Afrin Journl of Biotehnology Vol. 9(35), pp. 5665-5675, 3 August, 21 Aville online t http://www.demijournls.org/ajb ISSN 1684 5315 21 Ademi Journls Full Length Reserh Pper Sulphur depletion ltered somti emryogenesis in Theorom o L. Biohemil differene relted to sulphur metolism etween emryogeni nd non emryogeni lli Minyk Emile 1,2,3,4 *, Niemenk Niols 2, Issli Emmnuel Auguste 3, Sngre Adourhmne 3 nd Denis Ndoumou Omokolo 3 1 Deprtment of Biohemistry, Fulty of Sienes, University of Doul P.O. Box 24157 Doul - Cmeroon. 2 Deprtment of Biologil Sienes, Higher Tehers Trining College, University of Younde I, P.O. Box 47 Younde - Cmeroon. 3 Lortoire Centrl de Biotehnologies, Centre Ntionl de Reherhe Agronomique 1 P.O. Box 174 Aidjn 1 - Côte d Ivoire. 4 Lortoire de génétique, UFR Biosienes, University of Coody/Aidjn, 22 P.O. Box 582 Aidjn 22 - Côte d Ivoire. Aepted 22 July, 21 Somti emryogenesis is useful tool for Theorom o improvement nd propgtion. Depending on ulture medium omposition, different morphogeneti strutures (inluding somti emryo) our in response to ltertion of genes expression ptterns nd iohemil hnges. The 2- effet of SO 4 ion defiieny in ulture medi on somti emryogenesis ws studied through sequentil replement of MgSO 4 nd K 2 SO 4 y MgCl 2 nd KCl, respetively, t different steps of somti emryogenesis. It ppers tht explnts grdully lost their emryogeni ompetene s the period of exposition to sulphur free medium inreses. These results suggest tht, sulphur vilility nd the durtion to sulphur exposition might modulte the expression of genes involved in somti emryo differentition in T. o. Cysteine, glutthione, reduing sugrs, ysteine synthse nd ysteine desulfurse tivities were nlysed in different morphogeneti strutures otined in vitro. Cysteine nd reduing sugrs ontents ppered to e higher in emryogeni lli thn their nonemryogeni homologues, wheres glutthione ontent ppers to e lower in emryogeni lli. Cysteine synthse tivities lso disriminte the emryogeni lli from non emryogeni lli. In the emryogeni lli, the rtio ysteine synthse/ysteine desulfurse tivities were ove unit. The ssimiltion of exogenous sulphur (sulphte) for the synthesis of ysteine might hene e ruil for somti emryogenesis in T. o. This explins the redution nd the sene of somti emryo response oserved during sulphur depletion in ulture medi. Sulphur nutrition is therefore ritil in o somti emryogenesis. Keys words: Co, emryo, sulphte, ysteine synthse, glutthione, defiieny. INTRODUCTION Theorom o (hoolte tree) is tropil plnt. It is soure of inome for mny developing ountries. T. o ultivtion still fes mny hllenges inluding the *Corresponding uthor. E-mil: minyke@yhoo.fr. lk of elite plnt mteril. Plnt tissue ulture plys n importnt role in griulturl iotehnology. It llows in vitro regenertion, geneti improvement nd lrge sle multiplition of plnts under rseni onditions. Somti emryogenesis, tissue ulture, vegettive nd miropropgtion proesses hve een studied in T. o. This rop ppers unfortuntely to e relitrnt. The
5666 Afr. J. Biotehnol. type of explnts, the genotype nd the ulture medium re some of the ftors influening T. o somti emryogenesis. The medium omposition is the most reported nd seems to e the prepondernt ftor (Tn nd Furtek, 23). Li et l. (1998) hieved etter somti response from mny o genotypes using DKW omplex slt thn Lopez-Bez et l. (1994) did using Murshige nd Skoog omplex slt with the sme explnts (petls nd stminodes). DKW omplex slt provides signifintly higher onentrtion of lium, sulphur nd mgnesium ompre to MS omplex slt. Therefore, these elements re proly essentil for T. o somti emryogenesis nd differentition. A promoting effet of MgSO 4 nd K 2 SO 4 supplementtion on somti emryogenesis ws reported (Minyk et l., 28). The relitrne to somti emryogenesis of mny genotypes of T. o ws overomed with the ddition of oth sulphte slts in ulture medi (Minyk, 29). The influene of sulphur depletion on T. o somti emryogenesis hs not een investigted yet. Furthermore, studies deling with iohemil hnges relted to sulphur metolism in emryogeni nd nonemryogeni lli of T. o re inexistent. Sulphur is n essentil nd one of the most undnt mronutrient in plnt (Leustek, 22; Sito, 24). In plnt sulphurous iomoleules, sulphur not only serves s struturl omponent, ut it lso plys roles in tlyti, eletrohemil or hrteristi funtions of these iomoleules in ells (Sito, 24). In growing onditions, sulphur defiieny hllenges plnts (explnts) to lter the metolism neessry for growth. Restrition in sulphur not only limits the synthesis of sulphurous mino