The role of γ-aminobutyric acid (Gaba) in somatic embryogenesis of Acca sellowiana Berg. (Myrtaceae)

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1 2009 rzilin society of plnt physiology DOI /S RESEARCH ARTICLE The role of γ-minoutyric cid (G) in somtic emryogenesis of Acc sellowin Berg. (Myrtcee) Mristel Ritz Booz 1, Gilerto B. Keruy 2, Miguel Pedro Guerr 3 nd Rosete Pescdor 1* 1 Deprtmento de Ciêncis Nturis, Universidde Regionl de Blumenu, Ru Antônio d Veig, 740, CEP CP Blumenu SC, Brsil. 2 Deprtmento de Botânic, Universidde de São Pulo, CP 11461, São Pulo, SP, Brsil. 3 Deprtmento de Fitotecni, Centro de Ciêncis Agráris, Universidde Federl de Snt Ctrin, Rod. Ademr Gonzg, Km 3, Florinópolis, SC CEP , Brsil. * Corresponding uthor: rosetep@fur.r Received: 27 June 2009; Accepted : 24 Ferury Astrct The γ-minoutyric cid (G) is non-protein mino cid found in prokryotes nd eukryotes. Its role in plnt development hs not een fully estlished. This study reports quntifiction of the levels of endogenous G, s well s investigtion of its role in different stges of somtic emryogenesis in Acc sellowin Berg. (Myrtcee). Zygotic emryos were used s explnts nd they were inoculted into the culture medium contined different concentrtions of G (0,2, 4, 6, 8 nd 10 μm). The highest concentrtions of endogenous G were detected etween the third nd nine dys fter inocultion, reching the vlue of μmol.g -1 FW. High frequency of somtic emryogenesis ws oserved in response to 10 μm G. This tretment lso resulted in lrge numer of norml emryos, nd the lowest percentge of formtion of fused somtic emryos, phenotypic chrcteristic of most deformed emryos in ll tretments. Also, ll tretments promoted the formtion of the somtic emryos with positive chrcteristics of development resumption, which however did not originte the seedlings. Key words: Amino cid, pinepple guv, plnt growth Resumo O ácido γ-minoutírico (G) é um minoácido não protéico encontrdo em procriontes e eucriontes. Seu ppel em plnts ind não está em estelecido. No presente estudo procurou-se quntificr os teores endógenos de G, em como investigr seu ppel nos diferentes estágios d emriogênese somátic em Acc sellowin Berg. (Myrtcee). Form empregdos emriões zigóticos como explntes e os mesmos form inoculdos em meio de cultur contendo diferentes concentrções de G: 0 (controle), 2, 4, 6, 8 e 10 µm. As miores concentrções de g endógeno form detectds no período compreendido entre o 3º e o 9º di pós inoculção, tendo lcnçdo, neste último di, o vlor de 12,77 μmol.g -1 FW. Alt freqüênci de emriogênese somátic foi oservd em respost 10 µm de G. Este trtmento tmém resultou em grnde número de emriões normis, em como o menor percentul de formção de emriões cupuliformes, crcterístic fenotípic d miori dos emriões deformdos em todos os trtmentos. Em todos os trtmentos ocorrerm emriões somáticos que presentrm crcterístics positivs qunto à retomd de desenvolvimento, ms que não resultrm n formção de plântuls. Plvrs-chve: minoácido, crescimento vegetl, goieir serrn

