Dynamics of physiological and biochemical changes during somatic embryogenesis of Acca sellowiana

Transcription

1 Universidde de São Pulo Biliote Digitl d Produção Inteletul - BDPI Deprtmento de Botâni - IB/BIB Artigos e Mteriis de Revists Científis - IB/BIB Dynmis of physiologil nd iohemil hnges during somti emryogenesis of A sellowin In Vitro Cellulr nd Developmentl Biology - Plnt, Heidelerg, v.5, n.2, p , Downloded from: Biliote Digitl d Produção Inteletul - BDPI, Universidde de São Pulo

2 In Vitro Cell.Dev.Biol. Plnt (214) 5: DOI 1.17/s EMBRYOGENESIS/SOMATIC EMBRYOGENESIS Dynmis of physiologil nd iohemil hnges during somti emryogenesis of A sellowin Griel C. Cnghul-Inoente & Vnildo Silveir & Clriss A. Cprestno & Eny I. S. Floh & Miguel P. Guerr Reeived: 2 April 213 /Aepted: 13 Septemer 213 /Pulished online: 3 Otoer 213 / Editor: J. Forster # The Soiety for In Vitro Biology 213 Astrt Feijo (A sellowin [O. Berg] Burret [Myrtee]) is ntive fruit speies of southern Brzil nd northern Uruguy. This speies is menle to somti emryogenesis nd therefore suitle s model system for omprtive studies of zygoti nd somti emryo development. In seed plnts, emryogenesis involves three min steps, whih re regulted y mny ftors, suh s hormones, proteins, polymines, nd trnsription ftors. In the present work, the dynmis of protein, sugr, strh, mino id, nd polymine umultion were ssyed during somti emryogenesis of A. sellowin. Protein, strh, mino id, nd polymine levels umulted unevenly during the indution phse of somti emryogenesis, while the sugr ontent remined stle. Throughout the different developmentl stges of somti emryogenesis, synthesis nd umultion of proteins nd mino ids showed ptterns similr to those reported previously during the development of zygoti emryos of this sme speies. Differentil ptterns of polymine umultion were oserved. This is importnt euse these ompounds ffet the synthesis of other endogenous growth regultors, suh s uxinindole-3-eti id tht is minly involved in the estlishment of emryo polrity. Tken G. C. Cnghul-Inoente: C. A. Cprestno : M. P. Guerr (*) Lortory of Plnt Developmentl Physiology, Grdute Group in Plnt Geneti Resoures, Federl University of Snt Ctrin, Florinópolis, SC, Brzil e-mil: mpguerr@.ufs.r V. Silveir Biotehnology Lortory, CBB-UENF, Cmpos dos Goytzes, Rio de Jneiro, Brzil E. I. S. Floh Deprtment of Botny, Institute of Biosienes, University of São Pulo, CP 11461, São Pulo, SP, Brzil together, the present work rings new insights to the physiologil nd iohemil dynmis tht our during somti emryogenesis of A. sellowin. Keywords Feijo. Protein. Strh. Sugr. Amino ids. Polymines Introdution A sellowin (O. Berg) Burret, syn. Feijo sellowin Berg, ommonly known s Feijo or pinepple guv, is fruit speies ntive to southern Brzil nd northern Uruguy. It hs often een employed s model system for omprtive studies of zygoti nd somti emryo development (Cnghul-Inoente et l. 29; ). Aording to Vsil (28), this model system of somti emryogenesis n improve our understnding of ellulr totipoteny in higher plnts. Somti emryogenesis (SE) in A. sellowin ws first desried y Cruz et l. (199). Sine then, severl studies hve een onduted to improve SE protools (Cnhoto nd Cruz 1996; Guerr et l. 21; Stefnello et l. 25), s well s detiling the histologil nd iohemil fetures relted to the development of somti emryos (Cnghul-Inoente et l. 24, 27, 29; Pesdor et l. 28, 212). Aumulting evidene suggests tht the ptterns of protein synthesis during development of zygoti emryos re repitulted during somti emryogenesis (Winkelmnn et l. 26). The umultion of proteins (totl) is ssoited with emryo development nd mturtion through regultion of ell expnsion nd other iohemil nd iophysil fetures (Jiménez 21). During emryogenesis, mino ids re importnt in nitrogen (N) metolism nd protein synthesis, s well s the trnsition from heterotrophy to utotrophy (Ortiz- Lopez et l. 2). Surose nd mino ids, minly sprgines nd glutmine (Gln), re primry soures of ron nd

