Yield and quality of maize following the foliar application of a fertilizer based on the byproduct shale water

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1 Vol.4, No.12A, (13) Agriulturl Sienes Yield nd qulity of mize following the folir pplition of fertilizer sed on the yprodut shle wter Rfel d Silv Messis 1,2*, Vness Glli 1,3, Mnoel Artigs Shirmer 2, Clenio Nilto Pillon 1, Sérgio Delmr dos Anjos e Silv 1, Crlos Augusto Posser Silveir 1, Cesr Vlmor Romldi 2 1 Emrp Clim Temperdo, Pelots, Brzil; * Corresponding Author: rfel.emrp@yhoo.om.r 2 Fuldde de Agronomi Eliseu Miel, Universidde Federl de Pelots, Pelots, Brzil 3 Centro de Biotenologi, Universidde Federl do Rio Grnde do Sul, Porto Alegre, Brzil Reeived 24 Septemer 13; revised 2 Novemer 13; epted 28 Novemer 13 Copyright 13 Rfel d Silv Messis et l. This is n open ess rtile distriuted under the Cretive Commons Attriution Liense, whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited. ABSTRACT The wter extrted from the shle rok (shle wter) through the pyrolysis proess to otin fuel oil nd other produts shows omposition sed on orgni ompounds nd wide rnge of minerls nd tre elements with n importnt role in plnt nutrition, suggesting its use s fertilizer. Thus, the influene of folir pplition of shle wter (SW), with or without the mironutrients zin (Zn), mngnese (Mn), opper (Cu), oro (B) nd molydenum (Mo), ws evluted regrding yield nd qulity of mize grins. The yield, the totl ntioxidnt tivity, nd the ontent of strh, phenoli ompounds nd rotenoids were improved in mize grins following the pplition of three doses of 7 L/h of SW, whih indites tht SW my influene the primry nd seondry metolisms. The pplition of SW with mironutrients resulted in the inrese of grin yield; however, did not result in the improvement of grin qulity. The folir fertilizer formultions lso hd n influene in the ontent of minerls nd minoids of the grin. The results indite tht SW hs potentil to e used in griulture to improve yield nd qulity of mize. Keywords: Mize; Shle Wter; Folir Applition; Grin Qulity; Biofortifition 1. INTRODUCTION Nowdys, the serh for food with nutritionl nd funtionl qulity, free of toxi residues, s well s environmentl friendly produed, is worldwide tendeny. Mjor erel rops re stple foods tht hve the role to provide gret mount of dietry mronutrients suh s rohydrtes, lipids nd proteins, mironutrients suh s minerls nd vitmins, s well s funtionl ompounds, whih n improve humn helth [1,2]. In prtiulr, mize is one of the mjor rops ultivted over the world, minly in developing ountries, with vried rnge of onsumed forms nd utiliztions. Beuse of this, the efforts for the iofortifition of this rop re of interest [3,4]. Mize grins re onsiderle soure of poliphenol ntioxidnts, espeilly phenoli ids suh s feruli, ffei nd ρ-umri ids [5]. This erel lso displys gret nturl vrition for rotenoid omposition, inluding the xnthophylls lutein nd zexnthin nd vitmin A preursor s α-rotene, β-rotene, nd β-ryptoxnthin [2,6]. Most of these ompounds hve n importnt role s ntioxidnts nd regultors of the humn immune system, resulting in the prevention of rdiovsulr diseses, s well s severl types of ner nd other ge-relted diseses [4,7]. Therefore, phenoli ompounds nd rotenoids re importnt trgets for iofortifition efforts, espeilly vitmin A preursors, euse the dietry defiieny of vitmin A is relted to eye diseses in mny developing ountries tht hve mize s their mjor dietry soure [8]. The inrese in griulturl prodution nd the purity of industril fertilizers hve resulted in depletion of soil nd redution of mironutrients phytovilility; therefore, the pplition of fertilizers hve eome n importnt iofortifition pproh to inrese minerl ontent, espeilly in erel grins, tht re reltively poor soures of minerl nutrients [3]. However, studies out minerl fertiliztion hve een foused in the improvement of rotenoids nd phenoli ompounds ontent re Copyright 13 SiRes.