2 272 M. R. Booz et l. Introduction Acc sellowin Berg (Goieir serrn) is ntive Myrtcee from the South Brzil nd North Uruguy. This species is cultivted in commercil orchrds in New Zelnd, USA, nd Europe (Ducroquet & Hickel, 1997). In Brzil domestiction progrm culminted with the relese of the first commercil vrieties (Ducroquet et l., 2007, 2008). Woody plnts re considered reclcitrnt to somtic emryogenesis, nd the results so fr otined in Acc sellowin regrding to somtic emryogenesis suggest its use s model system for the induction nd control of this in vitro morphogenetic route (Guerr et l., 2001). In this wy somtic emryogenesis hs long een considering s interesting system for studies of fundmentl spects of plnt emryo development (Hlperin, 1995). A. sellowin somtic emryogenesis ws firstly reported y Cruz et l., (1990). High frequency of somtic emryos ws further otined (Cnhoto & Cruz, 1994, 1996). In Brzil the first results on somtic emryogenesis in this species were reported y Guerr et l. (1997) which showed tht this morphogenetic route ws dependent on the culture medium composition nd the genotype employed. Dl Vesco & Guerr (2001) showed tht different nitrogen sources in the culture medium ffected the numer of somtic emryos developed from zygotic emryos. Guerr et l. (2001) demonstrted tht somtic emryogenesis in this species ws ffected y 2,4-D pulses, nd histologicl studies reveled normlities on the development of somtic emryos. Stefnello et l. (2005) reported emryogenic competence from florl tissues, nd Cnghul-Inocente et l. (2007) included the technology of synthetic seeds in the somtic emryogenesis protocol. Somtic emryogenesis in A. sellowin seems to recpitulte the zygotic emryogenesis. Both of these processes re complex nd modulted y genetic, iochemicl nd physiologicl fctors. Among these fctors, mino cids represent the first step in the nitrogen ssimiltion (Ortiz- Lopez et l., 2000), nd ply key role in the development of somtic emryos (Merkle et l., 1995). The cid γ-minoutíric cid (G) is non protein mino cid found in prokryotes nd eukryotes. Although role of G in plnts hs not een fully defined, it seems to e involved in developmentl processes including signling, defense nd stresses s well s the induction of somtic emryogenesis (Bown & Shelp, 1997). It hs een shown tht G ccumultes in severl plnt tissues under different stress conditions, such s hypoxi, temperture, wter nd some plnt growth regultor s is the cse of 2,4-D (Snedden & Fromm, 1998). The im of the present work is to quntify the endogenous levels of mino cids nd G in the different developmentl stges of A. sellowin somtic emryos. The effect of different levels of G on the induction nd development of somtic emryos ws lso exmined. Mteril nd Methods Extrction nd quntifiction of totl mino cids nd G: Mtures zygotic emryos (0.4 mm length) excised from seeds under stereomicroscope were inoculted in test tues ( mm) contining 10 ml of culture medium composed of LP micro nd mcronutrients, Morel vitmins, sucrose (30 g.l -1 ), 2,4-D (20 µm), nd glutmine (4 mm). The ph of culture medium ws djusted to 5.8 prior to ddition of 0.7 % gr (Guerr et l., 1997). The tues were covered with metllic cps nd mintined in culture room in the drk t 25±1ºC. For mino cid nlysis the smples of zygotic emryos were used s explnts every three dys until 30th dy. After 70 dys in culture, somtic emryos in different developmentl stges were oserved nd collected corresponding to gloulr, hert, torpedo, nd cotyledonry stges. All these smples were frozen in liquid nitrogen nd then stored t -20 º C. For mino cid extrction solution of methnol, chloroform nd wter (MCW) ws used in the proportion of 12:5:3. Smples of 200 mg of fresh mss in triplicte were grinded in 10 ml of MCW with liquid nitrogen. After three dys the mcerte ws centrifuged t g for 10 min nd the superntnt ws collected. The residue ws extrcted gin with 5 ml of MCW nd centrifuged using the sme prmeters. The superntnts were pooled nd gitted with 5 ml of MCW (Bielski & Turner), nd centrifuged t g. After seprtion the cquous phse ws collected nd lyophilized under vcuum. G ws detected using HPLC (High Performnce Liquid Chromtogrphy) with Novopck column PLC C18 ( mm). The conditions for seprtion were: 1 ml.min -1 solvent flow (solvent A: 93.9% sodium cette

