Bio EZ Cangahuala Et Al 09

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ArticleTitle Dynamics of biochemical and morphophysiological changes during zygotic embryogenesis in Acca

sellowiana(Berg.) Burr.

Article Sub-Title

Article CopyRight Springer Science+Business Media B.V.

(This will be the copyright line in the final PDF)

Journal Name Plant Growth Regulation

Corresponding Author Family Name Guerra

Particle

Given Name Miguel Pedro

Suffix

Division Graduate Program in Plant Genetic Resources, Plant Developmental

Physiology and Genetics Laboratory

Organization Federal University of Santa Catarina

Address 88040-900, Florianpolis, SC, Brazil

Email [email protected]

Author Family Name Cangahuala-Inocente

Particle

Given Name Gabriela Claudia

Suffix






Author Family Name Silveira

Particle

Given Name Vanildo

Suffix

Division Biotechnology Laboratory

Organization CBB, State University of North Fluminense (UENF)

Address Av. Alberto Lamego, 2000, 28013-600, Campos dos Goytacazes, Rio de

Janeiro, Brazil


Author Family Name Caprestano

Particle

Given Name Clarissa Alves

Suffix





Email


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Author Family Name Ducroquet

Particle

Given Name Jean Pierre Henry Joseph

Suffix

Division

Organization Epagri, So Joaquim Experimental StationAddress 88600-000, So Joaquim, SC, Brazil


Author Family Name Floh

Particle

Given Name E. I. S.

Suffix

Division Laboratory of Plant Cell Biology, Department of Botany

Organization IB-University of So Paulo

Address 05422-970, So Paulo, SP, Brazil


Schedule

Received 10 December 2008

Revised

Accepted 9 June 2009

Abstract Acca sellowiana(Berg.) Burr. is a native Myrtaceae from southern Brazil and Uruguay, now the subject of a

domestication and breeding program. Biotechnological tools have been used to assist in this program. The

establishment of a reliable protocol of somatic embryogenesis has been pursued, with a view to capturing and

fixing genetic gains. The rationale behind this work relies on the fact that deepening comprehension of the

general metabolism of zygotic embryogenesis may certainly improve the protocol for somatic embryogenesis.

Thus, in the present work we studied the accumulation of protein, total sugars, starch, amino acids, polyamines

(PAs), IAA and ABA, in different stages ofA. sellowianazygotic embryogenesis. Starch is the predominant

storage compound during zygotic embryo development. Increased synthesis of amino acids in thecotyledonary stage, mainly of asparagine, was observed throughout development. Total free PAs showed

increased synthesis, whereas total conjugated PAs were mainly observed in the early developmental stages.

IAA decreased and ABA increased with the progression from early to late embryogenesis. Besides providing

basic information on the morphophysiological and biochemical changes of zygotic embryogenesis, the results

here obtained may provide adequate strategies towards the modulation of somatic embryogenesis in this

species as well as in other woody angiosperms.

Keywords (separated by '-') Embryo development -Feijoa sellowiana - IAA - Polyamine - Starch - Storage protein

Footnote Information


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correctly identified.

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Journal: 10725

Article: 9393
mailto:[email protected]:[email protected]:[email protected]


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O RI G I N A L P A P E R1

2 Dynamics of biochemical and morphophysiological changes

3 during zygotic embryogenesis in Acca sellowiana (Berg.) Burr.

4 Gabriela Claudia Cangahuala-Inocente Vanildo Silveira

5 Clarissa Alves Caprestano Jean Pierre Henry Joseph Ducroquet

6 E. I. S. Floh Miguel Pedro Guerra

7 Received: 10 December 2008 / Accepted: 9 June 20098 Springer Science+Business Media B.V. 2009

9 Abstract Acca sellowiana (Berg.) Burr. is a native

10 Myrtaceae from southern Brazil and Uruguay, now the11 subject of a domestication and breeding program. Bio-

12 technological tools have been used to assist in this pro-

13 gram. The establishment of a reliable protocol of somatic

14 embryogenesis has been pursued, with a view to capturing

15 and fixing genetic gains. The rationale behind this work

16 relies on the fact that deepening comprehension of the

17 general metabolism of zygotic embryogenesis may cer-

18 tainly improve the protocol for somatic embryogenesis.