2 R. S. Messis et l. / Agriulturl Sienes 4 (13) sre in the literture. Moreover, one ottlenek of this gronomi intervention is the ost nd environmentl impt of solid fertilizers, sine gret prt of them is lehed [9]. Thus, new investigtions on lterntive nd nturl soures of fertilizers nd/or iostimulnt produts, le to del with the grdul poorness of soil, nd to mximize plnt minerl uptke resulting in enefits in grin qulity nd yield, re of gret interest. Folir fertiliztion with mironutrients hs een intensively used in the lte yers euse this prtie llows the pplition of minerls t the pproprite time during plnt development (ording to plnt needs), it llows uniformity in nutrient distriution nd inrese in the nutrient sorption, nd onsequently it voids losses in the environment [1]. Nowdys, the use of iotive sustnes, suh s hitosn, enzothidizole, glyphoste, sliyli id nd some phytohormones, whih n e pplied onomitntly with the folir fertilizer, hve lso shown promising results in influening plnt growth nd development, plying role on gene expression, plnt defense metolism, nd in the minerl nutrients uptke [11]. The hemil omposition of shle wter (SW), the onstituent wter of shle pyroituminous rok, whih is extrted during the pyrolysis proess to otin, for instne, fuel oil, sulfur nd gs (LPG), ws reently desried [12]. The omposition of SW is sed on severl orgni ompounds, minly phenols, s well s minerls nd tre elements of interest for plnt nutrition. The pplition of SW in lettue [12] nd pepper [13] plnts resulted in the inrese of yield nd minerl ontent, suggesting its potentil s folir fertilizer. Furthermore, previous study ws performed in order to evlute the food sfety of using SW in griulture. To this purpose, SW ws pplied vi folir in the lettue rop tht ws onsidered n exellent iologil inditor of food qulity, euse it is usully eten fresh ; nd environmentl qulity, euse of its high suseptiility to ontminnts. As result, no hrmful element or ompound ws deteted in the lettue leves treted with SW, inditing tht the use of SW s folir spry in griulture repre- sents n environmentl friendly lterntive for the disposl of this y-produt [12]. Therefore, in the present study we evluted the effet of folir pplition of formultions sed on SW, delivered with or without mironutrients enrihment, on vegettive growth, yield nd qulity of mize grins. 2. MATERIAL AND METHODS 2.1. Experimentl Design nd Tretments The ommeril mize hyrid 3F53 (Pioneer ) ws grown on field in four rows per lok, 1 m long, ontining plnts per row. Plots were sped.25 m prt within rows, with rows sped.8 m prt. During sowing, 28 kg/h of the formul 1--1 (N-P-K) ws pplied, nd 3 dys fter germintion n pplition of ure (9 kg/h) ws performed. The experimentl lok design ws rndomized, with five replitions nd five tretments, s follow: C, ontrol (distilled wter); DW-M, mironutrients diluted in distilled wter; SW-M, mironutrients diluted in shle wter; SW-1, 2.5 L/h of shle wter; nd SW-2, 7 L/h of shle wter. The mironutrients used in the tretments DW-M nd SW-M were: 8% zin (Zn) in the form of ZnSO 4, 1.5% mngnese (Mn) in the form of MnSO 4, 1.5% opper (Cu) in the form of CuSO 4,.5% oro (B) in the form of H3BO3 nd.5% molydenum (Mo) in the form of (NH 4 )6Mo 7 O 24. All tretments were supplied with surftnt plus minerl oil s djuvnt nd djusted to ph using hloridri id (Vete, Rio de Jneiro, Brsil). The minerl nd orgni omposition of the SW used in the experiment ws previously desrie [12] nd is presented in Tle 1. The pplitions of the formultions were performed with the use of pressurised spryer (CO 2 ) djusted to n equivlent nozzle flow rte of 1 L/h. Three pplitions of the tretments were performed: the first two pplitions ourred during the vegettive growth period (V6-V8 nd V9-V1 stges, ording to Hnwy [14]), nd the third pplition ourred during the reprodutive phse (sprying flowering nd pollintion, stge R1, ording to Hnwy [14]). In these Tle 1. Chemil omposition of the shle wter used in the experiment. Minerl Composition mg/l µg/l S N Si Cl As Se C K Mg B Fe Cu Zn Hg Mn Orgni Composition mg/l Sulphte Totl Cresols Cynide Toluene Etilenzene m.p-xilene 4-Metilphenol Phenol 2-Metilphenol 2.4-Dimetilphenol Copyright 13 SiRes.