ids, ut will lso limit the proteins synthesis nd the rte with whih ll mino ids re inorporte into protein (Leustek, 22). The present reserh work is trying first of ll to investigte the mehnism tht n help to solve the relitrne, seondly to understnd the importne of sulphur metolism in o somti emryogenesis. MATERIALS AND METHODS Explnts preprtion The tissue ulture system used in this investigtion ws the sme s the system previously desried y Minyk et l. (28). Flowers uds used in this work were hrvested from S6 genotype (Forstero) t CNRA (Centre Ntionl de Reherhe Agronomique) experimentl frm in Bengerville (Côte d Ivoire). Flowers were olleted erly in the morning in old wter. They were surfesterilized y immersion for 2 min in 1% (w/v) lium hypohloride followed y three 2 min rinses in sterilized distilled wter. Stminodes nd petls were exised with slpel nd pled on ulture medi (in distint set) into Petri plte (thirty-five stminodes nd thirty-five petls per Petri plte). Study of the effet of sulphur defiieny on somti emryogenesis expression All medi were defined using DKW (Driver nd Kuniyuki Wlnut medium) sl slts of Driver nd Kuniyuki (1984). In order to test the effet of sulphur defiieny on T. o somti emryogenesis, sulphur ws sequentilly dded into ulture medi s MgSO 4 nd K 2SO 4. The explnts were first ultured in primry llus growth medium with norml (K 2SO 4 nd MgSO 4) sulphte onentrtion nd without sulphte (MgSO 4 nd K 2SO 4, respetively, repled y MgCl 2 nd KCl). Primry llus growth medium ws supplemented with 25 mgl -1 glutmine, 1 mgl -1 myoinositol, 1 ml L -1 DKW vitmin stok (1 mgml -1 myoinositol, 2 mgml -1 thimine-hcl, 1 mgml -1 niotini id nd 2 mgml -1 glyine), 2 gl -1 gluose, 18 µm 2,4 dihlorophenoxyeti id (2,4-D) nd 45.4 nm thidizuron (TDZ). Medi were dispensed into sterilized Petri pltes fter utolving for 2 min t 1 r pressure nd 121 C. Eh Petri plte ontined 35 stminodes nd 35 petls in two seprte sets. Experiments were repeted five times with three replite Petri plte t eh ulture initition. Petri pltes were inuted in the drk t 25 ± 1 C for 14 dys. After 14 dys inution in primry llus growth medium, the explnts from the medium with MgCl 2 nd KCl were sudivided into two thes. One th ws trnsferred in seondry llus growth medium with norml (.3 mm MgSO 4 nd 8.946 mm K 2SO 4) sulphte onentrtion nd the other th in the seondry llus growth medium with MgSO 4 nd K 2SO 4, respetively, repled y MgCl 2 nd KCl. Explnts from primry llus growth medium with norml sulphte onentrtion were trnsferred in seondry llus growth medium with norml sulphte onentrtion. Seondry llus growth medium onsisted of DKW (exept where MgSO 4 nd K 2SO 4 were respetively, reple y MgCl 2 nd KCl) sl slts, supplemented with.5 mll -1 DKW vitmin, 2 gl -1 gluose, 9 µm 2,4-D, 25 µgl -1 kinetin nd.2% (w/v) phytgel. Cultures were lso inuted t 25 ± 1 C for 14 dys in drkness. Explnts from seondry llus growth medium without sulphte were sudivided into two thes. One th ws ultured in emryo development medium without sulphte. The other th ws ultured in emryo development medium with 6. mm MgSO 4 (two fold ompred to norml) nd 8.946 mm K 2SO 4 (norml) onentrtion. Cultures from seondry llus growth medium were lso trnsferred in emryo development medium with two fold (6. mm MgSO 4 nd 8.946 mm K 2SO 4) sulphte onentrtions. Emryo development medium ws mde of DKW sl slt supplemented with 1 ml DKW vitmin, 3 gl -1 surose, 1 gl -1 gluose nd.2% (w /v) phytgel. Cultures were inuted t 25 ± 1 C in drkness for 21 dys. After 21 dys, ultures from emryo development medium without sulphte were sudivided in two thes, one ws ultured in presene of sulphte (6. mm MgSO 4 nd 8.946 mm K 2SO 4), the other in the sene of sulphte. Twenty one dys lter, the explnts ultured in sene of sulphte were lso sudivided in two thes, one trnsferred in presene of sulphte, the other in the sene of sulphte (Tle 1). This experiene ws done five times (ultures) with three replites eh time. Biohemil nlysis of emryogeni nd non emryogeni lli (white lli, nerosis lli, whithe lli) At the end (91st dy) of eh experiene ( giving ulture), eh type of mophogeneti struture (emryogenesis lli, white lli, lli with roots nd nerosis lli) ws olleted (in three different smples) nd nlyzed independently. Amino ids nd solule sugr extrtion Amino ids nd solule sugrs were extrted ording to the modified method of Bu et l. (22). A grm of iologil mteril (emryogeni, non emryogeni lli) ws ground in 3 ml of 8% ethnol nd entrifuge t 6 g for 2 min. The superntnt ws olleted nd used for mino ids nd sugr nlysis.