19 Thus, in the present work we studied the accumulation of

20 protein, total sugars, starch, amino acids, polyamines

21 (PAs), IAA and ABA, in different stages of A. sellowiana

22zygotic embryogenesis. Starch is the predominant storage

23compound during zygotic embryo development. Increased24synthesis of amino acids in the cotyledonary stage, mainly

25of asparagine, was observed throughout development.

26Total free PAs showed increased synthesis, whereas total

27conjugated PAs were mainly observed in the early devel-

28opmental stages. IAA decreased and ABA increased with

29the progression from early to late embryogenesis. Besides

30providing basic information on the morphophysiological

31and biochemical changes of zygotic embryogenesis, the

32results here obtained may provide adequate strategies

33towards the modulation of somatic embryogenesis in this

34species as well as in other woody angiosperms.

35

36Keywords Embryo development Feijoa sellowiana

37IAA Polyamine Starch Storage protein

38

39Introduction

40Acca sellowiana (Berg.) Burr. is a native fruit species from

41southern Brazil and northern Uruguay. In New Zealand,

42Australia, the USA and even certain countries in Europe, it

43has been commercially cultivated since the beginning of

44the twentieth century (Thorp and Bieleski 2002). Recently,

45efforts have been made in Brazil to launch a domestication

46program with this species.

47Conventional techniques for the vegetative propagation

48of A. sellowiana based on cuttings and grafting, are diffi-

49cult due to the negative effects of phenolic compounds (Dal

50Vesco and Guerra 2001). Micropropagation techniques

51have been employed to overcome such problems, with

52studies being focused on the establishment of reliable

53protocols for somatic embryogenesis (Canhoto and Cruz

541996; Dal Vesco and Guerra 2001; Stefanello et al. 2005;

A1 G. C. Cangahuala-Inocente C. A. Caprestano

A2 M. P. Guerra (&)

A3 Graduate Program in Plant Genetic Resources,

A4 Plant Developmental Physiology and Genetics Laboratory,

A5 Federal University of Santa Catarina, Florianopolis,

A6 SC 88040-900, Brazil

A7 e-mail: [email protected]

A8 G. C. Cangahuala-Inocente


A10 V. Silveira

A11 Biotechnology Laboratory, CBB, State University of North

A12 Fluminense (UENF), Av. Alberto Lamego, 2000, Campos dosA13 Goytacazes, Rio de Janeiro 28013-600, Brazil


A15 J. P. H. J. Ducroquet

A16 Epagri, Sao Joaquim Experimental Station, Sao Joaquim, SC

A17 88600-000, Brazil


A19 E. I. S. Floh

A20 Laboratory of Plant Cell Biology, Department of Botany,

A21 IB-University of Sao Paulo, Sao Paulo, SP 05422-970, Brazil


123Journal : Large 10725 Dispatch : 17-6-2009 Pages : 13

Article No. : 9393h LE h TYPESET

MS Code : GROW1340 h CP h DISK4 4

Plant Growth Regul

DOI 10.1007/s10725-009-9393-9


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55 Cangahuala-Inocente et al. 2007). One of the main limi-

56 tations of these protocols is associated with the low rate of

57 somatic embryo conversion to plantlets. These constraints

58 are mainly a result of the lack of knowledge on the phys-

59 iological and biochemical changes that occur during

60 development and maturation of the zygotic embryo.

61 Zygotic embryogenesis is a complex and highly orga-

62 nized process that plays a central role in the life cycle of63 higher plants. It can usually be divided into two main steps,

64 namely, (1) an initial morphogenetic phase which is char-

65 acterized by cell division and the onset of cell differenti-

66 ation, followed by (2) a maturation phase associated with

67 the accumulation of major storage products and preparation

68 for seed desiccation, dormancy and germination (Mord-

69 horst et al. 1997; Sallandrouze et al. 2002).

70 Both the synthesis and accumulation of storage com-

71 pounds play a central role in zygotic embryogenesis

72 (Merkle et al. 1995). Storage proteins are the source of

73 amino acids for seed germination (Misra et al. 1993). A

74 special class of storage compounds, the LEA proteins, play75 an important role in seed dehydration (Wise and Tunnac-

76 liffe 2004). Apart from proteins, carbon is stored in lipids

77 and starch. The levels of protein, lipids, and starch vary

78 between angiosperms species (Dam et al. 2009). Soluble

79 sugars, such as glucose and sucrose, are involved with the

80 regulation of developmental processes occurring from

81 embryo development to seed maturation (Gibson 2005).