3 DYNAMICS OF PHYSIOLOGICAL AND BIOCHEMICAL CHANGES 167 nitrogen ville to the developing soyen emryo (Rinird et l. 1984). Gln is the min nitrogen soure for developing seeds, nd while developing otyledons show limited ssimiltion pity for other forms of nitrogen, they essentilly show no pity for ssimiltion of inorgni nitrogen (Hg nd Sodek 1987). Some of these studies hve shown n inrese in the strh ontent during the mturtion of somti emryos in onentrtions higher thn those normlly found in zygoti emryos, suggesting metoli differenes etween the two developmentl proesses (Merkle et l. 1995). Polymines (PAs) re onsidered relile mrkers of metoli hnges tht our during mturtion of somti emryos sine they ply importnt roles in plnt developmentl proesses, suh s ell division, regultion of morphogenesis, emryogenesis, florl nd fruit initition nd development, fruit ripening, lef senesene, root growth, nd tueriztion (Bron nd Stsoll 28). In plnts, PAs re ommonly ssoited with ioti nd ioti stresses (for review, see Kkkr nd Swhney 22; Alázr et l. 21). They re lso ssoited with the development nd morphogenesis of somti emryos s they re known to regulte endogenous nitri oxide levels (Snt-Ctrin et l. 27), mintin the growth nd polrity of somti emryos (Silveir et l. 26), nd inrese endogenous sisi id (ABA) levels (Steiner et l. 27). A. sellowin is onsidered to e model system of SE in woody ngiosperms, whih re relitrnt to this in vitro morphogeneti route (Guerr et l. 213). Comprtive studies mong zygoti (Cnghul-Inoente et l. 29) nd somti emryogenesis (Cnghul-Inoente et l. 29) in this speies provide etter understnding on the ottleneks underlying developmentl pthwys. Thus, the present work imed to evlute the dynmi hnges in the endogenous protein ontent, totl sugrs, strh, mino ids, nd PAs during somti emryogenesis of A. sellowin. Mteril nd Methods Plnt mteril. Zygoti emryos were exised from seeds of mture fruits olleted from plnts elonging to the germplsm olletion of the Experimentl Sttion of EPAGRI (Snt Ctrin Stte Agriulturl Reserh Ageny), whih is loted in the muniiplity of São Joquin, Snt Ctrin Stte, in southern Brzil. Indution of SE ws rried out ording to Cnghul-Inoente et l. (27). Briefly, the seeds were kept in mixture of wter nd 1.% (v/v) sodium hypohlorite (NOCl) overnight. The seeds were then disinfested in flow inet with 2.% (v/v) NOCl for 2 min, followed y three rinses with sterile wter. Afterwrds, zygoti emryos were exised from seeds nd inoulted in test tues (22 15 mm) ontining 1 ml of LPm ulture medium (von Arnold nd Eriksson 1981), supplemented with Morel vitmins (Morel nd Wetmore 1951), glutmi id (8 mm), 2,4-dihlophenoxieti id (2,4-D; 2 μm), mltose (3%; w/v), nd Difo gr (.7%; w/v). The ph of the ulture medium ws djusted to 5.8 prior to utolving t 121 C, 12 kp for 15 min. Cultures were mintined in ulture room in the drk t 25 C. Smple preprtion nd hemils. Emryogeni ultures nd somti emryos t different developmentl stges were otined following the methodologies desried y Cnghul-Inoente et l. (24). More speifilly, smples were otined t regulr intervls (3 d) from to 3 d of ulture nd inluded the whole explnt nd the ompetent emryogeni ellulr msses, s well s somti emryos when present. Alterntively, fter 7 d ulture, somti emryos were olleted nd lssified ording to the developmentl stges: gloulr (G), hert (H), torpedo (T), preotyledonry (PC), nd otyledonry (CT). Somti emryos were then stored in Eppendorf tues nd kept t 2 C. For eh olletion time, nine replites were stored until the smples were proessed. Sodium dodeyl sulfte (SDS), phenylmethylsulfonyl fluoride (PMSF), nd Coomssie Brillint Blue G25 were purhsed from Bio-Rd (Herules, CA, USA). Methnol, ethnol, hloroform, nd perhlori id were purhsed from Synth (Diprol Com. Ltd., São Pulo, Brzil). Anthrone ws purhsed from VETEC (Rio de Jneiro, Brzil), nd other hemils were otined from Sigm Aldrih (St. Louis, MO). Protein mesurement. Totl proteins were evluted from three stored smples t eh olletion time-point. All mnipultions were rried out t 4 C. Totl solule protein extrtion ws performed ording to Cnghul-Inoente et l. (29). Three repetitions of smples (3 mg fresh weight [FM] for eh time-point) were merted t 4 C with 1 ml of extrtion uffer (ph 7.) ontining 5 mm sodium phosphte disi,.2 M β-merptoethnol, 17.3 mm SDS, nd 1 mm PMSF nd were entrifuged t 4 C for 2 min t 8, 3 g. The superntnt ontining totl solule proteins ws removed nd the pellet stored t 2 C. Solule proteins were sedimented t C y dding two volumes of 1% ethnol into the superntnt nd then entrifuged t 4 C for 2 min t 12,85 g. The protein sediment ws soluilized in 5 mm sodium phosphte disi (ph 7.). Protein ontent ws determined y the Brdford (1976) method, using ovine serum lumin s stndrd. Totl sugr nd strh determintion. The extrtion of totl solule sugrs ws performed ording to Shnnon (1968). The pellet from the protein extrtion ws merted using 2 ml methnol hloroform wter (MCW; 12:5:3; v/v) nd

4 168 CANGAHUALA-INOCENTE ET AL. entrifuged for 1 min t 5 g. The superntnt ws reovered nd the pellet re-extrted using 2 ml MCW. One prt hloroform nd 1.5 prts wter were dded for eh four prts of superntnt, followed y entrifugtion step t 5 g for 1 min, from whih two phses were otined. The upper queous phse ws removed for dosge using nthrone t.2% (w/v) following the proedure of Umreit nd Burris (196). The extrtion nd determintion of strh levels were sed on the proedure of MCredy et l. (195). The pellets used in the totl solule sugr extrtion were ground with 1 ml of 3% perhlori id nd entrifuged for 15 min t 12,85 g. The superntnt ontining strh ws removed, nd the pellets were re-extrted twie. The superntnts were omined nd the pellets eliminted. For dosge, nthrone t.2%w/v ws used. The sugr nd strh onentrtions were lulted using gluose s stndrd. The sorne ws red in UV VIS UV-123 spetrophotometer (Shimdzu) t 62 nm. Amino id nlysis. Amino ids determintion ws rried out ording to Snt-Ctrin et l. (26). Smples (2 mg) were ground in 6 ml of 8% (v/v) ethnol nd onentrted in SpeedV. Smples were resuspended in 2 ml Milli-Q wter nd entrifuged t 2, g for 1 min. The superntnt ws filtered through 2-μm memrne. Amino ids were derivtizted with o-phthldildehyde (OPA) nd identified y high-performne liquid hromtogrphy (HPLC), using C-18 reverse phse olumn (SUPELCOSIL TM,5μm prtile size, L I.D. 25 m 4.6 mm). The grdient ws developed y mixing inresing proportions of 65% (v/v) methnol with uffer solution (5 mm sodium ette, 5 mm sodium phosphte, 2 ml L 1 methnol, nd 2 ml L 1 tetrhydrofurnt ph 8.1 djusted with eti id). The grdient of 65% methnol ws progrmmed to 2% over the first 32 min, from 2% to 1% etween 32 nd 71 min nd 1% etween 71 nd 8 min, t 1 ml min 1 flow t 4 C. Fluoresene exittion nd emission wvelengths of 25 nd 48 nm, respetively, were used for mino id detetion. Pek res nd retention times were mesured y omprison with known quntities of stndrd mino ids. All the nlytil grde regents nd solvents used in the mino id determintion were purhsed from Sigm-Aldrih or Merk nd were used or prepred s reommended y the produer. Anlysis of polymines. Putresine (Put), Spermidine (Spd), nd Spermine (Spm) were determined ording to the proedures desried y Silveir et l. (24). All mnipultions were rried out t 4 C, nd nlyses were performed in triplite. Smples (2 mg FM) for eh olletion time were ground in 3 ml of 5% (v/v) perhlori id. After 1 h, the extrted smples were entrifuged for 2 min t 15, g t C. The superntnts ontining free PAs were