Bradyrhizobium improves nitrogen assimilation, osmotic adjustment and growth in contrasting cowpea cultivars under drought

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1 AJCS 7(13): (213) ISSN: Brdyrhizoium improves nitrogen ssimiltion, osmoti djustment nd growth in ontrsting owpe ultivrs under drought Mri Antoni Mhdo Bros 1, Alln Klynger d Silv Loto 1*, Dniel Ken Yuen Tn 2, Géli Dinh Monteiro Vin 1, Kelly Nyr Nsimento Coelho 1, José Rirdo Sntos Bros 1, Mrele de Cássi Henriques dos Sntos Mores 1, Roerto Cezr Loo d Cost 1, Benedito Gomes dos Sntos Filho 1, Cândido Ferreir de Oliveir Neto 1 1 Núleo de Pesquis Bási e Aplid d Amzôni, Universidde Federl Rurl d Amzôni, Prgomins, Brzil 2 Fulty of Agriulture nd Environment, University of Sydney, Sydney, NSW 26, Austrli *Corresponding uthor: llnlloto@yhoo.om.r Astrt This study investigted the effet of inoultion with Brdyrhizoium spp. on nitrogen metolism nd growth of two ontrsting Vign unguiult ultivrs exposed to oth inoultion nd wter defiit. The study ws rried out in Universidde Federl Rurl d Amzôni (1 27 S nd W). The plnts remined in greenhouse environment. The experimentl design used ws ompletely rndomised with 2 ultivrs (drought tolernt nd sensitive) omined with 2 wter regimes (wter defiit nd ontrol), nd 2 inoultion tretments (inoulted nd non-inoulted), totlizing 8 tretments. The vriles evluted were lef reltive wter ontent, nitrte redutse tivity, nitrogen ompounds, nd growth prmeters. Results of this study showed tht oth ultivrs presented inreses in nitrte nd proline, ompred with non-inoulted plnts. Wter defiieny indued more intense redutions in shoot dry mtter of non-inoulted plnts, ompred with inoulted plnts. This investigtion onfirmed the hypothesis tht inoultion with Brdyrhizoium improves nitrogen ssimiltion, osmoti djustment nd growth prmeters in Vign unguiult plnts. Keywords: Nitrogen metolism; Vign unguiult [L.] Wlp.; leguminous; iologil fixtion of nitrogen; inoultion; wter defiit. Arevitions: N 2 _nitrogen; H + _hydrogen; NH 4 + _mmonium; E.C _nitrte redutse; GS_glutmine synthetse; ATP_ denosine-5'_triphosphte; GOGAT_glutmte synthse; P5CR_pyrrolline-5-roxylte redutse; NO 3 - _nitrte; NO 2 - _nitrite. Introdution Wter defiit is n ioti ftor tht ffets griulturl prodution, influening spets relted to plnt development, suh s derese in photosynthesis rte, redution in lef re (Fontn et l., 1992), nd stomt losing (Sntos nd Crlesso, 1998). As in other rops, performne in owpe is ffeted y wter defiieny, whih n use lower growth nd development, with progressive redution in lef dry mtter (Cost et l., 211) nd onsequent redution in yield nd yield prmeters suh s the numer of grins nd pods per plnt. Cowpe (Vign unguiult (L.) Wlp.) plnts re onsidered tolernt to wter defiit, nd importnt mehnisms hve een developed y this speies to tolerte indequte wter supply. For exmple, iohemil modifitions in ron metolism, suh s inrese in surose (Loto et l., 29); s well s interferene in nitrogen metolism, suh s redution of solule proteins (Cost et l., 211) nd inrese in totl mino ids (Loto et l., 28) ontriute to osmoti djustment of Vign unguiult plnts. The iologil fixtion of nitrogen is the pity of distint group of miroorgnisms to divide the moleule of nitrogen (N 2 ) nd to omine hydrogen toms (H + ), forming mmonium (NH 4 + ) (Alino nd Cmpo, 21), under symiosis with root system. The Brdyrhizoium gender is desried s soil teri tht hve ility to infet root hir of leguminous plnts, nd it n indue nodule formtion, with susequent fixtion of nitrogen (Mernte et l., 22). Negtive effets of wter defiit re desried in physiologil (Ismil et l., 24), iohemil (Hmidou et l., 27), morphologil (Loto et l., 28), nd gronomil (Leport et l., 1998) spets, nd these modifitions