3 58 R. S. Messis et l. / Agriulturl Sienes 4 (13) stges the mjor nutritionl demnd of mize plnts ours [15] Yield of Mize Grins At physiologil mturity (R6 stge, ording to Hnwy, 1966), the os from the useful re (two entrl rows) were hrvested, dried t 65 C until pproximtely 13% moisture ontent, nd weighed in order to evlute yield of mize grins Amino Aids Composition nd Crohydrte Content of Mize Grins The free mino ids were extrted from 5 mg of lyophilized mize grins smples (t physiologil mturity) with 25% etonitrile in.1 N hloridri id solution, using the Ez:fst kit from Phenomenex (Torrne, CA). After entrifugtion t g, the superntnt ws filtered in.45 μm PTFE syringe nd 2 μl ws injeted in gs hromtogrphy (GC-FID, Shimdzu, Jpn) with pillry olumn ZB-AAA. The rrier gs used ws N, with the flow of the olumn djusted to 2 ml/min nd pressure of 6 kp. The flow rte vlve ws split 1:17 nd the injetion nd detetion temperture ws 28 nd 3 C, respetively. The strh nd the totl solule sugr (TSS) ontent of mize grins t physiologil mturity were quntified y the method with nthrone [16]. All determintions were mde in three nlytil replites nd five field replites Minerl Composition of Mize Grins The minerl omposition ws determined in dried mize grins smples olleted t physiologil mturity. The nitrogen (N) nd sulfur (S) ontent ws rried out on CHN-S nlyzer oupled (LECO, St. Joseph, MI). The ontent of phosphorus (P), potssium (K), lium (C), mgnesium (Mg), opper (Cu), iron (Fe), mngnese (Mn) nd zin (Zn) ws quntified y tomi sorption spetrometry (AAS) (Vrin AA24FS, Snt Clr, CA) from 25 mg of smple digested with 1 ml of H 2 O 2 nd 5 ml of HNO 3 in mirowve, ording to D Silv [17]. Mens vlue from three nlytil nd five field replites were expressed s g/kg DW for mronutrients nd mg/kg DW for mironutrients Assessment of Seondry Metolism Antioxidnt Ativity, Phenoli Compounds nd Crotenoid Content For the nlyses of seondry metolism, lyophilized grins olleted t physiologil mturity were rushed with mortr nd pestle using liquid nitrogen. The ntioxidnt tivity ws determined ording to Arno et l. [18]. The phenoli ompounds were quntified y the method from Adom nd Liu [19]. Totl rotenoids ontent ws quntified y the method from Rodriguez- Amy nd Kimur []. Dt were lulted sed on β-rotene urve Expression of Genes Involved in Crotenoid Biosynthesis To evlute the expression of genes phytoene synthse (PSY1), rotenoid ε-hydroxylse (CYP97C) nd β- rotene hydroxylse (HYD3), the entire mize grins olleted t R4 stge [14], orresponding to the pek of expression of these genes [21], were rushed with mortr nd pestle using liquid nitrogen. Three replites of eh smple were sujeted to totl RNA isoltion using CTAB modified protool, previously dpted [22]. RNA qulity ws evluted y eletrophoresis on 1% grose gel stined with ethidium romide nd y spetrometry, using A26/A28 nd A26/A23 rtio nd RNA onentrtion ws mesured in Quit fluorometer (InvitrogenTM, São Pulo, Brsil). One μg of totl RNA ws digested with 1 U DNAse, nd used for reverse trnsription using M-MLV enzyme, ording to mnufturer s instrutions (Invitrogen, São Pulo, Brsil). For RT-qPCR retions, speifi primers were designed on