Emile et l. 5667 Tle 1. Culture monitoring during sequentil supply of sulphte in the ultures medi. Culture medi PCG SCG ED 1 st ED 2 nd ED th Inution time (dys) 14 14 21 21 21 Cultures ges (dys) -14 14-28 28-49 49-7 7 91 *Referene medium (positive ontrol) PCG 1S SCG 1S ED 2S ED 2S ED 2S **Dely of sequentil sulphte supply s PCG -S - SCG -S - ED -S -ED -S -ED 2S MgSO 4 nd K 2SO 4 in ulture medi PCG -S - SCG -S - ED -S -ED 2S ED 2S PCG -S -SCG -S -ED 2S ED 2S ED 2S PCG - S -SCG 1S ED 2S ED 2S ED 2S ***Referene medium (negtive ontrol) PCG -S PCG - S -SCG - S PCG -S -SCG -S - ED -S PCG -S - SCG -S - ED -S -ED -S PCG -S - SCG -S - ED -S -ED -S -ED -S * PCG 1S: Primry llus growth medium with norml MgSO 4 nd K 2SO 4 onentrtion; SCG 1S: Seondry llus growth medium with norml MgSO 4 nd K 2SO 4 onentrtion; ED 2S: Emryo development medium with two fold MgSO 4 nd K 2SO 4 onentrtion. ** PCG - S -SCG 1S: MgSO 4 nd K 2SO 4 dded into ulture medium on the 14 th dy; PCG -S -SCG -S -ED 2S: MgSO 4 nd K 2SO 4 dded into ulture medium on the 28 th dy; PCG -S - SCG -S - ED -S -ED 2S : MgSO 4 nd K 2SO 4 dded into the ulture medium on the 49 th dy; PCG -S - SCG -S - ED -S -ED -S -ED 2S : MgSO 4 nd K 2SO 4 dded into ulture medium on the 7 th dy. *** MgSO 4 nd K 2SO 4 respetively repled y MgCl 2 nd KCl during the 91 dys of ulture. Totl mino ids, ysteine nd reduer sugrs nlysis The mino ids ontent ws ssyed using the method desried y Yemm nd Cooking (1955)..5 ml itrte uffer (.2 M, ph 5.), 1. ml etone ninhydrin KCN regent, 5 µl ethnoli extrt (smple) nd.5 ml ethnol 8% were mixed. The mixture ws heted (1 C, 15 min) nd ooled in ie. 8 ml of distilled wter ws susequently dded nd the sorne ws red t 57 nm. To mesure ysteine onentrtion in extrts, the method of Gitonde (1967) ws used. An liquot (.15 ml) of the smple ws dded to.35 ml idi ninhydrin regent (1.3% ninhydrin (w/v) in 1:4 on. HCl:HOA). These were heted t 1 C for 1 min to llow olour development, followed y ooling in ie nd ddition of.7 ml ethnol 95%. The sorne t 55 nm ws mesured. Reduing sugrs were ssyed using Müller regent (1% 3,5-dinitrosliili id (w/v), 1.6% NOH (w/v), 3% sodium potssium trtrte (w/v). The retion mixture ontined 2 µl smple,.5 ml Müller regent nd 1.5 ml distilled wter. The mixture ws heted t 1 C for 1 min nd ooled t room temperture. The sorne ws mesure t 575 nm. Glutthione extrtion nd ssy Glutthione ws extrted y grinding 1 g of morpho- geneti strutures in 3 ml extrtion uffer (Tris-HCl ph 7.4, 5 mm) in presene of.1 g polyvinylpyrilidone phosphte. These were entrifuged (1 g, 1 min, 4 C). The superntnt ws olleted nd used to ssy totl glutthione using Ellmn (1959) method. 5 µl phosphte uffer (1 mm, ph 6.8) supplemented with DTNB (8 mm) nd EDTA (19 mm) ws dded to 1 µl extrt (smple) nd 1 ml Tris-HCl uffer (ph 8., 5 mm). The mixture ws homogenized nd inuted t room temperture for 25 min. Asorne ws determined t 412 nm. Cysteine synthse nd ysteine desulfurse extrtion nd nlysis For ysteine synthse nlysis, morphogeneti strutures were olleted nd ysteine synthse ws immeditely extrted using the method of Wrrilow et l. (1998). A grm of morphogeneti strutures (emryogeni lli nd non emryogeni lli) ws ground in mortr t 4 C with 2 ml of 5 mm sodium phosphte, ph 8 uffer ontining.1% (v/v) Triton X-1,.1 % (w/v) dithiothreitol, nd.2% (w/v) sodium sorte. After entrifugtion t 1, g for 2 min, the superntnt ws used for ysteine synthse nlysis. The ysteine synthse ssys ontined 1.5 mm O-etylserine, 3 mm sodium sulphide, 1 mm dithiothreitol, 12.5 mm sodium phosphte, ph 8 (.2 ml volume) nd 1 µl enzyme extrt. The retion ws initited y the ddition of sodium sulphide nd ws inuted for 1 