removed, nd the pellets were re-extrted. Superntnts were then omined nd the pellets disrded. Free PAs were determined diretly from the superntnt, nd onjugted PAs were extrted y hydrolyzing 2 μl of superntnt with 2 μl of 12 N HCl for 18 h t 11 C. The smples were dried under liquid nitrogen. The onjugted PAs were soluilized in 2 μl of5%(v/v) perhlori id. Free nd onjugted PAs were derived y dnsyl hloride mixed with etone t onentrtion of 5 mg ml 1. A 4-μL liquot of smple ws dded to 1 μl of dnsyl hloride, 2 μl of.5 mm diminoheptne, nd 5 μl ofsturted sodium ronte. The smples were then inuted in the drk for 5 min t 7 C. After 3 min inution, dnsylted PAs were extrted with 2 μl of toluene. The toluene phse ws olleted nd dried under liquid nitrogen. The dnsylted PAs were soluilized in 2 μl ofetonitrile. Twenty miroliters of the dnsylted PAs ws seprted y reverse phse HPLC in C-18 reverse phse olumn (Shimdzu SHIM-PACK CLC ODS, 5 μm prtile size, L I.D. 25 m 4.6 mm). The grdient ws developed y mixing inresing proportions of solute etonitrile to 1% etonitrile in wter (ph 3.5). The grdient of solute etonitrile ws progrmmed to 65% over the first 1 min, from 65% to 1% etween 1 nd 13 min nd 1% etween 13 nd 21 min. The flow ws 1 ml min 1 t 4 C. The fluoresene detetor ws set t 34 nm (exittion) nd 51 nm (emission). A mixture of Put, Spd, nd Spm ws used s stndrd. All the nlytil grde regents nd solvents used for determintion of PAs were from Sigm-Aldrih or Merk nd were used or prepred s reommended y the produer. Dt nlysis. Dt were nlyzed y ANOVA (P <.5) followed y the Student-Newmn-Keuls (SNK) test, using Sttisti version 7. softwre. When ANOVA nd SNK tests ould not e used, the men of three replites, s well s stndrd error, ws pplied to nlyze the dt. Results nd Disussion Biohemil hnges during the first 3 d of indution of somti emryogenesis. During the first 3-d ulture, orresponding to the indution phse of somti emryogenesis, the totl protein levels deresed nd remined onstnt ompred to levels oserved in explnts t the inoultion time (.84 mg g 1 FM; Fig. 1). This redution in protein levels over time ould e ttriuted to their onsumption in order to tivte the ellulr metolism for the estlishment of emryogeni ompetene s reported y Gutmnn et l. (1996) for emryogeni ultures of hyrid Lrix leptoeuope. The growth of emryogeni ultures is usully ssoited with hnges in the synthesis nd moiliztion of proteins, rohydrtes, nd lipids. The levels of these sustnes re

5 DYNAMICS OF PHYSIOLOGICAL AND BIOCHEMICAL CHANGES 169 [ ] mg/g FM protein sugr strh vrile t the different stges of ell ulture (Lulsdorf et l. 1992), where they t in the signl trnsdution pthwy or in the supply of sustrtes nd energy required for ell growth nd morphogenesis (Nomur nd Kommine 1995). In the present work, the strh ontent in the emryogeni ultures rpidly deresed then stilized. Sugr levels showed slight inrese fter 2 d ulture with rpid inrese towrds the end of the ulture period (Fig. 1). In Medigo rore, the ontent of reduing sugrs nd strh distinguished emryogeni from non-emryogeni llus; while high levels of sugrs nd low levels of strh were oserved in emryogeni ultures, the opposite ws oserved in non-emryogeni ultures (Mrtin et l. 2). Totl free mino id levels peked t the 6th d in ulture (Tle 1), period in whih the otyledons of the zygoti emryo used s explnt egn their expnsion. Following this, mino id levels deresed, peking gin t the 15th d of ulture (Tle 1), whih oinided with epiderml ell prolifertion (Fig. 7). Following this, mino id levels deresed up to the 24th d, remining onstnt until the 3th d of ulture (Tle 1). Similr results were reported in emryogeni ultures of Oote thrinensis, whih showed n inrese in the levels of totl free mino ids in the first wk in ulture, followed y signifint derese y the fifth wk (Snt- Ctrin et l. 