82 Amino acids are important in nitrogen metabolism and

83 protein synthesis, as well as in the transition from hetero-

84 trophy to autotrophy (Ortiz-Lopez et al. 2000).

85 The polyamines, aliphatic amines with a positive charge

86 in neutral pH, play a basic role in cell proliferation and

87 differentiation (Bouchereau et al. 1999; Puga-Hermida

88 et al. 2003; Baron and Stasolla 2008), and in protein syn-

89 thesis and responses to water stress in plants (Bais and

90 Havishankar 2002; Kusano et al. 2007). Taken together,

91 these substances are reliable biochemical markers of

92 zygotic and somatic embryo quality.

93 From a physiological point of view, auxins are involved

94 in cell division and expansion, and differentiation of the

95 vascular system (Liu et al. 1993). These hormones are also

96 associated with regulation of the embryonic patterns of

97 histodifferentation (Fischer-Iglesias and Nauhaus 2001;

98 Bassuner et al. 2007). ABA is another hormone which plays

99 a major role in the embryo, by preventing precocious ger-

100 mination (Kermode 1995) and promoting the accumulation

101 of storage compounds (Cailloux et al. 1996), and thus,

102 embryo maturation (Black1991). In somatic embryogenesis

103 ABA reduces the frequency of embryo malformation (Eti-

104 enne et al. 1993) and the occurrence of secondary or

105 repetitive embryogenesis (Nuutila et al. 1991).

106 The present work aimed to study the dynamics of biochemi-

107 cal and physiological changes during the development

108ofA. sellowiana zygotic embryos, such as the levels of total

109proteins, amino acids, carbohydrates, polyamines, IAA and

110ABA. The accumulation patterns of certain of these sub-

111stances in specific stages of seed development were also

112assayed.

113Materials and methods

114Plant material

115Biological material was compiled together with the A. sel-

116lowiana germplasm collection at the Epagri Sao Joaquim

117Experimental Station (28180 Latitude S, 49560 Longitude

118W), Santa Catarina, in the south of Brazil, from November

1192004 to March 2005. About 3,800 flowers were emascu-

120lated followed by manual pollination. Ovules from non-

121pollinated flowers were considered as the zero-time limit.

122Fruits were collected after 21 and 30 days, then every

12315 days until reaching fruit maturation which occurred124after 120 days (Fig. 1). The fruits were transported in

125plastic boxes with dry ice. Using a stereoscope (Olympus

126SZH10) the seeds were extracted from the fruits, and stored

127at -20C.

128Histological analysis

129Samples were fixed for 24 h in a 0.2 M phosphate buffer

130(pH 7.3) containing 2.5% paraformaldehyde. After fixation,

131these were dehydrated in a graded ethanol series and

132embedded in historesin (Leica), as described by Arnold

133et al. (1975). 5 lm thick sections were sliced with a rotary

134microtome (Slee Technik Cut 4055) and fixed onto slides

135by heating. The sections were stained with 0.5% toluidine

136blue O (TBO) (C.I. 52040) in a 0.2 M phosphate buffer

137(pH 6.8) for 1 min (OBrien et al. 1965). Photographs were

138taken with a standard Olympus BX 40 microscope and

139Olympus SZH10 stereoscope.

140Soluble proteins

141For each collecting date 300 mg of seeds were extracted.

142The seeds were stored in eppendorff tubes and stored at

143-20C until the processing. Total soluble protein extraction

144was performed according to Cangahuala-Inocente et al.

145(2009). Three repetition of samples (300 mg fresh weight-

146FW each) were macerated at 4C with 1 ml of extraction

147buffer (pH 7.0) containing 50 mM sodium phosphate

148dibasic, 0.2 M b-mercaptoethanol, 17.3 mM sodium dode-

149cyl sulfate (SDS) and 1 mM phenylmethylsulfonyl fluoride

150(PMSF), to then be centrifuged to 4C by 20 min at

1518,000 rpm. The supernatant containing total soluble pro-

152teins was removed and the pellet stored at -20C. Soluble

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153 proteins were sedimented at 0C by adding two volumes of

154 100% ethanol into the supernatant and then centrifuging

155 4C by 20 min at 10,000 rpm. The sedimented proteins

156 were solubilized in 50 mM sodium phosphate dibasic (pH

157 7.0). Protein content was determined by the Bradford (1976)

158 method, using bovine serum albumin as standard.