min t 26 C fter whih,.35 ml of idi ninhydrin regent (1.3% ninhydrin (w/v) in 1:4 on HCl:HOA) ws dded to determine ysteine onentrtion (Gitone, 1967). These were heted t 1 C for 1 min to llow olour development follow y ooling in ie nd ddition of.7 ml ethnol. The sorne t 55 nm ws determined. One unit of enzyme is defined s the formtion of 1 µmol of ysteine per min under the stte ssy onditions. Cysteine desulfurse ws extrted using Riemenshneider et l. (25) method. The retion mixture for ysteine desulfurse tivity ontined Tris-HCl uffer (ph 8., 1 mm),.25 mm dithiotreitol,.8 mm ysteine nd 1 µl enzyme extrt. After 15 min inution t 37 C, the remining ysteine ws ssyed using Gitonde method (1967). The enzyme tivity is defined s the trnsformtion of 1 µmol of ysteine per min under the stte ssy onditions. Dt nlysis Dt were sujeted to sttistil nlysis using SPSS softwre version 1.. Anlysis of vrine ws performed where pplile nd differenes etween mens were determined using Student Newmn nd Kell s multiplernge test.
5668 Afr. J. Biotehnol. Tle 2. Influene of sulphur depletion in ulture medi on the perentge of explnts produing llus. Non emryogeni lli Dte of sulphte supply (dys) 1 14 28 49 7 91* % of stminodes-derived non emryogeni lli 8 ± 17 85 ± 14 89 ± 18 91 ± 12 1 - % of petls-derived non emryogeni lli 71 ± 15 86 ± 11 88 ± 13 93 ± 19 1 - Eh vlue is men of five identil experiments. Individul experiment inluded three replite Petri dishes with 35 stminodes nd 35 petl-derived morphogeneti strutures. Signifine ws determined t P <.5 using ANOVA. In the sme line, vlues tht re signifintly different (t P <.5) re indited with different letters. *In the sene of sulphte in ulture medium (91 dys), ll explnts died (nerosis). RESULTS In order to test the effet of sulphur defiieny on o somti emryogenesis, MgSO 4 nd K 2 SO 4 were sequentilly repled y MgCl 2 nd KCl, respetively, in ulture medi t different steps of T. o somti emryogenesis (indution to expression steps). The results showed tht the frequeny non-emryogeni morphogeneti strutures (white lli, nerosis lli nd lli with roots) inrese with the durtion of sulphur strvtion in ulture medi (Tle 2). All explnts died (nerosis) when they re ultured for 91 dys in the sene of sulphur. White lli without emryo were mostly oserved when sulphur ws supplied on 49th dy of ulture. Clli with roots were undnt when sulphur ws supplied on the 28th in ulture medi. The perentge of explnts produing somti emryo is highly ltered y sulphur defiieny in ulture medi. There ws no emryo differentition when MgSO 4 nd K 2 SO 4 were repled y MgCl 2 nd KCl, respetively, in ulture medi during the whole ulture period (91 dys). The presene of MgSO 4 nd K 2 SO 4 in ulture medi t ll steps (91 dys) leded to 29.4 ±.4 nd 2 ± 3.6% of stminodes nd petls produing emryo, respetively. The ddition of sulphur lter on in ulture medi progressively nd signifintly redued the perentge of explnts produing emryo. Hene, when explnts styed in ulture medium for the 14 first dys of ulture followed y their trnsfer in medi with MgSO 4 nd K 2 SO 4, just 14% of explnts produed emryo. This represents pproximtely 5% redution of explnts produing emryo ompred to the positive ontrol. And when the explnts were ultured in sene of sulphte slts for 28 first dys (then trnsferred in presene of sulphte), the perentge of explnts produing emryo ws 1%. The sene of sulphte in ulture medi for the 7 dys of ulture inhiited emryo differentition (Figure 1). Curves tendeny linking the perentge of explnts produing emryo to the dtes of sulphte supply in ulture medi presented negtive slopes. With stminodes the stright line eqution ws y = -4.51x + 