the retion would e in the reverse order. Tht mens t the solution-root interfe, solution ph would pproh the vlue of 4. Hene, the stress on rie due to low ph ondition ws somewht llevited. Citri, oxli nd mli ids were relesed y MR 219 nd MR 253 under stress. Aording to Yng et l. (2013), the effiy of orgni ids in the lleviting Al nd Fe toxiity ws in the order of itri > oxli > mli. This finding is onsistent with the study of Bin et l. (2013) nd Miysk et l. (1991) who found tht luminum-tolernt rie ultivr relesed more itrte thn oxlte. The seretion of orgni ids ould hve ourred immeditely fter the positively-hrged Al 3+ ions touhed the negtively-hrged memrne surfes nd petin ell wlls (Horst et l., 2010). One this hppened, the orgni ids so relesed from the root ells ound the Al 3+ to form orgno-al-omplex, preventing it from umulting in the poplst tht ould dmge the root ells. Hene, the Al 3+ ions were detivted nd no longer toxi to the rie seedlings. In the urrent study, we found tht orgni ids exuded y MR 253 ws higher thn tht of MR 219, whih mens tht MR 253 ws le to redue more Fe toxiity, mking the seedlings grow etter ompred to tht of MR 219. However, this hs not een the se. We oserved tht the root length nd root surfe re of MR 219 were higher nd hd higher ritil Fe vlue ompred to tht of MR 253 even though the seretion of orgni ids for MR 219 showed otherwise. In onlusion, oth MR 219 nd MR 253 rie vrieties were ffeted signifintly y low ph nd high Al nd/or Fe onentrtion, shown y the derese in root length nd root surfe re with inresing Al nd/or Fe onentrtion. At very high Al nd/or Fe onentrtion, the ellulr strutures of the rie roots were severely dmged. The respetive ritil Al onentrtion for MR 219 nd MR 253 rie vrieties ws 8.91 nd µm, while tht of Fe ws nd 8.76 µm. On the ontrry, the root length inresed s the ph of the wter inresed. The est ph for the growth of oth rie vrieties ws out 6. Under dverse onditions, rie hs mehnisms to protet itself ginst Al nd/or Fe toxiity. When the onentrtion of Al nd/or Fe ws high its roots sereted orgni ids tht helted Al nd Fe, rendering them unville for the uptke y the rie roots. Aknowledgements We wish to knowledge Universiti Putr Mlysi nd the Ministry of Edution, Mlysi (the reserh is funded y LRGS Food Seurity) for finnil nd tehnil supports. 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