Vetor NTI 11 Progrm (Invitrogen, São Pulo, Brsil) for the mplifition of CYP97C (F: 5 -GTTGACATT- GGATGTGATTGG-3, R: 5 -AACCAACCTTCCAG- TATGGC-3 ), PSY1 (F: 5 -GACAGATGAGCTTGTAG- ATGGGC-3, R: 5 -TCAGAGAGAGCGGCATCAAG- CA-3 ), HYD3 (F: 5 -GGGGATTACGCTGTTCGG-3, R: 5 -GTGGTGTATCTTGTGCGAGG-3 ), ACT (F: 5 - CATGGAG-AACTGGCATCACACCTT-3, R: 5 -CTG- CGTCATTTTCTCTCTGTTGGC-3 ), GAPDH (F: 5 - ACTGTTCATGCCATCACTGC-3, 5 -GAGGACAG- GAAGCACTTTGC-3 ) nd UBI (F: 5 -GTTTAAG- CTGCCGATGTGCCTG-3, R: 5 -GACACGACTCAT- GACACGAACAGC-3 ). All mplions were designed to e less thn 15 p. ACT nd UBI were used s referene genes nd C tretment s referene smple. The effiieny of these primers ws previously verified in rel time PCR, whih ws lose to 1%. The PCR onditions used were s follows: finl volume of µl ontining 15 ng of DNA, 3-1 ρmol of eh primer, 3-5 mm MgCl 2, 1 µl Pltinum Syr UDP Mix (Invitrogen, São Pulo, Brsil). The mplifition ws stndrdized in 75 Fst therml yler (Applied BiossystemsTM, Crlsd, Clifórni) using the following onditions: 5 C for s, 95 C for 1 s, followed y 4 yles of 15 s t 95 C nd 6 s t 6 C. Melt urve onditions used ws: 15 s t 95 C, 6 s t 6 C, 3 s t 95 C nd 15 s t 6 C. The PCR produt ws sujeted to eletrophoresis on 1% grose gel for verifition of unspeifi nds. The housekeeping genes tin (ACT), Copyright 13 SiRes.

4 R. S. Messis et l. / Agriulturl Sienes 4 (13) uiquitin (UBI) nd glyerldehyde 3-phosphte dehydrogense (GAPDH) were used s referene genes. The Cq vlues otined were nlyzed ording to 2 Ct method nd presented s fold hnge [23] Sttistil Anlysis The results were sujeted to nlysis of vrine etween tretments with onfidene p-level of 1% or 5% y Tukey verge test. Spermn s test ws used to orrelte tretments. All sttistil nlysis ws onduted on Systems Anlysis Softwre nd grphs were prepred in GrphPd Prism RESULTS 3.1. Evlution of the Folir Applition of SW on Grin Yield The yield of mize following the pplition of three doses of 7 L/h of SW (SW-2) ws sttistilly higher thn the ontrol nd similr to the yield of mize following the pplition of mironutrients (DW-M), suggesting tht the SW my e used s n lterntive. In ontrst, the pplition of three doses of 2.5 L/h of SW (SW-1) did not inrese the weight nd yield vlues, inditing dose dependent effet (Figure 1()). The enrihment of SW with mironutrients (SW-M) lso resulted in yield improvement ompred to ontrol. This is in greement with studies showing tht mironutrients my ply role in the synthesis nd trnslotion of photossimiltes [3,24]. The SW-M tretment showed similr yield s SW-2 tretment; therefore, the pplition of SW-M represents n lterntive strtegy insted of SW-2, if yield is the prmeter of interest, euse the tretment SW-M hs lower dose of SW thn SW-2 tretment Evlution of the Folir Applition of SW on Strh nd TSS Aumultion in Mize Grin The tretments with doses of SW (SW-1 nd SW-2) showed sttistilly more strh umultion thn the others t physiologil mturity (Figure 1()), whih is in greement with the results of yield for these tretments (Figure 1()). However, only SW-2 tretment showed high strh/tss rtio, wheres SW-1 tretment showed gret mounts of TSS, whih ould hve negtive effet euse grins with