24.53. The determining oeffiient ws 93.2%. With pe-tls, the stright line eqution ws y= -6.55x + 31.96. Determining oeffiient ws 91.61%. The omprison of oth slopes shows tht, s emryo expression is onerned, petls re more sensitive to sulphur defi-ieny thn stminodes (Figure 1). The in vitro morphogenesis of T. o presented different morphogeneti strutures inluding, white lli, nerosis lli, lli with roots nd emryogeni lli. The emryos otined were le to germinte nd generte plntlet (Figure 2). Biohemil nlyses relted to sulphur metolism were onduted in these strutures. Totl mino ids, ysteine, reduer sugrs, glutthione, ysteine synthse nd ysteine desulphurse were nlyzed. The mino id ontent in morphogeneti strutures vried from 2942.23 ± 496 µg g -1 FW (emryogeni lli) to 3951.97 ± 615 µg g -1 FW in nerosis lli (dt not shown). Cysteine ontents in white lli (31.29 ± 1.88 µg g -1 FW) nd in lli with roots (3.74 ± 1.57 µg g -1 FW) were not signifintly different. However, ysteine ontents in nerosis nd emryogenei lli were signifintly higher ompred to the ontent of this metolite in lli with roots nd white lli. Additionlly, emryogeni lli presented the highest ysteine ontent (Figure 3). In the morphogeneti strutures nlysed, ysteine ontent pttern ws not mthed to glutthione ontent pttern. The glutthione ontent ws lower in em-ryogeni lli (9.94 ± 1.54 µg g -1 FW) thn the glutthione ontents in white lli (12.38 ± 2.68 µg g -1 FW), nerosis lli (14.25 ± 3.1 µg g -1 FW) nd lli with roots (14.5 ± 1.2 µg g -1 FW) (Figure 4). Reduing sugrs disriminte morphogeneti strutures. The lowest redu-ing sugrs ontent ws oserved in white lli (36.95 ± 2.33 µg g -1 FW) while the highest ontent ws mesured in emryogeni lli (14.3 ± 4.18 µg g -1 FW). Reduing sugrs ontent ws signifintly higher in lli with roots (14.85 ± 5.78µg g -1 FW) thn in nerosis lli (71.34 ± 3.77 µg g -1 FW). It ppers tht, ells differentition is hrterized y high reduing sugrs synthesis (Figure 5). Cysteine synthse tivities presented ptterns relted to type of morphogeneti struture. The tivity ws 2., 1.7 nd 4.3 fold higher in emryogeni lli thn the tivities in white lli, nerosis lli nd lli with roots, respetively (Figure 6). Cysteine desulphurse disriminted emryogeni lli from non emryogeni lli. In emryogeni lli, ysteine desulphurse tivity presented the highest tivity. There ws no signifint differene etween ysteine desulphurse tivities in white lli nd nerosis lli. However, the ysteine
Emile et l. 5669 35 Stminodes Liner (Stminodes) Petls Liner (Petls) 3 Perentge of exolnts produing emryo 25 2 15 1 y = -4.5137x + 24.531 R 2 =.932 y = -6.5476x + 31.962 R 2 =.9161 5 D1 D14 D28 D49 D7 Dte of sulphte supply s MgSO4 nd K2SO4 (dys) Figure 1. Influene of sulphur depletion in ulture medi on the perentge of explnts produing somti emryos. Vertil rs represent stndrd error. Eh plot ws drwn from mens of five identil experiments. Individul experiment inluded three replite Petri dishes with 35 stminodes nd 35 petl-derived morphogeneti strutures. Signifine ws determined t P <.5 using ANOVA. de-sulphurse tivity ws signifintly higher in nerosis lli thn in white lli nd lli with roots (Figure 7). The rtio ysteine synthse/ysteine desulphurse ws estimted. This rtio signifintly vried from one morphogeneti struture to nother. This rtio ws not similr to the tivity pttern of oth enzymes. Clli with roots presented the lowest rtio nd emryogeni lli the highest rtio (Figure 8). DISCUSSION Stminodes nd petls were sequentilly sujeted to sulphur defiieny. It ppered tht explnts grdully lost their potentil to differentite emryo s the period in medium without sulphur inreses. These results suggest tht, sulphur