159 Total sugar and starch

160 The extraction of total soluble sugars was performed161 according to Shannon (1968). The pellet from the protein

162 extraction was macerated using 2 ml methanolchloro-

163 formwater (MCW) (12:5:3), centrifuged for 10 min at

164 2,000 rpm. The supernatant was recovered and the pellet

165 was re-extracted using 2 ml MCW. One part chloroform and

166 1.5 part water were added for each four parts of the super-

167 natant, followed by centrifuging for 10 min at 2,000 rpm,

168 from which two phases were obtained. The upper aqueous

169 phase was removed for dosage using anthrone at 0.2%, in

170 accordance with Umbreit and Burris (1960).

171 The extraction and determination of starch levels were

172 based on the procedures of McCready et al. (1950). The

173 pellets used in the total soluble sugar extraction were

174 ground with 1 ml of 30% perchloric acid and centrifuged

175 for 15 min at 10,000 rpm. The supernatant containing

176 starch was removed and the pellets were re-extracted

177 twofold. The supernatants were combined and the pellets

178 eliminated. For dosage was using anthrone at 0.2%. The

179 sugar and starch concentrations were calculated using

180 glucose as standard. The absorbance was read in UVVIS

181 UV-1203 spectrophotometer (Shimadzu) at 620 nm.

182Amino acid

183Amino acid determination was carried out according to

184Santa-Catarina et al. (2006). Three biological samples

185(200 mg FW) of each developmental stage were ground,

186individually, in 6 ml of 80% (v/v) ethanol and evaporated

187in a speed vac centrifuge concentrator (ThermoSavant,

188Milford, USA). Samples were re-suspended in 2 ml of

189MilliQ type water and centrifuged at 20,000g for 10 min.

190The supernatant was filtered through a 20 lm membrane.191Amino acids were derivatizated with o-phthaldialdehyde

192(OPA) and identified by HPLC using a C-18 reverse phase

193column (SUPELCOSILTM, 5 lm particle size, L9 I.D.

19425 cm 9 4.6 mm). The gradient was developed by mixing

195increasing proportions of 65% methanol with a buffer

196solution (50 mM sodium acetate, 50 mM sodium phos-

197phate, 20 ml l-1 methanol, 20 ml l-1 tetrahydrofuran and

198pH 8.1 adjusted with acetic acid). The gradient of 65%

199methanol was programmed to 20% over the first 32 min,

200from 20 to 100% between 32 and 71 min, and 100%

201between 71 and 80 min, at a 1 ml min-1 flow and 40C.

202Fluorescence excitation and emission wavelengths of 250

203and 480 nm, respectively, were used for amino acid

204detection. Peak areas and retention times were measured by

205comparison with known quantities of standard amino acids.

206Polyamines

207Putrecine (Put), Spermidine (Spd) and Spermine (Spm)

208were defined according to Silveira et al. (2004a). Three

209biological samples (300 mg FW) of each developmental

Fig. 1 Development of the seed

of A. sellowiana after

pollination directed. 0 DAP:

floral button at the stage

balloon, 21 DAP: formation of

the zygotic embryo with

successive cell divisions

causing a pro-embryo, 30 DAP:

zygotic embryo at the globular

stage, 45 DAP: zygotic embryo

at the heart stage, 60 DAP:

zygotic embryo at the torpedo

stage with endosperm still

liquid, 75 DAP: zygotic embryo

at the cotyledonary stage with

presence of endosperm, 90, 105

and 120 DAP: zygotic embryo

at the cotyledonary stage

without presence of endosperm.

Bar= 1 mm

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210 stage were ground, individually, in 3 ml of 5% (v/v) per-

211 chloric acid. After 1 h, extracted samples were centrifuged

212 for 20 min at 15,000g and 0C. In each case, the super-

213 natant containing free polyamines was removed and the

214 pellets re-extracted. Supernatants were then combined and

215 the pellets eliminated. Free polyamines were determined

216 directly from the supernatant. Conjugated polyamines were

217 extracted by hydrolyzing 200 ll of supernatant with 200 ll218 of 12 N HCl for 18 h at 110C. The samples were dried

219 under nitrogen. The conjugated polyamines were solubi-

220 lized in 200 ll of 5% Perchloric acid.