24). In rrot, rpid inrese in mino id ontent ws reported during ell prolifertion nd in the erly developmentl stges of SE (Kmd nd Hrd 1984). Amino ids re the min form of nitrogen trnsport in ells, eing lso used for protein synthesis in order to support growth nd development in tissues with intense metoli tivity (Ortiz-Lopez et l. 2). In most somti emryogenesis systems, mino ids supplemented in ulture medium re soures of orgni nitrogen for the stimultion, indution, nd mintenne of this morphogeneti route. Glutmine, for exmple, is ommonly supplemented in Dys Figure 1. Totl protein, sugr, nd strh levels during the indution phse of A. sellowin somti emryogenesis (men±stndrd devition, n =3). Right y xis (red line) orresponds to strh levels. Left y xis orresponds to levels of protein nd sugr. Different letters indite differenes ording to the SNK test (P <.5). The sene of letters indites no signifint differenes were oserved mong tretment mens [ ] mg/g FM ulture medium s soure of orgni nitrogen (Frnklin nd Dixon 1994). In the present work, glutmine, rginine, nd sprgine showed the highest level mong the mino ids evluted (Tle 1). The high ontent of glutmine ould e sried to the supplementtion of glutmi id in the ulture medium. Glutmi id is preursor of glutmine, s well s the min donor of nitrogen, during nolism (Bohinski 1991). Similrly, emryogeni llus of Pinus nksin showed high levels of glutmine nd sprgine (Durzn nd Chlup 1976). In O. thrinensis, these mino ids showed intermedite vlues, while rginine, γ-minoutyri id (GABA), lysine nd glutmi id showed higher levels (Snt-Ctrin et l. 24). In our work, group of mino ids, inluding lnine, rginine, GABA, glutmine, glyine, methionine, phenyllnine, ornithine, nd tyrosine showed enhned levels t the 6th d in ulture. Another group, sprgine, sprti id, nd glutmi id predominted y the 15th d. The other mino ids presented their mximum vlues in oth evluted periods (6th nd 15th d; Tle 1). During the SE indution phse, PA levels inresed, peking t the 9th d of ulture, nd then deresed (Fig. 2). In the indution phse of Solnum melongen emryogeni ultures, n intense ell prolifertion ws deteted nd ws ssoited with enhned PA levels (Ydv nd Rjm 1997). In Querus ilex, totl PAs were more undnt in emryogeni llus nd in oth somti nd zygoti immture emryos. Spm ws more undnt in emryogeni llus nd in immture zygoti emryos thn in mture emryos (Muri nd Mnzner 211). High PA levels re ommonly oserved in tissues undergoing somti emryogenesis (Louknin nd Thorpe 28). In our work, the PA umultion minly resulted from high levels of free Put. Free PA inresed during the first d in ulture then deresed (Fig. 3A). In Citrus sinensis, inresed levels of Spd nd Spm were deteted in the ultures during the first 2 d, period oiniding with the indutive phse of somti emryogenesis (Wu et l. 29). Conjugte PAs were not deteted from the 6th to the 15th d in ulture, ut did eome evident strting from the 18th d. Conjugte Put ws the predominnt form of onjugte PA (Fig. 3B). The lne mong free nd onjugted PA forms my e ritil in different developmentl proesses (Torrigini et l. 1987), nd when the onjugted PA levels re high, orgnogenesis proeeds (Srmgli et l. 1999). On the other hnd, high levels of Put t the eginning of somti emryogenesis re relted to the ility of ompetent ells to develop somti emryos (Nieves et l. 23). Tken together, results for PAs otined in the present study reveled tht synthesis nd umultion of these ompounds ourred t the onset of somti emryogenesis. In this sme speies, n intensive prolifertion of epiderml ells ws ssoited with the umultion of phenoli ompounds with further orgniztion of meristemti enters nd prolifertion of pro-emryoni ell msses