n influene yield omponents suh s grins per pod nd pod per plnt. (Szilgyi, 23; Showemimo nd Olrewju, 27). In other hnd, the responses in morphologil prmeters hve not lwys een ssoited with nitrogen ompounds suh s inrese in proline nd redution in proteins (Cost et l., 211). Benefiil effets provided y the inoultion on growth prmeters s lef, stem nd root re well known in leguminous plnts (Rmos et l., 1999; Silveir et l., 21; Mores et l., 21), ut informtion on the speifi spets of this symioti proess on essentil ompounds suh s mino ids nd proteins re limited. Figueiredo et l. (1999) reported tht inoultion using Brdyrhizoium n llevite the negtive onsequenes in Vign unguiult plnts indued y wter defiieny, ut Serrj nd Sinlir (1996) reveled tht wter supply limits on symioti effiieny. This study imed to investigte if inoultion with Brdyrhizoium spp. n improve the plnt performne linked to nitrogen 1983

2 Nitrte (µmol NO - 3 g DM -1 ) Nitrte redutse tivity (µmol NO - 2 g -1 h -1 ) Lef reltive wter ontent (%) ssimiltion, osmoti djustment, nd growth prmeters of two ontrsting Vign unguiult ultivrs exposed to oth inoultion nd wter defiit. Results 1 8 wter defiit ontrol A Lef reltive wter ontent, nitrte redutse tivity, nd nitrte 6 e The inoulted plnts hd higher vlues linked to lef reltive wter ontent, ompred with sme tretments in noninoulted plnts (Fig 1 A), in oth well-wtered nd wter defiit tretments. Drought tolernt plnts hd lower redution in lef wter ontent under wter defiit, ompred with drought sensitive ultivrs. Wter defiit redued nitrte redutse tivity (Fig 1B) in ll tretments, when ompred to respetive ontrol. The inoultion produed n inrese in nitrte of oth ultivrs (Fig 1C), ompred to the sme tretments without inoultion. Both non-inoulted ultivrs hd lower nitrte onentrtions under wter restrition ompred with the well-wtered ontrols B Totl solule mino ids, totl solule proteins, nd proline.6.4 The onentrtion of totl solule mino ids in plnts sujeted to inoultion ws higher only in tolernt plnts, ompred with the sme tretments on non-inoulted plnts (Fig 2 A). Wter defiit promoted signifint inrese in this vrile to ll tretments, with exeption in tolernt plnts under inoultion. Wter defiieny used signifint derese in totl solule proteins oth ultivrs in inoulted nd non-inoulted plnts, with slightly greter derese for sensitive plnts (Fig 2 B). For proline the inoulted plnts presented higher vlues, ompred with sme tretments in non-inoulted plnts (Fig 2 C). The two ultivrs demonstrted higher vlues under wter limittion, ompred with their respetive ontrols de ed C Shoot dry mtter, plnt dry mtter, nd lef numer The inoultion used n inrese in shoot dry mter, ompred with the sme tretments in non-inoulted plnts (Fig 3 A). Wter defiit resulted in signifint derese in this prmeter espeilly for the sensitive ultivr. The inoultion used slight inrese in plnt dry mtter, with exeption of the sensitive ultivr under irrigtion (Fig 3 B), ompred with sme tretments in non-inoulted plnts. Wter defiieny indued derese in plnt dry mtter for ll tretments, ompred with ontrol plnts. Aross ll tretments, sensitive ultivrs hd lower plnt dry mtter ompred with tolernt ultivrs. Lef numer inresed slightly fter inoultion, with exeption of sensitive plnts under wter defiieny (Fig 3 C). Wter defiit redued lef numer for ll plnts, ompred with well-wtered ontrol plnts. The tolernt ultivr hd higher lef numer, ompred with the sensitive ultivr. Disussion The inoultion promoted ttenution of the negtive effets indued y wter limittion on lef reltive wter ontent. The derese of this vrile n e explined y the redution in wter vilility in sustrte (Lwlor nd Corni, 22), euse the wter defiieny indues turgor loss nd onsequent stomtl losing, whih will