3 The role of g-minoutyric cid (G) in somtic emryogenesis of Acc sellowin Berg. (Myrtcee) 273 uffer + 6% de cetonitrile + 0.1% de triethilmine, nd solvent B: de cetonitrile + ultrpure wter). G ws progressively eluted t 45ºC ccording to the increse in the orgnic frction ccording to Endres (2001). The G levels were clculted y mens of comprison of the re with the stndrd of known concentrtion. G Suplementtion in culture medium: Seeds of A. sellowin were otined from plnts mintined in the Germplm Colection of EPAGRI s São Joquim Experimentl Sttion, São Joquim, Snt Ctrin Stte, South of Brzil. The experiments were performed t the FURB Biotechnology Lortory, Blumenu, Snt Ctrin Stte. Mture zygotic emryos were used s explnts. The seeds were previously treted with 2.5% sodium hypochlorite solution for 40 min nd then wshed three times in sterile distilled wter. The emryos were excised under stereomicroscope in sterile chmer nd inoculted in test tues ( mm) contining 10 ml of culture medium nd closed with metllic cps. The culture medium ws sed on LP slts (Von Arnold & Eriksson, 1981), Morel vitmins (Morel nd Wetmore 1951) supplemented with sucrose (30 g.l -1 ), 2,4-D (20 µm), glutmine (4 µm), gr (7 g.l -1 ), nd different concentrtions of G: 0 (control), 2, 4, 6, 8 nd 10 µm. The ph of the culture medium ws djusted to 5.8. A totl of 360 zygotic emryos were inoculted in ech test tue with 30 replictes for ech tretment. The cultures were mintined in the drk t 25 ± 2ºC. The evluted prmeters used were the numer of somtic emryos in the different developmentl stges: gloulr, hert, torpedo nd cotyledonry oserved fter 20 nd 35 dys in culture. Mlformtion of somtic emryos ws exmined fter 64 dys in culture. After 70 dys in culture the somtic emryos were isolted nd seprted ccording to the developmentl stge. Afterwrds they were trnsferred to the sme culture medium free of 2,4-D nd G nd with the level of sucrose lowered to of the initil concentrtion. The cultures were mintined during 20 dys in chmer room with 16h light period, 50 µmol.m 2.s -1 light intensity, nd 25 ± 2ºC. The experimentl design ws rndomized with 15 replictes for ech tretment nd ech developmentl stge. The evluted prmeters were the numer of somtic emryos progressing to the next developmentl stge nd the numer of necrosed, quiescent nd regrown somtic emryos. The dt were sumitted to ANOVA nd men seprtion crried out y Tukey test (5%) ResultS Quntifiction of mino cids nd G levels: The quntifiction of the endogenous levels of mino cids in culture reveled more thn 6-fold increse from the inocultion time to the third dy, followed y slow decrese until 24 dy in culture (Figure 1). New rise in these levels ws oserved fterwrds for next 6 dys, till culture completed 30 dys. The decrese in the mino cids levels from 3rd to 24th dy ws concomitnt with the period of intense cell prolifertion oserved from nine to eighteen dy nd the formtion of gloulr somtic emryos which took plce in culture from 15th to 24th dy. 60 Totl Amino cids (µmol.g -1 FW) Dys Figure 1. Vritions on totl mino cids levels during Acc sellowin somtic emryogenenesis. The dt re the men of 3 experiments ±SE. As shown in Figure 2, continuous decrese in the mino cid levels ws oserved in different developmentl stges of A. sellowin somtic emryos. The gloulr stge exhiited the totl mino cids concentrtion of 42.6 mmol.g -1 FM, while cotyledonry stge showed slight reduction y ~29%.

4 274 M. R. Booz et l Totl Amino cids (µmol.g -1 FW) G (µmol.g -1 FW) gloulr hert torpedo cotyledonry Emryonic phses Figure 2. The levels of totl mino cids in different developmentl stges of Acc sellowin somtic emryos. The dt re the men of 3 experiments ±SE gloulr hert torpedo cotyledonry Emryonic phses The highest levels of G ws detected from the third to nine dy, when the levels peked t µmol.g -1 fm (Figure 3). Agin, this period coincided with the intense cell prolifertion in the explnts. Similr to mino cids content, decrese in the G levels occurred during the different developmentl stges of somtic emryos (Figure 4). However G concentrtions dropped ~90% in cotyledonry-stged somtic emryos (0.08 µmol.g -1 fm) compring to gloulr ones. The contriution of G to the totl free mino cids in emryogenic cllus is sustntil compred to non emryogenic cllus (Nieenk et l., 2008) nd further developmentl stges of cco somtic emryos. This finding fits the evidence tht cquisition of emryogenic competence is stress process. G is commonly found ssocited with stress conditions in plnts (Bown & Shelp 1997; Mesnrd et l., 2000). It is produced in plnts s result of the ctivity of glutmte decroxylse nd rpidly ccumultes under vrious stress conditions (Shelp et l., 1999; Bouché et l., 2003; Bouché & Fromm, 2004). G (µmol.g -1 FW) Dys Figure 3. G levels during Acc sellowin somtic emryogenesis. The dt re men of 3 experiments ±SE Figure 4. Levels of G in different developmentl stges of Acc sellowin somtic emryos. The dt re the men of 3 experiments ±SE. Induction nd development of somtic emryos is ffected y G: In the present work 100% of zygotic emryos were used s explnts for producing the somtic emryos. The presence of G in cultures resulted in the induction of somtic emryos fter 20 dys indicting tht G plyed effective role in conferring emryogenic competence to explnts. The tretment with 10 µm G significntly enhnced the induction rte of gloulr somtic emryos (Figure 5). After 35 dys in culture, ll developmentl stges of somtic emryos were present, i.e. gloulr, hert, torpedo nd cotyledonry (Figure 6). Somtic Emryos 80% 70% 0% Figure 5. Formtion of somtic emryos from Acc sellowin zygotic emryos in response to exposure to different G concentrtions. The numer of somtic emryos ws determined fter 20 dys in culture. Different letter in the rs show different meningful sttistic, ccording to Tukey (p = 0.05).