221 Free and conjugated polyamines were derivatizated by

222 dansyl chloride made up in acetone at a concentration of

223 5 mg ml-1. A 40 ll aliquot of the sample was added to

224 100 ll of dansyl chloride, 20 ll of 0.05 mM diaminohep-

225 tane (internal standard) and 50 ll of saturated sodium

226 carbonate. In sequence, the samples were incubated in the

227 dark for 50 min at 70C. The toluene phase was collected

228 and dried under nitrogen. Finally, dansylated polyamines

229 were solubilized in 200 ll of acetonitrile.230 Twenty ll of the dansylated polyamines were separated

231 by reverse phase HPLC in a C-18 reverse phase column

232 (Shimadzu SHIM-PACK CLC-ODS, 5 lm particle size,

233 L9 I.D. 25 cm 9 4.6 mm). The gradient was developed

234 by mixing increasing proportions of absolute acetonitrile to

235 10% acetonitrile in water (pH 3.5). The gradient of abso-

236 lute acetonitrile was programmed at 65% over the first

237 10 min, from 65 to 100% between 10 and 13 min and at

238 100% between 13 and 21 min. The flow was 1 ml min at

239 40C. The fluorescence detector was set at 340 nm (exci-

240 tation) and 510 nm (emission). A mixture of putrescine

241 (Put), spermidine (Spd) and spermine (Spm) was used as

242 standard.

243 IAA and ABA determination

244 IAA and ABA contents were determined according to

245 Santa-Catarina et al. (2006). Three biological samples

246 (1,000 mg FW) of each developmental stage were ground,

247 individually, in a 5 ml extraction buffer (80% ethanol ?

248 1% polyvinylpyrrolidone-40). [3H]IAA and [3H]ABA were

249 added to the samples as internal radioactive standards. After

250 90 min of incubation, samples were centrifuged during

251 15 min at 15,500g, and 4C. Supernatants were concen-

252 trated in a speed vac centrifuge concentrator (Ther-

253 moSavant, Milford, USA) at 45C, until reaching 20% of

254 the initial volume (1 ml). Volumes were then raised (Mil-

255 liQ water type) to 3 ml and the pH adjusted to 2.5 using

256 HCl (1 N). The samples were partitioned twice by using

257 ethyl ether as an organic solvent. Organic layers were

258 combined and the aqueous residue eliminated by freezing

259 the base of the tubes during 5 s in liquid nitrogen, with

260 transference of the organic phase to a clean tube. The

261organic phase containing IAA and ABA was dried in a

262speed vac at 45C, dissolved in 200 ll of 100% methanol,

263transferred to micro tubes and after stored at -80C until

264analysis. Aliquots (40 ll) of stored samples were analyzed

265by reverse phase HPLC, by using a C-18 reverse phase

266column (Shimadzu SHIM-PACK CLC-ODS, 5 lm particle

267size, L9 I.D. 25 cm 9 4.6 mm). The gradient was devel-

268oped by mixing increasing proportions of absolute methanol269to 10% methanol plus 0.5% acetic acid in water. The IAA

270content was defined by using a fluorescence detector at

271280 nm (excitation) and 350 nm (emission) and the ABA

272content by means of a UVVIS detector at 254 nm. A

273mixture of IAA and ABA was used as a standard. Fractions

274containing IAA and ABA were collected and analyzed in

275the Packard Tri-Carb liquid scintillation counter to estimate

276losses and data validation.

277Data analysis

278Data were analyzed by ANOVA (P\ 0.05) followed by279the Student-Newman-Keuls (SNK) test, using Statistica

280version 7.0 software. In the event of ANOVA and SNK

281tests not being used, the mean of three replicates and

282standard error were then applied to analyze data.

283Results and discussion

284Formation of zygotic embryos

285 A. sellowiana ovules are anatropous, developing asyn-

286chronously (Fig. 2a) with axillary placenta. The nucellus

287presents layers of cells with prominent nuclei (Fig. 2b).

288Members of the order Myrtales, which includes the Myrt-

289aceae, have a crassinucellate ovule, the micropyle being

290formed by external and internal teguments, with two layers

291of cells, ephemeral or absent antipodal cells and a nuclear-

292type endosperm (Tobe and Raven 1983).

293Twenty one days after pollination (DAP), the first cel-

294lular division, leading to the formation of the pro-embryo

295(Fig. 2c) with embryonal and suspensor cells (Fig. 2d),

296was noted. According to Dodeman et al. (1997), several

297structural alterations occur in the pro-embryo, mostly

298associated with vacuole organization, nucleus location, the

299presence of organelles and cell wall changes. After 30 DAP

300the apical cells of the pro-embryo originated a spherical

301mass of cells, corresponding to the globular stage (Fig. 2e, f).