high TSS ontent re more suseptile to inset ttks during storge. Furthermore, the pplition of SW supplemented with mironutrients (SW-M) did not result in strh umultion improvement, whih suggests tht SW ontriution ws higher thn mironutrient supplementtion regrding strh umultion Evlution of the Folir Applition of SW on Free Amino Aids Content in Mize Grins The evlution of free mino ids in mize grins t R4 stge showed tht folir pplition with the highest dose of SW (SW-2) resulted in the improvement of lnine, proline, vline nd sprgines ontent, ompred to ontrol (Figure 2). However, the sme result ws not oserved in the SW-1 tretment, whih inresed only tyrosine nd vline ontent, inditing tht the umultion of some mino ids is dependent of the dose of SW pplied. When SW ws enrihed with minerls (SW-M), the ontent of sprti id, treonine, lnine nd glyine ws improved, ompred to DW-M tretment, showing the influene of the intertion etween SW nd minerls supplied Evlution of the Folir Applition of SW on Minerl Content in Mize Grins The folir fertiliztion of mize with SW-2 resulted in inrese in the umultion of K, Cu, Fe, Mn nd Zn in grins, ompred to the ontrol (Tle 2). No signifint differene in minerl umultion ws oserved etween the two doses of SW pplied (SW-1 nd SW-2). ton/h A B () Strh (mg/g) Strh TSS () Figure 1. ) Yield of mize grins; ) Strh nd totl solule sugrs (TSS) ontent in mize grins t hrvest. Different letters represent signifint differene t 5% onfidene level y Tukey test etween tretments for the sme dependent vrile TSS (mg/g) Copyright 13 SiRes.

5 6 R. S. Messis et l. / Agriulturl Sienes 4 (13) Proline Glutmi Aid Phenillnine Tyrosine Leuine Vline 25 Asprgine Isoleuine Lysine Asprti Aid Threonine Serine Glyine Alnine Histidine Figure 2. Free mino ids ontent in mize grins olleted t R4 stge. Dt shows mens of three field nd nlytil replites. Different letters represent signifint differene t 5% onfidene level y Tukey test for the sme dependent vrile. Copyright 13 SiRes.

6 R. S. Messis et l. / Agriulturl Sienes 4 (13) Tle 2. Minerl omposition of mize grins treted with folir fertilizer formultions. Minerl N P K C Mg S Cu Fe Mn Zn Tretment 1 g/kg mg/kg REF Control 1.3 Cd A B.69 A.912 A A D C 3.35 D D DW-M 1.95 B A A.44 B.816 A A C 14.5 B 4.6 B B SW-M A 3.13 A A.66 A.89 A 1.33 A 3.3 B A 5.23 A A SW A A A.7 A.865 A A 3.55 A B 5.15 A C SW B A A.55 A.848 A 1.34 A 3.78 A A 5.71 A C 1 Control ontrol without folir spry; DW-M formultion with mironutrients in distilled wter (2.5 L/h); SW-M formultion with the sme mironutrients onentrtion of DW-M in shle wter (SW) (2.5 L/h); SW-1 nd SW-2 shle wter pplied t rtes equivlent to 2.5 L/h nd 7 L/h, respetively. 2 All vlues from USDA Ntionl Dtse for Stndrd Referene. Aville in: 3 Men of five field nd two nlytil replites nd sttistil signifine t 5% level of onfidene y Tukey test for the sme dependent vrile. The enrihment of folir tretments with the mironutrients Zn, Cu, Mn, Mo nd B (DW-M nd SW-M) signifintly inresed the levels of Zn, Cu nd Mn on mize grins, ompred to ontrol, nd the pplition of SW- M tretment resulted in the umultion of Zn in higher onentrtion thn SW delivered lone (SW-1 nd