6 17 CANGAHUALA-INOCENTE ET AL. Tle 1. Free mino ids (μg g 1 FW) during the indution of A. sellowin somti emryogenesis throughout the first 3 d ulture Dys fter indution All periods Alnine 9.7± ± ± ± ± ± ± ±8.9 3.±.4 8.3±.2 9.± C Arginine 75.1± ± ± ± ± ± ± ± ± ± ± AB γ-aminoutyri id (GABA) 17.9± ± ± ± ± ± ± ± ± ± ± C Glutmine 6.2 ± ± ± ± ± ± ± ± ± ± ± A Glyine 4.2± ± ± ± ± ± ± ±1.6.6±. 1.3±.2 1.3± C Methionine.3±.5 5.9± ±.6 3.7±.5 1.6± ±2. 3.2± C Phenyllnine 2.4±.6 1.7± ±.9 6.4±.5 8.4± ±3. 4.1± ±.6 1.8±.5 1.4±.3 1.5± C Ornithine.6±.1 1.6±.3 2.4±.2.8±.3.6±.5 1.2±.3.8± ±.3 1.±.5.9C Tyrosine 1.4±.1 2.9±.7 12.±.7 2.7±.4 2.4±.9 3.4±.4 1.2±.4 1.3±.3.8±.2.8±.2.9±.1 2.7C Asprgine 22.2± ± ± ± ± ± ± ± ± ± ± B Asprti id 4. ± ± ± ± ± ± ± ±.4 1.8± ± ± C Glutmi id 1.7±.4 4.2± ± ± ± ± ± ± ± ± ± C Histidine 28.3± ± ± ± ± ± ± ± ± ± ± C Isoleuine 2.2±.4 8.1± ± ± ± ±6.4 5.± ±.8.8±.2 1.1±..9± C Leuine 2.± ± ± ± ± ± ± ± ±.1 2.2±.2 1.9± C Lysine 5.7± ± ± ± ± ± ± ± ±. 3.7±.8 3.2± C Serine 2.9± ± ± ± ± ± ± ± ±.4 7.8±.6 7.5± C Threonine 3.7±.9 5.9± ±.9 6.7± ± ± ± ±.6 1.3±.1 1.8±.3 2.4± C Tryptophn.8±.2 2.7±.6 9.6±.5 2.6±.6 6.± ± ±.7 7.7±.4 2.9±.2 2.9±.5 3.3±.5 4.9C Vline 3.5± ± ± ± ± ± ± ±1. 1.7±.2 2.2±. 1.6± C Totl 194.6E C A CD C B 838.6DE CD 335.5E 376.4E 415.9E Dt re presented s men±stndrd error (n =3). Different letters indite differenes ording to the SNK test (P <.5)

7 DYNAMICS OF PHYSIOLOGICAL AND BIOCHEMICAL CHANGES 171 Totl PAs (ug/g FM) ef e (Cnghul-Inoente et l. 24). Aruri ngustifoli emryogeni ultures sumitted to two tretments (ontrol nd 5 mm GSH) showed the sme levels of endogenous PAs during 3 d of inution. On the other hnd, totl PA ontent showed signifint derese in Put nd Spd levels fter 15 d in ulture, remining stle until dy 3 (Vieir et l d ef ef f f Dys of indution Figure 2. Totl polymines (PAs) during the indution phse of A. sellowin somti emryogenesis (men±stndrd devition, n =3). Different letters indite differenes ording to the SNK test (P <.5). The sene of letters indites no signifint differenes were oserved mong tretment mens. A Free PAs(µg/g FM) B Conjugte PAs (µg/g FM) d e d e e d PUT SPD SPM d d e ef d e f d e f d e f d Dys of indution Figure 3. Polymines (PAs): (A) free nd(b) onjugte during the indution phse of A. sellowin somti emryogenesis (men±stndrd devition, n =3). Different letters indite differenes ording to the SNK test (P <.5). The sene of letters indites no signifint differenes were oserved mong tretment mens. Biohemil hnges during development of somti emryos. The developmentl stges of somti emryos were estlished, tking into ount the following riteri: () gloulr (G), trnsluent nd spheril gloulr somti emryos.5 1 mm in dimeter; () hert (H), hert-shped somti emryos, 1 2 mm long; () torpedo (T), torpedoshped elongted somti emryos pproximtely 3 mm long, with histologil nlysis reveling well-defined promium nd onspiuous shoot nd root pil meristems; (d) preotyledonry (PC), white-trnsluent somti emryos longer thn 3 mm showing inipiently elongted otyledonry leves; (e) otyledonry (CT), white somti emryos with expnded otyledonry leves. Signifint differenes were found in the totl protein levels mong somti emryos t different developmentl stges ( mg g 1 FW; Fig. 4). As for the totl sugr levels, signifint differenes were oserved mong the different developmentl stges of somti emryos. Strh ontent peked t the gloulr stge, progressively deresed, nd then inresed gin in the otyledonry stge (Fig. 4). In the sme speies, otyledonry-stged zygoti emryos olleted 9 d fter pollintion showed the highest strh ontent (Pesdor et l. 28; Cnghul-Inoente et l. 29). In somti emryos of Medigo stiv, the high ontent of sugr ws sried to the ron soure in the ulture medium (Horowiz et l. 1995). In soyen somti emryos, the solute mounts of protein, lipid, nd solule sugr orrelted with the umultion of fresh mss, with mrked derese when the ultures eme senesent (He et l. 211). In this work, erly-stge somti emryos (gloulr