5 The role of g-minoutyric cid (G) in somtic emryogenesis of Acc sellowin Berg. (Myrtcee) Averge Numer of Emryos gloulr hert torpedo cotyledonry Figure 6. Numer of Acc sellowin somtic emryos in different developmentl stges in response to different G concentrtions fter 35 dys in culture. Different letter in the rs show different meningful sttistic, ccording to Tukey (p = 0.05), only for gloulr stge. Morphology of somtic emryo in response to G: Exmintion of somtic emryos in the cotyledonry stge identified t lest four different morphologies. Figure 7 shows the difference etween norml somtic emryo nd those exhiiting some errnt mlformtion s the sence of cotyledon, fused cotyledons or the presence of more thn two cotyledons. Fused somtic emryos were the most frequent followed y norml ones. The tretments with G resulted in the sme four types of somtic emryo morphology, however the elevtion in G concentrtions promoted decrese in the rte of fused somtic emryos nd n increse in the rte of norml somtic emryos (Figure 8).

6 276 M. R. Booz et l. c d 1mm 1mm 1mm 1mm Figure 7. Morphology of Acc sellowin somtic emryos in cotyledonry stge: norml emryo; emryos with fused cotyledons; c one cotyledon emryo; d emryo with three cotyledons. Arrows indicte cotyledons % Somtic Emryos c c c c c c Asence of cotyledon Fused cotyledons More thn two cotyledons Norml Figure 8. Norml nd norml somtic emryo rte of Acc sellowin in response to different G concentrtions fter 64 dys in culture. Different letters in the rs show different meningful sttistic ccording proportion test (p = 0.05).

7 The role of g-minoutyric cid (G) in somtic emryogenesis of Acc sellowin Berg. (Myrtcee) 277 Somtic emryo regrowth fter G tretment: Somtic emryos were clssified in three ctegories: necrosed, quiescent nd regrown. Somtic emryos in gloulr stge were le to regrowth in ll G tretments (Figure 9). From the other hnd, only control nd tretments with 6 nd 8 µm G resulted in quiescent somtic emryos. Low levels of necrosis were oserved in the control nd 2 µm G contining culture ( nd, respectively) while higher concentrtions incresed necrosis. These sme tretments lso leded to enhnced re-growth of somtic emryos ( nd 80%, respectively). In the hert stge (Figure 9) ll tretments provided three ctegories of somtic emryos. G t 8 µm stimulted the rte of regrowth of somtic emryos () while 4 nd 6 µm G exhiited low rtes () of necrosed somtic emryos. Figure 9c summrizes the dt on torpedo stge. Quiescent somtic emryos were oserved under ll tretments. G t 4 to 10 µm induced the rte of somtic emryos showing regrowth () nd 4 e 8 µm G leded to low rtes of necrosed somtic emryos (). In cotyledonry somtic emryos (Figure 9d), the tretment with 10 µm G promoted the highest rte of somtic emryos showing regrowth (80%). This is significnt increse s compred to the control culture. The tretments with 6 nd 8 µm G resulted in of quiescent somtic emryos. Control tretment nd 2 µm G reveled higher rtes of somtic emryos with necrosis ( nd, respectively). 100% 100% Emryos 80% Emryos 0% 0% Quiescent Necrosis Regrowth Quiescent Necrosis Regrowth c 100% d 100% Emryos 0% Emryos 0% 70% 80% Quiescent Necrosis Regrowth Quiescent Necrosis Regrowth Figure 9. Rte of Acc sellowin somtic emryos in response to different levels of G fter 20 dys in culture of mturtion. Stges: ) gloulr ) hert c) torpedo d) cotyledonry. DISCUSSION In plnts free mino cids re importnt for the growth regultion nd their levels re strictly controlled t cytoplsm (Brniex & Cusin, 1996). Present study reports vrition in the mino cid levels were found during Acc sellowin emryos development. These results re in greement with dt otined using Vigni mungo (Sen et l., 2002) on vritions in the totl mino cid levels during specific stges of orgnogenesis nd somtic emryogenesis. Modifictions in those levels during the induction nd development of Archis