ffet other physiologil proesses suh s photosynthesis nd tolernt sensitive tolernt sensitive inoulted non-inoulted Fig 1. Lef reltive wter ontent (A), nitrte redutse tivity (B), nd nitrte (C) in two ontrsting Vign unguiult plnts under wter defiit nd sujeted to inoultion. Mens followed y the sme letter re not signifintly different y the Sott-Knott test t 5% of proility. The rs represent the men stndrd error. trnspirtion (Nogueir et l., 1998). This redution ws lso shown y Loto et l. (29) studying two Vign unguiult ultivrs exposed to wter restrition, orroorting the dt from this reserh. The lower nitrte redutse tivity in inoulted plnts ould e due to nitrogen eing sored in the N 2 form nd trnsformed into NH 4 in the symioti proess medited y nitrogen fixing teri (Gerhty, 1992; Tiz nd Zeiger, 24), using the nitrogense enzyme (Burris, 1999). In ddition, Silveir et l. (21) desried n intense tivity of the nitrte redutse in root nd/or nodules n ompenste the derese of this enzyme in leves. But further studies will e neessry to understnd the regultory mehnisms of this metoli pthwy. The redution of this enzyme during wter defiit is explined y Mhdo et l. (1992); y negtively ffeting 1984

3 Proline (µmol g DM -1 ) Totl solule proteins (mg g DM -1 ) Totl solule mino ids (µmol g DM -1 ) the nitrogen metolism, s well s the nitrte redutse eing extremely sensitive to wter vilility (Cost, 1999). Serrj nd Sinlir (1996) reported tht severl tropil leguminous suh Vign unguiult re prtilly ffeted y the wter restrition. Ferreir et l. (22) lso otined lower tivity of this enzyme with wter defiieny in two Ze mys genotypes. In this study, the nitrte onentrtion ws inresed y the inoultion, nd this effet might e onsidered indiret. The inrese in onentrtion of orgni nitrte in inoulted plnts under wter defiit is response indued y derese in nitrte redutse (NR) tivity, euse the nitrte ts s sustrte in retion tlyzed y nitrte redutse enzyme, nd enzyme is responsile for the redution of nitrte (NO 3 - ) from nitrite (NO 2 - ) (Ferreir et l., 22). In other words, the fll in nitrte redutse tivity (Figure 1 B) promotes umultion of orgni nitrte. The redution in nitrte in non-inoulted plnts nd under drought is ssoited with the lower ssimiltion of nitrogen in nitrte form. Similr results on the inrese of nitrte in leves were desried y Delfini et l. (21) investigting different tissues in Arhis hypoge plnts indued y inoultion of Brdyrhizoium sp. Inoulted drought tolernt plnts showed inrese in mino ids, nd this n e ttriuted to iologil fixtion of nitrogen. The nitrogense enzyme promotes the nitrogen sorption in form of nitrogen gs (N 2 ) nd onversion to mmonium (NH 4 + ). In ddition, the inresed formtion of mino ids is proly linked to n inrese in tivity of the enzyme glutmine synthetse (GS), whih depends ATP (denosine-5'- triphosphte), nd glutmte synthse (GOGAT). In ddition, the inrese in mino ids of plnts exposed to inoultion is due to greter flux nd etter ssimiltion of nitrogen in form of mmonium, onomitntly with higher tivity of GS nd GOGAT enzymes. Rmos et l. (25) evluting the responses of Glyine mx plnts under wter defiit nd inoultion of Brdyrhizoium jponium lso oserved n inrese in onentrtion of totl solule mino ids. The onentrtion of totl solule mino ids in plnts under wter defiit inresed in ll tretments. This inrement proly ourred due to inrese in tivity of protese enzymes, responsile for the rekdown of proteins to djust the plnt osmotilly (Cost et l., 26). For tolernt plnts, this prmeter ws higher in inoulted thn non-inoulted tretment, however n opposite tendeny showed in sensitive plnts exposed to non-inoultion nd drought, eing explined y the sensiility of this ultivr under ondition of wter restrition. Similr dt on