302The endosperm of the nuclear position was constituted of

303a cell layer with conspicuous nuclei (Fig. 2e). In Arabid-

304opsis thaliana the embryo in the globular stage stems

305from a sequence of divisions of embryonal cells, the

306embryo soon acquiring a heart shape (Mansfield and Bri-

307arty 1991).

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308 Forty five DAP, histological sections at heart stage

309 embryos showed a well-defined proto-dermal layer

310 (Fig. 3a, b). Sixty days after pollination, the embryo was in

311 the torpedo-stage with an extended apicalbasal axis

312 (data not shown). According to Goldberg et al. (1994),

313 dramatic changes occur in the transition from globular to

314 torpedo-stage embryos. Among other features, cotyledons

315 arise from two lateral domains, this resulting in bilateral

316 symmetry.

317 The early cotyledonary stage was observed at 75 DAP

318 (Fig. 3c) showing rudimentary cotyledonary leaves sur-

319 rounded by the endosperm. At 90 DAP, the endosperm

320 present in seed had almost been consumed (Fig. 3d). In

321 addition, at this stage cells in the apical (Fig. 3e) and basal

322 (Fig. 3f) meristems were observed at the end of the

323 hypocotylsradicle axis. Starting from 105 to 120 days,

324 fruit development was concluded with maturation. Con-

325 comitantly, embryos showed a thickened cotyledons with

326 storage compounds (see Fig. 1).

327Protein and carbohydrates

328Total protein levels during embryo development ranged

329from 0.24 to 0.39 mg g FW-1 (Fig. 4). Storage proteins

330provide free amino acids to be used for early embryonal

331stages until reaching plantlet autotrophy (Prewein et al.

3322006). During zygotic embryogenesis of Ocotea catharin-

333ensis, proteins were only detected at high concentrations in

334the late developmental stages, peaking at the mature

335embryo stage (Santa-Catarina et al. 2006). In Araucaria

336angustifolia zygotic embryogenesis, the embryonic axis

337protein content increased until the cotyledonary stage, with

338further stabilization in the mature seed (Silveira et al. 2008).

339Total soluble sugars ranged from 1.45 to 2.23 mg g

340FW-1 during zygotic embryo development (Fig. 4). Starch

341content remained quite stable during embryo development,

342the highest value (24.5 mg g FW-1) being observed at 21

343DAP (Fig. 4). This phase is coincident with fertilization and

344zygote formation. Similar results were reported by Pescador

Fig. 2 Histological sections of

the process of zygotic

embryogenesis ofA. sellowiana.

a Ovule of the immature fruit 0

DAP, stained with AT-O, b the

opening of the micropyle detail

and the presence of the

teguments protection,

c formation of the pro-embryo

in the immature fruit, d cells in

the suspensor and pro-embryo,

e immature fruit at 30 DAP,

observing the zygotic embryo in

globular stage, f details of the

zygotic embryo in globular

stage and the presence of the

outer tegument of protection.

Bar= 10 lM. Mi micropyle,

teg tegument, ca chalaza, nu

nucellus, fu funicular, ex-teg

outer tegument, in-teg internal

tegument, en endosperm, ze

zygotic embryo, sus suspensor,

pro-en pro-embryo, glo globular

stage

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345 et al. (2008) with this same species. Starch and sugar con-

346 tents play a major role in seed development through the

347 supply of compounds to be consumed during transition

348 from seed heterotrophy to plantlet autotrophy (Merkle et al.

349 1995). In Quercus robur, starch accumulated gradually in

350 the late embryonal developmental stages (Prewein et al.

351 2006). Studies using an Arabidopsis mutant showed the role

352of sugars in the regulation of genetic expression, prolifer-

353ation and cell death, in seedling-growth, leaf expansion and

354senescence, and in seed development (Smeekens 2000;

355Gibson 2005; Rolland et al. 2006).

356Histochemical analysis of mature A. sellowiana zygotic

357embryos (120 DAP) revealed a low starch-content and a

358high content of protein and lipidic bodies in cotyledon cells

Fig. 3 Histological sections of

the process of zygotic

embryogenesis ofA. sellowiana.

a Immature fruit at 45 DAP

looking up the zygotic embryo

in stage cordiforme, b zygotic

embryo in detail the heart stage

and the presence of protoderm,

c longitudinal section of the

zygotic embryo at the initial

cotyledonary stage to 75 DAP,

d longitudinal section the

zygotic embryo at the

cotyledonary stage at 90 DAP,

e details of the root meristem,

f detail of the apical meristem.