SW-2). SW-M tretment lso inresed N, C, Cu nd Fe ontent ompred to DW-M, whih ws proly n effet of the presene of SW in the formultion Evlution of the Folir Applition of SW on the Content of Seondry Metolites in Mize Grins The liposolule frtion of ntioxidnt tivity nd the ound phenoli ompounds ontent ws higher thn hidrosolule frtion of ntioxidnt tivity nd the free phenoli ompounds ontent in ll tretments (Figures 3() nd ()). The liposolule frtion of ntioxidnt tivity orresponded to out 8% of the totl ntioxidnt tivity nd showed.51 (p <.1 y Spermn s test) of orreltion with totl rotenoids (Figure 4()), inditing n importnt role of these ompounds on ntioxidnt tivity of mize grins. The folir pplition of SW inresed the levels of totl phenoli ompounds in mize grins, ompred to the other tretments (Figure 3()), due to the inrese in ound phenoli ontent. This result ws oserved when oth doses of SW ws pplied (SW-1 nd SW-2), inditing no dose dependent effet. However, totl ntioxidnt tivity ws signifintly higher in grins from the SW-2 tretment, due to the higher tivity of liposolule frtion (Figure 3()), proly relted to the inrese in totl rotenoids lso oserved in this tretment (Figure 4()). The expression of PSY1, key gene of rotenoid metoli pthwy, ws 1.48, nd fold higher thn the ontrol in the tretments SW-M, SW-1 nd SW-2, respetively. CYP97C nd HYD3 genes were lso upregulted in the DW-M, SW-1 nd SW-2 tret- ments ompred to ontrol, nd they were more expressed in SW-2 tretment thn the other tretments. In SW-2 tretment, there ws lso HYD3/CYP97C expression rtio of 4.5 folds (Figure 4()), inditing greter llotion to the rotenoids from the β,ε-rnh of rotenoid metoli pthwy (β-rotene, β-ryptoxnthin nd zexnthin), whih represent n inrese of vitmin A preursors. The greter llotion to rotenoids from the β,ε-rnh ws lso oserved in the other tretments; however the HYD3/CYP97C expression rtio vlues were lower thn in the SW-2 tretment (C fold; DW-M fold; SW-M fold; SW fold). In summry, there ws lose reltionship etween trnsript umultion nd rotenoids ontent, inditing tht trnsription nd physiologil response ours simultneously. In ddition, this metolism ws signifintly stimulted y the pplition of three doses of 7L/h SW. 4. DISCUSSION Folir sprying of fertilizers nd/or iostimulnts hs een onsidered n importnt strtegy to iofortify rops, euse it hs shown to improve sorption nd/or trnslotion of minerls vi xylem, it enhnes vegettive growth nd yield nd it my stimulte the synthesis of nutritionl nd funtionl ompounds [3,9,11]. In the present study, the folir pplition of three doses of 7L/h shle wter (SW-2), distriuted throughout the rop yle (see mteril nd methods setion), resulted in the inrese of grin yield nd the improvement in the umultion of minerl nutrients s well s nutritionl nd funtionl ompounds. The minerls tht were inresed in grins following the pplition of SW-2 (ompred to the ontrol) inlude K, Cu, Fe, Mn nd Zn (Tle 2). Some of these minerls, suh s K, Cu nd Mn, ply n importnt role in photosynthesis [4,25], whih ould ontriute to explin the etter performne to generte energy nd to onvert it into struturl nd storge plnt ompounds, Copyright 13 SiRes.