nd hert) showed signifint vrition in the totl ontent of free mino ids, in omprison with somti emryos t more dvned stges, i.e., torpedo nd otyledonry, showing enhned levels of sprgine nd rginine (Tle 2). In tissues with intense metoli tivity, mino ids re moilized in order to support growth nd development (Ortiz-Lopez et l. 2). Thus, in the present work, we suggest tht the low levels of totl mino ids in the erly developmentl stges resulted from demnd to support hystodifferentition events ulminting in the full development of somti emryos. [ ] mg/g FW protein strh sugr H T PC C stge of development Figure 4. Totl protein, strh, nd sugr levels (mg g 1 FW) t different stges of A. sellowin somti emryos (men±stndrd devition, n =3). H hert, T torpedo, PC preotyledonry, CT otyledonry. Different letters indite differenes ording to the SNK test (P <.5). d

8 172 CANGAHUALA-INOCENTE ET AL. Tle 2. Free mino ids (μgg 1 FW) t different developmentl stges of A. sellowin somti emryos 18 Stges G H T CT Asprti id 1.3± ± ± ±3.5 Glutmi id 14.9±2.9 1.± ± ±2.2 Asprgine 126.2± ± ± ±8.9 Serine 4.9± ± ± ±9. Glutmine 6.8± ± ± ±12. Histidine 18.1± ± ± ±1.8 Glyine.9±.3.9±. 9.1± ±7.1 Arginine 55.5± ± ± ±51.7 Threonine.6±.1 1.3±.1 11.± ±.5 Alnine 4.±.5 3.± ± ±2.4 γ-aminoutyri 3.4±.9 5.2± ± ±21.4 id (GABA) Tyrosine...7±.1 2.5±.1 Tryptophn 3.9± ±.8 15.±.7 4.6±7.1 Methionine. 4.5±... Vline 3.3±.9 2.± ± ±1.3 Phenyllnine 7.2± ± ± ±7. Isoleuine 2.1±.6 1.2±. 9.± ±1.6 Leuine 5.5± ±. 19.3± ±4.9 Ornithine.... Lysine 6.± ±. 21.9± ±6.7 Totl 273.8C 24.8C 915.2B 2,37.4A Dt re presented s men±stndrd error (n =3). Different letters indite differenes ording to the SNK test (P <.5) G gloulr, H hert, T torpedo, CT otyledonry Amino id levels, s determined in the present study, re oinident with ptterns oserved in zygoti emryos t the sme developmentl stges (Cnghul-Inoente et l. 29), ut t reltively lower levels. However, it should e stressed tht in the se of zygoti emryos the presene of teguments nd endosperm tissues my hve ffeted the umultion ptterns of these ompounds. During A. sellowin zygoti emryogenesis, in the olleting times orresponding to the evluted developmentl stges, the predominnt mino ids were sprgine nd glutmine (Cnghul-Inoente et l. 29). However, in the present work, the predominnt mino ids in A. sellowin somti emryos were rginine nd sprgine. In plnts, sprgine is normlly ssoited with nitrogen trnsport, nd its ontent in some speies re stritly regulted y light (Lm et l. 1998). Surose nd mino ids, minly sprgine nd glutmine, were the primry ron nd nitrogen soures ville for soyen emryo germintion (Rinird et l. 1984). A positive orreltion ws oserved etween free sprgine nd the storge protein ontent in Totl PAs (ug/g FM) 12 6 soyen seeds (Hernández-Sestià et l. 25). In the present work, ornithine ws not deteted t ny evluted developmentl stge, suggesting tht whole synthesized ornithine ws onverted into rginine. It is lso importnt to stress tht sine the ulture medium ws supplemented with glutmi id, this mino id ould e onverted to ornithine vi etyltion of derivtives of glutmte, s desried y Thompson (198). It hs een shown tht rginine ts s rrier moleule of N, G H T PC CT Stges of development Figure 5. Totl Polymines (PAs) t different developmentl stges of A. sellowin somti emryos. Ggloulr, H hert, T torpedo, PC preotyledonry, CT otyledonry (men±stndrd devition, n =3). Different letters indite differenes ording to the SNK test (P <.5). A Free PAs (ug/g FM) B Conjugte PAs (ug/g FM) G H T PC CT PUT SPD SPM G H T PC CT Stges of development Figure 6. Polymine (PA) levels: (A) free nd (B) onjugte t different developmentl stges of A. sellowin somti emryos. G gloulr, H hert, T torpedo, PC preotyledonry, CT otyledonry (men±stndrd devition, n =3). Different letters indite differenes ording to the SNK test (P <.5).