vilility nd the durtion to sulphur exposition might modulte the expression of genes involved in somti emryo differentition in o. It is known tht somti emryo differentition in o is genes ontrolled proess (Sntos et l., 25; Alemnno et l., 28). Thus, it will e interesting in the future investigtions to nlyze key genes trnsriptome involved in sulphur metolism nd emryogenesis (somti nd zygoti). Additionlly, the nlysis of sulphur trnsripttome relted to sulphur defiieny in Aridopsis thlin reveled the ltertion of the expression of genes implited in sulphur ssimiltion (Nikiforov et l., 23). Moreover, there ws differene etween slopes of stright lines explining the loss of emryogeni potentil for stminodes nd petls. This differene underlines the explnt effet on emryogeni response of ultured tissues. This set of results suggests tht, when genetilly genotype of o is emryogeni, the limit ftor is the vilility of sulphur whih ontrol the expression of genes implited in o somti emryogenesis. During somti emryogenesis, different ptterns of genes expression re oserved from the quisition to the expression of somti emryogeni ompetene (Zimmermn, 1993). The vrition of genes expression
567 Afr. J. Biotehnol. Figure 2. Different morphogeneti strutures. ) Stminodes nd petls in Petri plte; ) llus (ß- white llus); ) nerosis llus; d) llus with roots; e) somti emryos t gloulr stte; f) somti emryo t otyledonry stte, nd g) plntlets. Br =.5 m. pttern, leds to the modifition of iohemil (metolites, enzymes tivities) nd physiologil sttutes of explnts from whih derived morphogeneti strutures (Shrp et l., 198). Amino ids, ysteine, glutthione, reduers sugrs, ysteine synthse nd ysteine desulfurse tivities were nlyzed in different morphogeneti strutures (white lli, lli with roots,nerosis lli nd emryogeni lli). Cysteine ontent ws higher in emryogeni lli thn their non emryogeni homologous. This result might underline the implition of this ysteine in o somti emryogenesis. The present result indites determinnt role of sulphur ssimiltion somti emryo differentition. And, this explins the improvement of somti emryo response nd the inrese in somti
Emile et l. 5671 Cysteine ontnt (µg.g -1 of FW) 4 36 32 28 24 2 16 12 8 4 white lli nerosis lli emryogenei lli lli with roots Morphogeneti strutures Figure 3. Vrition of ysteine ontents in morphogeneti strutures. Dt re presented s mens of five identil experiments with three replite. Vlues tht re signifintly different t the 5% level of signifine re indited with different letters. 16 14 Glutthione ontent (µmol.ḡ 1 of FW) 12 1 8 6 4 2 white lli nerosis lli emryogeni lli lli with roots morphogeneti strutures Figure 4. Vrition of glutthione ontent in morphogeneti strutures. Dt re presented s mens of five identil experiments with three replite. Vlues tht re signifintly different t the 5% level of signifine re indited with different letters.
5672 Afr. J. Biotehnol. 16 d Reduers sugrs ontent (µg.g -1 of FW) 14 12 1 8 6 4 2 white lli nerosis lli emryogenei lli lli with roots Morphogeneti strutures Figure 5. Vrition of reduing sugrs ontent in morphogeneti strutures. Dt re presented s mens of five identil experiments with three replite. Vlues tht re signifintly different t the 5% level of signifine re indited with different letters. 1,2 d ysteine synthse tivity (µmol.min -1 ) 1,8,6,4,2 white lli nerosis lli emryogeni lli lli with roots morphogeneti strutures Figure 6. Vrition of ysteine synthse tivities in morphogeneti strutures. Dt re presented s mens of five identil experiments with three replite. Vlues tht re signifintly different t the 5% level of signifine re indited with different letters.