inrese in mino ids were otined y Cost et l. (1996) investigting Vign unguiult plnts. Delfini et l. (21) evluting the responses of two Arhis hypoge ultivrs inoulted with Brdyrhizoium sp. showed signifint inrese in mino ids. The inrese in totl solule proteins indued y inoultion suggests tht teri tion resulted in inrese in nitrogen supply through seondry route, tht is regulted y the nitrogense, euse sine there ws no orresponding inrese in tivity of nitrte redutse fter inoultion in this study. Hristozkov et l. (26) evluting the responses in Pisum stivum plnts under inoultion nd molydenum pplition lso otined n inrese in protein levels. The derese in protein levels promoted y the wter defiit is ssoited with derese of the protein synthesis omined with n inrese of proteolyti enzymes, responsile y rekdown of solule proteins in plnts (Lehinoshi et l., 27). Cost (1999) otined similr results studying Vign unguiult sujeted to wter defiit, orroorting our results. The inrese of proline levels indued y the inoultion ws proly linked to etter wter defiit d d d ontrol d tolernt sensitive tolernt sensitive inoulted non-inoulted Fig 2. Totl solule mino ids (A), totl solule proteins (B), nd proline (C) in two ontrsting Vign unguiult plnts under wter defiit nd sujeted to inoultion. Mens followed y the sme letter re not signifintly different y the Sott-Knott test t 5% of proility. The rs represent the men stndrd error. mino id utilistion suh s glutmi id nd rginine, glutmi id eing the preursor of the proline, while rginine n suffer from retion medited y the enzyme, pyrrolline-5-roxylte redutse (P5CR) nd onsequently lierte proline (Rmos et l., 25). Kohl et l. (1991) lso oserved higher mounts of proline in Glyine mx plnts inoulted with Brdyrhizoium jponium, supporting this study. The inrese of proline in plnts under wter defiit is response to loss of ell turgidity (Oliveir et l., 26). Nogueir et l. (1998) stted tht proline umultion ws relted to drought tolerne in higher plnts, tuting s osmoregultor gent to keep wter in plnt tissues (Muhow nd Crerry, 1993). Similr ehviour ws desried y González et l. (1998) working with Pisum stivum plnts under wter restrition. Shoot dry mtter ws mximised fter the inoultion, nd this ft n e explined y inrese in e d e A B C

4 Shoot dry mtter (g) Lef (numer) Plnt dry mtter (g) wter defiit e d ontrol tolernt sensitive tolernt sensitive inoulted ed non-inoulted Fig 3. Shoot dry mtter (A), plnt dry mtter (B), numer of leves (C) in two ontrsting Vign unguiult plnts under wter defiit nd sujeted to inoultion. Mens followed y the sme letter re not signifintly different y the Sott- Knott test t 5% of proility. The rs represent the men stndrd error. totl solule proteins, with onsequent etter supply of nitrogen, in whih is essentil to dequte plnt development (Epstein nd Bloom, 26; Souz et l., 28). Similr dt linked to shoot dry mtter were reported y Figueiredo et l. (1999) with Vign unguiult plnts exposed to Brdyrhizoium inoultion. Drought redued the prodution of shoot dry mtter due to inhiition in iohemil proesses suh s iologil fixtion of nitrogen (Cost et l.,1996), indiretly modifying the prtitioning of photo-ssimiltes in root nd shoot, nd onsequently resulting in derese in umultion of shoot iomss (Correi nd Nogueir, 24). Similr results were found y Mendes et l. (27) working with two Vign unguiult ultivrs sujeted to wter defiieny during two stges. The inoultion inresed totl dry mtter, nd this my e linked to etter development A B C nd effiieny of root system, providing higher nitrogen sorption through the nodultion proess. In ddition, the higher nitrogen fixtion will produe n inrese in mino ids nd proteins (Delfini et l., 21), nd lso photossimilte vilility. Similr responses were