7 62 R. S. Messis et l. / Agriulturl Sienes 4 (13) () Figure 3. () Antioxidnt tivity of liposolule nd hydrosolule frtions of mize grins hrvested t R4 stge. Dt re presented in mirogrm equivlent Trolox per grm of grin (µg/g). () Free nd ound phenols of mize grins hrvested t R4 stge. Dt re presented in milligrms of hlorogeni id equivlents per 1 grms of grin (mg/1). Dt shows mens of five field nd two nlytil replites. Different letters represent signifint differene t 5% onfidene level y Tukey test for the sme dependent vrile. () Figure 4. () Totl rotenoids ontent nd the reltive expression of rotenogeni genes. The rotenoid ontent is presented s µg of β-rotene equivlents/g. The Control tretment ws used s referene tretment to otin the reltive expression of the genes enoding for phytoene synthse (psy1), ε-rotene hydroxylse (Cyp97) nd β-rotene hydroxylse (Hyd3) in mize grins spryed with six folir fertiliztion nd hrvested t R4 stge. () Reltive expression of Cyp97 nd Hyd3 ompred to psy1 expression in mize grins spryed with folir fertiliztion SW-2 (7 L/h SW). Different letters represent signifint differenes t 5% onfidene level y Tukey test. wheres signifintly inrese in yield (Figure 1()) with positive orreltion with strh ontent in grins (Figure 1()) ws oserved in plnts treted with SW-2. Aumultion of K is lso ssoited with the improvement of nutrients sorption, euse it is responsile for the orret osmoti grdient, nd plys role in the trnsport of photossimiltes in plnts [4]. Moreover, Mn hs shown positive influene in grins yield [24,26], nd its folir pplition hs lso improved seed qulity [27]. Despite yield remins the mjor im of gronomi efforts, qulity of mize grins is of gret importne, prtiulrly for people whose min soure of food is this erel. SW-2 pplition, esides improving yield, ws le to indue the umultion of essentil minerl nutrients (Tle 2), severl mino ids, nd strh (Figure 1()), whih is importnt to provide energy in diet, ting in the redution of holesterol nd stimulting the immune system, mong others [28]. Totl ntioxidnt tivity ws lso signifintly inresed due to the pplition of SW-2 tretment (Figure 3()) y induing high levels of ound phenoli ompounds (Figure 3()) nd totl rotenoids ontents (Figure 4()), oth relted to the prevention of hroni diseses [7]. Studies towrds the iofortifition of rotenoids in mize, espeilly those with provitmin A tivity (α nd β-rotene nd ryptoxnthin), hve lso een fous of gret interest euse of the nutritionl effets provided y its ingestion [21,29]. The pplition of SW-2 tretment resulted in the improvement of totl rotenoids umultion in mize grins, whih ws shown to e diretly relted to the upregultion of key genes in this metolism (Figure 4()). As fr s we know, this is the first survey on the evlution of the influene of fertiliztion on indution of these genes, despite the ft tht they re widely known nd studied in mize [21]. The indution of trnsript levels (pproximtely two fold higher thn ontrol) ourred t the eginning of this metoli pthwy, inresing PSY1 expression (oding for the enzyme phytoene synthse) nd t the end, inresing the expression of genes enoding for enzymes diretly ssoited to the synthesis of β-rotene, β- ryptoxnthin nd zexnthin (HYD3), nd lutein synthesis (CYP97C) [21]. In ll tretments there ws greter llotion to the prodution of ompounds from Copyright 13 SiRes.