Emile et l. 5673 ysteine desulphurse tivity (µmol.min -1 ) 1,8,6,4,2 white lli nerosis lli emryogeni lli lli with roots morphogeneti strutures Figure 7. Vrition of ysteine desulphurse tivities in morphogeneti strutures. Dt re presented s mens of five identil experiments with three replite. Vlues tht re signifintly different t the 5% level of signifine re indited with different letters. 1,4 d Rtio ysteine synthse tivity/ysteine desulphurse tivity 1,2 1,8,6,4,2 white lli nerosis lli emryogenei lli lli with roots morphogeneti strutures Figure 8. Vrition of ysteine synthse/ysteine desulphurse rtio in morphogeneti strutures. Dt re presented s mens of five identil experiments with three replite. Vlues tht re signifintly different t the 5% level of signifine re indited with different letters.
5674 Afr. J. Biotehnol. emryogenesis of mny o genotypes oserved when the medi were supplied with sulphte s reported y Minyk et l. (28). Sulphur in ulture medi ws supplied s MgSO 4 nd K 2 SO 4. Both sulphte slts were uptken up y explnts ells. Within ell, sulphte is susequently tivted into denosine 5 -phosphosulfte (5 -denylyl-sulfte [APS]) for further onversion. The mjor ssimiltory pthwy is redution of APS to sulfite (SO 3 2- ) nd then sulfide (S 2- ). Sulphide is then oupled with O-etyl- serine (OAS) tht is formed from serine, yielding ysteine (Leustek, 22). Cysteine is the entrl ompound for prodution of vriety of metolites ontining redued sulfur, suh s methionine, glutthione, phytoheltins, nd gluosinoltes (Sito, 2). In ontrst to ysteine ontent, glutthione ontent ws lower in emryogeni lli thn non emryogeni lli. It seems tht in emryogeni lli, glutthione is tolised in order to lierte ysteine for emryo development or ysteine synthesized from sulphte is used for nother metoli pthwy ut not for the synthesis of glutthione. The umultion of glutthione in white lli, nerosis lli nd lli with roots (ll non emryogeni) ould e explined when we ssume tht most ysteine is used for the synthesis of glutthione; ut no for metoli sequenes neessry for somti emryo development. Reduing sugrs ontent ws prtiulr to eh morphogeneti struture. The umultion of this group of rohydrte ppered to e ssoited to the differentition of roots or somti emryos. However, the ontent of reduer rohydrtes ws higher in emryogeni lli thn lli with roots. This might onsolidte the ft tht reduer rohydrtes re importnt for lli formtion nd ell differentition (An et l., 1997). Hene, it ppers tht, in o, somti emryogenesis is ssoited with high synthesis of ysteine, redution in rohydrtes, nd glutthione tolism. Cysteine synthse tivities disriminte emryogeneti lli from their non emryogeneti homologous. The ssoition of emryogeni response, the tivities of ysteine synthse nd ysteine ontent in emryogeni lli onfirmed the use of exogenous sulphur (sulphte) for the synthesis of ysteine ruil for somti emryogenesis in o. This therefore explins the redution nd the sene of somti emryo response oserved during sulphur depletion in ulture medi. Xu nd Moller (24) reported tht ysteine defiieny inhiits zygoti emryogenesis in A. thlin from the gloulr stte. Cysteine desulphurse tivities were highest in nerosis lli (whih frequently differentite emryo) nd emryogeni lli. Additionlly, the rtio ysteine synthse tivities/ysteine desulphurse tivities were determined in eh type of morphogeneti strutures in order to evlute the differene etween the flux of synthesized ysteine nd ysteine used (s preursor for the synthesis of other sulphurous metolites). This rtio ws ove one in emryogeni lli. This might suggest tht, in emryogeni lli there is very high ysteine synthesis, however, frtion of ysteine synthesized is used in other