desried y Sssi et l. (21) investigting two Phseolus vulgris ultivrs sujeted to inoultion with teri of Rhizoium genus. Plnts under wter defiit frequently hve the prodution of dry mtter redued, due to the derese in severl metoli proesses suh s wter nd nutrient sorption, whih re fundmentl to mintin dequte growth nd development rtes. Nsimento (29) lso reported tht wter deprivtion ffets the osmoti mehnism, nd y onsequene redues the CO 2 supply, essentil for the photosyntheti proess. Similr results were found y Leite nd Virgens Filho (24) studying Vign unguiuld plnts exposed to wter limittion. The inrese in lef numer promoted y inoultion my e proly due to the higher numer of nodules in root, nd onsequently due to the etter iologil fixtion of nitrogen (Ferreir et l., 211). Arújo et l. (29) studying Vign unguiult nd Leuen leuoephl plnts lso reported n inrese of this vrile, onfirming our dt. The lower lef numer fter wter defiieny is used y the proess of lef sission, due to insuffiient wter nd nutrient to the plnt (Sntos nd Crlesso, 1998). Correi nd Nogueir (24) otined similr results with Arhis hypoge plnts under wter defiit. Mterils nd methods Growth onditions The study ws rried out in Instituto de Ciênis Agráris (ICA) of the Universidde Federl Rurl d Amzôni (UFRA), Belém ity, Prá stte, Brzil (1 27 S nd W). The plnts remined in greenhouse environment under nturl sunlight onditions, with ir temperture minimum nd mximum of 24.1 nd 38.2 C, respetively. Air reltive humidity osillted etween 72 nd 89%. The photoperiod ws 12 h of light. Plnt mterils, sustrte nd pot The Vign unguiult (L.) Wlp. seeds used in this study were Pitiu nd Pérol ultivrs, in whih re tolernt nd sensitive to wter defiit, respetively (Loto et l., 29). The sustrte used for the plnt growth ws omposed y mixture of snd nd sili in the proportion of 2:1, respetively, nd it ws utolved t 12 C tm -1 for 4 min. The ontiner used for the plnt growing ws Leonrd pot with 2 L pity, dpted in Lortório de Fisiologi Vegetl Avnçd (LFVA). Experimentl design The experimentl design used ws ompletely rndomised with 2 ultivrs (drought tolernt nd sensitive), 2 wter regimes (wter defiit nd ontrol), nd 2 inoultion tretments (inoulted nd non-inoulted), mking up 8 tretments, with 6 replites nd 48 experimentl units, in whih eh experimentl unit onsisted of 1 plnt pot -1. Inoultion nd ondution of plnt Three seeds per pot were sowed nd thinned out to 1 seedling fter germintion. The seedlings were inoulted with 1 ml of 1986

5 Brdyrhizoium spp. (BR 3256) suspension with onentrtion of CFU y 3 times, nd t regulr intervls on the 5 th, 1 th nd 15 th dy fter the experiment strted. The ontrol nd wter defiit tretments reeived mro nd mironutrients from nutrient solution of Hoglnd nd Arnon (195), for 3 dys, nd the nutrient solution ws hnged with 2 dys of intervl, lwys t 9: h. The ph of the nutrient solution ws djusted to 6. ±.1 with ddition of HCl or NOH. On the 3 th dy fter the experiment strted, the plnts of the tretment under wter defiit were sumitted to period of 5 dys without nutrient solution, in whih the defiit ws simulted from the 3 th until 35 th dy fter of the experiment strted. Numer of leves At the end of the experimentl period, the plnts were hrvested nd numer of leves ounted, inluding the first pir of leves. Susequently, lef fresh mtter ws used to determine lef reltive wter ontent nd nitrte redutse tivity. Lef reltive wter ontent Lef reltive wter ontent ws evluted on lef disks of 1 mm dimeter from eh plnt; 4 disks were removed nd the lultion ws done using the formul proposed y Slvik (1979): LRWC = [(FM DM)/(TM DM)] 1 Where: FM is fresh mtter, TM is turgid mtter evluted fter 24 h sturtion in deionised wter t 4 C in drk, nd DM is the dry mtter determined