8 278 M. R. Booz et l. hypoge somtic emryos were lso descried (Murch et l., 1999). In the present work the increse in the totl mino cids levels oserved immeditely fter the inocultion indictes n increse in the metolic ctivity which could e stimulted y 2,4-D present in the culture medium s suggested y Fehér et l., (2003). The decrese in the totl mino cid levels oserved in the course of A. sellowin somtic emryogenesis my e relted to the protein synthesis. The synthesis of LEA proteins (Merkle et l., 1995) occurs in the lte emryogenic stges, more precisely in the cotyledonry stge (Rock & Qutrno, 1995; Devic et l., 1996; Hirner et l., 1998). The decrese in the totl mino cids levels in the cotyledonry stge could lso e ssocited to the enzymes synthesis, minly those relted to the rffinose sugr (Konrádová et l., 2003), which is ccumulted during emryo mturtion nd desicction cquisition tolernce (Keller & Ludlow, 1993). The evidences indicte tht G ccumultes in severl plnt tissues under stress conditions, including tht cused y 2,4-D (Snedden & Fromm, 1998). The results of the present study suggest tht the increse in G levels could e ssocited with the high levels of 2,4-D (20 µm) supplemented to the culture medium for the somtic emryogenesis induction. In nimls G is ssocited with neurotrnsmission (Mody et l., 1994). The role of G in plnts is not fully understood ut it hs een suggested tht its ccumultion might e prt of n dptive response ssocited to cytoplsmtic cidosis (Crwford et l., 1994). However, the decrese in the cytoplsmtic ph is not pre-requisite for the G synthesis (Oh & Choi, 2001). Severl stressing fctors which re ssocited with the G synthesis re lso involved in the increse in the levels of cytosolic C 2+ (Bown & Shelp, 1997). Kmd & Hrd (1984) reported n increse in the totl mino cids levels, specificlly G, during the cell prolifertion nd development of somtic emryos in Ducus crot. An increse in G levels ws lso oserved in Archis hypoge emryogenic cultures (Murch et l, 1999). The induction of somtic emryogenesis relies on severl fctors such s crohydrte sources, mino cids, minerl nutrients nd hormones (Emons, 1994). Severl studies pointed out the eneficil effects of exogenous mino cid supplementtion in somtic emryogenesis. In A. sellowin the supplementtion of Glu, Asp nd Arg to the culture medium enhnced the rte of somtic emryogenesis induction (Dl Vesco & Guerr, 2001). In this sme species G induced high frequency of somtic emryogenesis (Booz & Pescdor, 2007). However it hs een postulted tht the supplementtion of mino cids to the culture medium my e positive or negtive to somtic emryogenesis (Merkle et l., 1995). The mlformtion of somtic emryos descried in the present work ws lso reported for this sme species y Cnhoto & Cruz (1994), nd Cnhoto et l. (2002). Such normlities were lso oserved in soyen (Lzzeri et l., 1987), pecn (Rodriguez & Wetzstein, 1994), linen (Dedicová et l., 2000), nd sweet potto (Mglhães, 2006). In A. sellowin s well s in the species listed ove the mlformtion of somtic emryos were minly ssocited to fused cotyledons nd ltertions in the stem picl meristem s is the cse of pecn (Mendes-d-Glóri, 1998). According to Cligri & Shohet (1993) long term mintennce of emryogenic cultures in culture medium with 2,4-D is ssocited with genetic modifictions tht my negtively ffect the emryogenic potentil. The present study provides the dt supporting the role of G in Acc sellowin somtic emryogenesis, which ws induced t high frequency in response to 10 µm G. This tretment lso enhnced the production of norml emryos, ut did not ffect the conversion rte of emryos into seedlings. These results dd new insights for future strtegies relted to the modultion of somtic emryogenesis in A. sellowin, nd evlution of physiologicl chnges ssocited with GABA. Acknowledgements: We thnk FAPESC for finncil support nd Alexndre Cohn d Silveir for the English corrections. References Brniex AJ, Cusin HF (1996) The centrl role of mino cids on nitrogen utiliztion nd plnt growth. J. Plnt Physiol. 149: Bielski LR, Turner NA (1966) Seprtion nd estimtion of mino cids in crude plnt extrcts y thin-lyer electrophoresis nd chromtogrphy. Anl. Biochem. 17:

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