iosynthesis pthwys. In plnt, ysteine is used s preursor of thimine nd iotine (Begley et l., 1999). Conlusion In in vitro ulture onditions, sulphur supply improves somti emryogenesis response in o. Contrrily, sulphur depletion in ulture medi grdully nd irreversily inhiits somti emryogenesis euse, sulphur depletion in ulture medi impirs ysteine (whih ppers to e ruil for somti emryogeneis in o) synthesis tlyzed y ysteine synthse. And the ysteine synthesized is prtilly used in other iosyntheti pthwys. Sulphur nutrition is therefore ruil in o somti emryogenesis. REFERENCES Alemnno L, Devi M, Niemenk N, Snier C, Guilleminot J, Rio M, Verdeil JL, Montoro P (28). Chrteriztion of lefy1-like during emryogenesis in Theorom o L. Plnt, 227: 853-866. An BM, Yolnd C, Hilrio G, Piedd G, Osr H, Luis M, An D, Nieves V (1997). Differenes in the ontents of totl sugrs, reduing sugrs, strh nd surose in emryogeni nd non-emryogeni lli from Medigo rore L. Plnt Si. 154: 143-151. Bu SSV, Shreef MM, Shetty PKA, Shetty TK (22). HPLC method for mino ids profile in iologil fluids nd inorn metoli disorders of minoidopthies. Indin J. Clin. Biohem. 17 (2): 7-26. Begley TP, Xi J, Kinslnd C, Tylor S, MLfferty F (1999). The enzymology of sulfur tivtion during thimin nd iotin iosynthesis. Curr. Opin. Chem. Biol. 3: 623-629. Ellmn G (1959). Tissue sulfhydryl groups. Arh. Biohem. Biophys. 82: 7-77. Gitonde M (1967). A spetrophometri method for the diret determintion of ysteine in the presene of other nturlly ourring mino ids. Biohem. J. 14: 627-633. Leustek T (22). Sulfte metolism. In CR Somerville, EM Meyerowitz, eds, The Aridopsis Book. Amerin Soiety of Plnt Biologists, Rokville, MD, doi/1.1199/t.9, http://www.sp.org/ pulitions/ridopsis/. 31 1 26. Li Z, Adoulye T, Mximov S, Aiken WM (1998). Somti emryogenesis nd plnt regenertion from florl explnts of o (Theorom o L.) using thidizuron. In vitro ell Dev. Biol. Plnt, 34: 231-238. Lopez-Bez OH, Bollon A, Eskes, Petird V (1994). Emryogenèse somtique de oyer Theorom o L. à prtir de pièes florles. C.R. Ad. Si. Pris Serie III. 316: 549-585. Minyk E (29). Métolisme du soufre et emryogenèse somtique hez Theorom o L (Mlvee). Thèse de Dotort/Ph.D, Université de Youndé I, Université de Coody/Aidjn. p. 132. Minyk E, Niemenk N, Fotso, Sngré A, Omokolo ND (28). Effet of MgSO 4 nd K 2SO 4 on somti emryo differentition in Theorom o L. Plnt Cell Tissue Orgn Cult. 94(2): 149-16. Nikiforov V, Freitg J, Kemp S, Admik M, Hesse H, Hoefgen R (23). Trnsriptome nlysis of sulfur depletion in Aridopsis thlin: interling of iosyntheti pthwys provides response speifiity. The Plnt J. 33: 633-65. Sito K (2). Regultion of sulfte trnsport nd synthesis of sulphur ontining mino ids. Curr. Opin. Plnt Biol. 3: 188-195. Sito K (24). Sulfur ssimiltory metolism. The long nd smelling rod. Plnt Physiol. 136: 2443-245. Sntos MO, Romno E, Yotoko KSC, Tinoo MLP, Dis BB A, Arg o FJL (25). Chrteristion of the o somti emryogenesis reeptor-like kinse (SERK) gene expressed during somti emryogenesis. Plnt Si. 168: 723-729. Tn CL, Furtek DB (23). Development of n in vitro regenertion
Emile et l. 5675 system for Theorom o from mture tissues. Plnt Si. 164: 47-412. Wrrilow AGS, Hwkesford MJ (1998). Seprtion, suellulr loliztion nd influene of sulphur nutrition on isoform of ysteine synthse in spinh. J. Exp. Bot. 327(49): 1625-1638. Xu M, Moller S (24). AtNAP7 is plstidie sufc-like ATP-uriding sette/atpse essentil for Aridopsis emryogenesis. Pro. Ntl. Ad. Si. 11(24): 9143-9148. Yemm E, Cooking W (1955). Determintion of mino ids with ninhydrin. Anlysis, 8: 29-213. Zimmermn JL (1993). Somti emryogenesis: model for erly development in higher plnts, Plnt Cell, 5: 1141-1423.