9 DYNAMICS OF PHYSIOLOGICAL AND BIOCHEMICAL CHANGES 173 Figure 7. Summry of iohemil hnges during the indution phse of somti emryogenesis of A. sellowin. nd s suh, it is n importnt form of N storge in plnts. In seeds, rginine omprises out 4% of the nitrogen stored in proteins, s shown y Millef nd Shelp (1989). Totl PA levels were found to signifintly inrese when ompring gloulr-, hert- nd torpedo-stged somti emryos with preotyledonry- nd otyledonry-stged somti emryos (Fig. 5). In zygoti emryos, PA levels were similr t ll developmentl stges (Cnghul-Inoente et l. 29). Thus, otyledonry-stged somti emryos displyed more totl free PAs thn otyledonry-stged zygoti emryos, inditing tht the metolism of PAs is quite different in these two developmentl pthwys. PAs re synthesized from ornithine, rginine, lysine, nd methionine (Bgni nd Tssoni 21). In the present work, orreltion mong PAs nd their preursor mino ids, rginine nd lysine, reveled tht oth showed the highest vlues in otyledonry-stged somti emryos. Put nd Spd re generlly the most undnt PAs in plnts, while Spm is only present in tre mounts (Bgni nd Tssoni 21). In A. sellowin, free Put inresed during ll developmentl stges of somti emryos, while n inrese in free Spd ws oserved t more dvned developmentl stges (torpedo, pre-otyledonry nd otyledonrystged somti emryos). For free Spm, the vlues were onsistently high t ll developmentl stges (Fig. 6A). In somti emryos of O. trinensis, high levels of PAs were present in gloulr emryos nd wheres low levels were reorded t otyledonry stges (Snt-Ctrin et l. 24). Figure 8. Summry of iohemil hnges during the developmentl stges of A. sellowin of somti emryos. G gloulr, H hert, T torpedo, PC preotyledonry, CT otyledonry. Asterisk mens not present.

10 174 CANGAHUALA-INOCENTE ET AL. Similrly, in Theorom o somti emryo differentition nd development, Spd levels were signifintly higher thn oth Put nd Spm levels (Niemenk et l. 212). Querus ilex showed high levels of Spd, whih deresed during mturtion nd germintion (Muri nd Mnzner 211). In plnts, PAs our s free moleules together with other moleules, suh s mides of hydroxy-innmi id or proteins (Bgni nd Tssoni 21). In the present work, only Put ws present s PA onjugte in the gloulr nd torpedostged somti emryo. Spd nd Spm onjugtes were present in hert- nd preotyledonry-stged somti emryos in different proportions (Fig. 6B). In zygoti emryos of A. sellowin, the onjugte PAs were present t ll evluted developmentl stges (Cnghul-Inoente et l. 29). Thus, the ptterns of synthesis nd umultion of PAs in A. sellowin somti nd zygoti emryos seems to follow different ptterns. In onlusion, this work sheds light into the dynmis of physiologil nd iohemil hnges ourring during somti emryogenesis of A. sellowin (Figs. 7 nd 8). Vritions in the levels of protein, strh, mino ids, nd polymines ourred during the indution phse of somti emryogenesis. The ptterns of synthesis nd umultion of proteins nd mino ids t different developmentl stges of somti emryos prllel the ptterns oserved during the development of zygoti emryos, s oserved in previous studies with this sme speies (Cnghul-Inoente et l 29). Differenes in the ptterns of synthesis nd umultion of PAs etween somti nd zygoti emryos re evident, nd these findings re importnt sine PA n ffet the synthesis of other endogenous ompounds, suh s the uxin IAA, whih is involved, mong others, in the estlishment of emryo polrity. Referenes Alázr R.; Altell T.; Mro F.; Bortolotti C.; Reymond M.; Konz C.; Crrso P.; Tiurio A. F. Polymines: Moleules with regultory funtions in plnt ioti stress tolerne. Plnt 231: ; 21. 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