Note that the cells of the leaves

cotyledonary are somewhat

thickened. Bar= 10 lM. ex-teg

outer tegument, he heart stage,

protoderprotoderm, cot

cotyledon, procam procambium,

ap-me apical meristem, ra-me

radicle meristem, cap root cap

cbcbcbcabcabcabc ab

a

a

aaab

bcab

bcdcddcd

cd

cddcdcd

bc

b

a

0,0

0,5

1,0

1,5

2,0

2,5

3,0

0 21 30 45 60 75 90 105 120

Days after pollination

Concentration(mg/gFW)

0

5

10

15

20

25

30

Concentration(mg/gFW)

Protein Sugar StarchFig. 4 Mean concentration of

total protein, starch and totalsugars (mg/g) of fresh weight

(FW) for the formation of the

zygotic embryo ofA. sellowiana

(n = 3). Values followed by

different letters, inside of

parameters indicating

significant differences

according to the SNK test

(P\ 0.05)

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359 (data not shown). It is commonly accepted that the main

360 storage compounds during seed development are proteins,

361 carbohydrates and lipids, and that the ratio of this com-

362 position is different in each species (Bewley and Black

363 1994). Thus, Caesalpinia peltophoroides seed cotyledons

364 presented about 50% of lipids, 32% of soluble carbohy-

365 drates, 7.7% of starch and 6.8% of soluble proteins (Corte

366 et al. 2006). On the other hand, the predominant reserve367 was starch in seed cotyledons of Anthyrium andraeaman,

368 whereas endosperm reserves were mainly constituted of

369 starch and proteins (Matsumoto et al. 1998).

370 Amino acids

371 Amino acids are important primary assimilation products

372 in nitrogen metabolism (Ortiz-Lopez et al. 2000). In

373 addition to the synthesis of proteins, amino acids directly

374 or indirectly control various aspects of plant growth and

375 development (Coruzzi and Last 2000).

376 In the present work, amino acid content was enhanced377 after 45 days from the pollination period, when the embryos

378 had already reached the heart stage (Table 1). At this stage,

379 amino acids may be used for the synthesis of specific pro-

380 teins associated with histodifferentiation, since this is the

381 moment when the embryo begins to establish its radial

382 symmetry, vascularization and formation of the cotyledons.

383 The total amino acid content peaked at 75 DAP

384 (Table 1), a period in which the zygotic embryo was in the

385 cotyledonary stage. These results confirm those obtained

386 during embryonic development of A. angustifolia (Astarita

387 et al. 2003a), Pinus taeda (Silveira et al. 2004b) and

388 O. catharinensis (Santa-Catarina et al. 2006).

389 A decrease in amino acid levels was observed from 90 to

390 105 DAP (Table 1). This could be ascribed to their con-

391 sumption for the synthesis of various, mainly reserve,

392 proteins. According to Weber et al. (1997) the transport of

393 amino acid to the cotyledons can be initially passive,

394 although an additional system of active absorption can be

395 established in late developmental stages, when large

396 amounts of proteins are stored to ensure seedling devel-

397 opment. A decrease in total amino acid levels in the mature

398 embryo was also observed in O. catharinensis (Santa-

399 Catarina et al. 2006).

400 Asparagine was the prevalent amino acid, with a peak

401 (Table 1) in the late embryonal stages (7590 DAP), when

402 the cotyledons were fully developed, fully suggesting its

403 involvement in the mobilization of embryo reserves. This

404 result confirms that reported by Feirer (1995) and Santa-

405 Catarina et al. (2006) during seed development of

406 P. strobus and. O. catharinensis, respectively. Asparagine

407 plays a central role in nitrogen storage and transport in

408 plants, which is further facilitated by its biochemical

409 properties (Lea et al. 2007).

410Glutamine, aspartic acid, c-aminobutyric acid (Gaba)

411and alanine are accumulated at lower levels than aspara-

412gine (Table 1). Glutamines and glutamic acid are precur-

413sors of other amino acids (Radwanski and Last 1995). In

414addition, exogenous glutamine is commonly supplemented

415to the basal culture medium in several protocols of somatic

416embryogenesis, including A. sellowiana (Dal Vesco and

417Guerra 2001). In the present work alanine showed high418levels at 45, 75 e 90 DAP, and the IAA levels showed an

419inverse proportion comparatively to alanine. This suggests

420that synthesis and degradation of this amino acid could be

421associated with the IAA synthesis when this auxin is higly

422required. The L-alanine and e-lysine conjugates were also

423found to be useful for induction and development of

424Oenothera leaf callus and in tomato cell-suspension cul-

425ture, two systems which require highly active sources of

426auxin (Magnus et al. 1992).