fter 48 h in oven with fored ir irultion t 8 C. In vivo nitrte redutse tivity redutse enzyme (E.C ) ws extrted from 2 mg of lef nd root smples. nd inuted in 5 ml of extrtion uffer (KH 2 PO 4 t.1 M, KNO 3 t 5 mm, isopropnol t 1% (v/v) nd ph 7.5) for 3 minutes t 3ºC, nd ll the proedures were rried out in the drk. The quntifition of the enzyme tivity ws in ordne to the method of Hgemn nd Huklesy (1971) with sorne t 54 nm using spetrophotometer (Quimis, model Q798DP). Shoot dry mtter, plnt dry mtter, nd smple preprtion Lef, stem nd root were divided nd dried in n oven with fored ir irultion t 7 ± 2º C y 96 h. After drying, shoot dry mtter (lef nd stem) nd plnt dry mtter (lef, stem nd root) were determined. Susequently, plnt tissues were triturted, with the resulting powder kept in glss ontiners. These ontiners remined in the drk t 15ºC until they were redy for iohemil nlysis. Nitrte, totl solule mino ids, nd proline In determintion of nitrte, mino ids nd proline were performed using 5 mg of lef dry mtter powder, nd inuted with 5 ml of sterile distilled wter t 1ºC for 3 minutes. After inution, the homogenised ws entrifuged t 2. g for 5 minutes t 2ºC nd superntnt ws removed. The quntifition of the nitrte ws rried out t 41 nm in ordne with Ctldo et l. (1975), with KNO 3 (Sigm Chemil) s stndrd. Quntifition of the totl solule mino ids ws rried out t 57 nm ording to the method of Peoples et l. (1989), nd L-sprgine + L- glutmine (Sigm Chemils) ws used s stndrd. The quntifition of proline ws performed fter mesuring the sorne t 52 nm ording to Btes et l. (1973) sed on L-proline (Sigm Chemils) s stndrd. Totl solule proteins Determintion of the totl solule proteins ws rried out with 1 mg of powder, inuted with 5 ml of extrtion uffer (Tris-HCl t 25 mm nd ph 7.6). This ws homogenised nd kept in gittion for 2 h, nd entrifuged to 2. g y 1 minutes t 2ºC. Quntifition of the totl solule proteins ws rried out t 595 nm in ordne with Brdford (1976), with lumin ovine (Sigm Chemils) s stndrd. Dt nlysis Dt were sujeted to vrine nlysis nd when signifint differenes were deteted, Sott-Knott test t 5% level of error proility ws pplied (Steel et l., 26). Stndrd errors were lulted for ll mens. All sttistil proedures were rried out with the SAS softwre (SAS, 1996). Conlusions Inoultion with Brdyrhizoium spp.indued inreses in shoot dry mtter, nd lso higher levels of nitrogen ompounds suh s nitrte nd proline, ompred with the sme tretments in non-inoulted plnts. This investigtion onfirmed the hypothesis tht inoultion promotes improved nitrogen ssimiltion, osmoti djustment nd growth prmeters in Vign unguiult plnts. Aknowledgments This reserh hd finnil support from Conselho Nionl de Pesquis (CNPq/Brzil) for R.C.L. Cost nd A.K.S. Loto. Referenes Alino UB, Cmpo RJ (21) Effet of soures nd levels of molydenum on Brdyrhizoium survivl nd on iologil nitrogen fixtion in soyen. Pesq Agrope Brs 36: Arújo ASF, Crneiro RFV, Bezerr AAC, Arújo FF. (29) Co-inoultion rhizoi nd Bilus sutilis in owpe nd Leuen: effets on nodultion, N 2 fixtion nd plnt growth. Cien Rurl 4: Btes LS, Wldren RP, Tere ID (1973) Rpid determintion of free proline for wter-stress studies. Plnt Soil 39: Brdford MM (1976). A rpid nd sensitive method for the quntittion of mirogrm quntities of protein utilizing the priniple of protein-dye inding. Ann Biohem 72: Burris RH (1999) Advnes in iologil nitrogen fixtion. J Ind Miroiol Biot 22: Ctldo DA, Hroon SLE, Yougs VL (1975) Rpid olorimetri determintion of nitrte in plnt tissue y nitrtion of sliyli id. Comm Soil Si Plnt Anl 6: Correi GK, Nogueir CMJR (24) Evlution of the growth of groundnut (Arhis hypoge L.) sujeted to wter defiit. Rev Biol Ciên Terr 4:

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