8 R. S. Messis et l. / Agriulturl Sienes 4 (13) metoli pthwy rnh ssoited to HYD3 expression insted of the ones ssoited to CYP97C expression (Figure 4()), proly euse yellow grins, suh s those from the hyrid vriety used in this study usully shows high zexnthin/lutein ontent rtio [29,3]. Therefore, there ws greter llotion to the prodution of provitmin A rotenoids in ll tretments, representing n effiient iofortifition pproh. It is interesting to note tht usully negtive orreltion is oserved etween primry metolites nd totl phenoli ompounds [31,32]. In this study, SW-M tretment showed inresed N umultion (Tle 2) nd yield (Figure 1()), nd did not inrese the levels of phenoli ompounds (Figure 3). This effet would our y ompetition with phenyllnine, preursor for oth phenoli ompounds nd protein [31]. Similrly, in the SW-2 tretment, the yield nd strh ontent inresed while the free phenoli ompounds ontent nd hydrosolule frtion of ntioxidnt tivity deresed. However, in the SW-2 tretment, the lipophyli frtion of ntioxidnt tivity nd the ontent of ound phenoli ompounds, whih orresponds to the mjor frtion of phenoli ompounds in mize grins, lso inresed (Figure 3). Therefore, the pplition of SW-2 tretment ws le to indue primry nd seondry metolisms in mize, possily euse third pplition of the tretments ws performed during the reprodutive phse of mize plnts, providing n extr mount of nutrients. These nutrients were then used in the prodution of seondry metolites. Similr effets were otined with folir fertiliztion omined with growth regultor tivity ompound [33] nd with the pplition of severl eliitors/iostimulnts [11]. The inrese of seondry metolites in mize grins following the pplition of SW my lso e result of moderte non-hrmful stress in plnts s ws evidened y the inrese of vline, sprgine, lnine nd proline (Figure 2) in mize grins following the pplition of SW-2. These lst two mino ids re relted to stress tolerne. Alnine ply role in the intrellulr ph regultion in hypoxi stress ondition [34]. The umultion of free proline in plnts is widely oserved in different ioti stresses suh s slinity nd drought, ting in the stomt opening regultion nd osmoti proesses. Studies show the influene of this mino ids in the synthesis of phenoli ompounds [35], orroorting the dt oserved in SW-2 tretment. The enrihment of SW with mironutrients ording to mize rop requirement ws lso investigted (SW-M tretment) in order to verify if the effet of SW ould e improved y this pproh. Indeed, supplementtion with mironutrients, inluding minly Zn, llowed inresed Zn umultion (Tle 2), representing gret nutritionl nd funtionl improvement, sine this min- erl is essentil s trnsript oftor nd plys role in the ntioxidnt defense [4]. However, is importnt to evlute its iovilility in humns due to the presene of phytte in mize grins. In plnts, Zn hs shown diret reltionship with yield [9], lso oserved in the tretment SW-M (Figure 1()). Besides Zn umultion nd yield, the ontent of some free mino ids ws lso improved in tretment SW-M (Figure 2). However, for most of the other prmeters, SW delivered lone in three doses of 7 L/h (SW-2) presented etter results thn SW-M. 5. CONCLUSION The results of this study showed tht the use of folir pplition of SW-2 influened minerl nd primry metolites umultion, whih resulted in signifint inrese in yield. This tretment lso resulted in the improvement of seondry metolites, suh s ound phenolis nd rotenoids, proly due to the lter pplition during the reprodutive stge. The enrihment of SW with minerls potentited the effet of SW delivered lone regrding the improvement of free mino ids nd some minerls, espeilly Zn; however, the umultion of severl minerls, mino ids, strh, rotenoids nd phenoli ompounds were higher when SW ws delivered without nutrient supplementtion. In this ontext, the pplition of three doses of 7 L/h SW results in gret improvement in grin yield nd qulity. However, further studies re neessry to optimize SW doses nd etter understnd SW mehnisms of tion. REFERENCES [1] Wng, L., Xu, C., Qu, M. nd Zhng, J. (8) Kernel mino id omposition nd protein ontent of introgression lines from Ze mys ssp. mexin into ultivted mize. Journl of Cerel Siene, 48, [2] Kuhnen, S., Lemos, P.M., Cmpestrini, L.H., Ogliri, J.B., Dis, P.F. nd Mrshin, M. (11) Crotenoid nd nthoynin ontents of grins of Brzilin mize lndres. Journl of the Siene of Food nd Agriulture, 91, [3] Gomez-Gler, S., Rojs, E., Sudhkr, D., Zhu, C., Pelho, A.M., Cpell, T. nd Christou, P. (1) Critil evlution of strtegies for minerl fortifition of stple food rops. Trnsgeni Reserh, 19, [4] Messis, R.S, Glli, V., Silv, S.D.A., Shirmer, M.A. nd Romldi, C.V. (13) Mironutrient nd funtionl ompounds iofortifition of mize grins. Critil Reviews in Food Siene nd Nutrition, in Press. [5] Sosulski, F., Krygier, K. nd Hogge, L. (1982) Free, esterified, nd insolule ound phenoli ids. Composition Copyright 13 SiRes.

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