427Unlike the rest, Gaba is a non-proteic amino acid which

428results from glutamic acid decarboxylation (Satya-Naraian

429and Nair 1990), normally being accumulated in response to4302,4-D, or under stress conditions (Snedden and Fromm

4311998). In the present work, Gaba was the fourth largest

432amino acid accumulated, mostly from the 75 to 90 DAP,

433decreasing thereafter (Table 1). The presence of these

434amino acids at such levels suggests its role in A. sellowiana

435embryogenesis. This amino acid was also accumulated in

436the early somatic embryogenesis of carrots as shown by

437Kamada and Harada (1984).

438Polyamines (PAs)

439The total PAs level was low at zero-time, increasing

440significantly up to 21 DAP. In subsequent stages (30, 45 and

44160 DAP), increased levels of conjugated PAs were

442observed, these progressively decreasing until embryos

443reached maturity (Fig. 5a). Conversely, total free PA levels

444increased according to progression to further developmental

445stages, peaking by 105 DAP (Fig. 5a). This variation was

446different from that observed in O. catharinensis where the

447levels of free PAs were higher than those of conjugated PAs,

448both types of PAs reaching high levels in mature zygotic

449embryos (Santa-Catarina et al. 2006). The metabolic role of

450conjugated PAs has not yet been fully elucidated. Bais and

451Havishankar (2002) suggested that free PA levels in plants

452can be regulated by the formation of reversible conjugated

453PAs. It has also been suggested that a combination of PAs

454with cinnamic acid and phenols may regulate the pool of

455free PAs in plant cells (Mader and Hanke 1997).

456The PA ratio [Put.(Spd ? Spm)-1] was low both at

457zero-time and 21 DAP (Fig. 5b), corresponding, respec-

458tively, to fertilization and formation of the zygotic embryo.

459Geoffriau et al. (2006) showed the involvement of PAs in

460the gynogenesis of Allium cepa, where the Put/Spd-Spm

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Table1

Aminoacidscontent(lg

g-

1

FW)inzygoticembryosduringseeddevelopmentinA.sellowiana

Daysafterpo

llination(DAP)anddevelopmentstages

0

21

30

45

60

75

90

105

120

Globular

Heart

Torpedo

Earlycotyle

donary

Cotyledonary

Maturecotyledonary

Asparticacid

22.5

0.7

24.1

8.2

27.5

7.8

75.5

6.4

19.9

4.3

95.2

6

.0

88.6

11.4

30.8

3.3

54.5

5.3

Glutamicacid

50.2

2.7

17.3

6.0

39.3

6.0

121.0

2.8

54.9

13.2

41.1

1

3.5

26.9

4.2

9.4

0.1

9.3

0.8

Asparagine

123.2

3.8

141.8

43.5

110.4

30.8

417.8

33.0

293.9

87.4

982.2

1

37.2

629.5

96.4

126.7

4.7

157.2

3.5

Serine

32.3

0.5

23.7

7.5

27.7

5.6

83.3

5.3

45.3

11.2

131.6

9

.9

81.8

8.9

21.6

3.5

33.8

7.3

Glutamine

34.3

1.9

16.9

5.5

35.7

12.0

357.7

79.3

229.5

44.8

708.7

2

7.2

481.8

77.4

78.2

8.4

62.2

8.6

Histidine

3.5

0.1

3.6

0.4

5.5

1.3

24.9

3.6

25.3

7.0

91.5

1

1.0

83.1

13.0

27.7

3.7

53.2

4.2

Glycine

2.5

0.1

3.7

0.9

4.8

0.3

12.7

2.0

5.9

1.3

16.0

2

.2

18.9

5.3

2.4

0.2

10.2

1.9

Arginine

14.9

1.4

27.8

7.1

14.5

2.4

13.9

3.0

5.3

1.4

123.9

1

5.2

324.9

54.1

28.7

5.0

22.8

3.3

Threonine

10.5

0.1

5.9

2.2

11.1

2.2

33.1

1.3

15.1

4.6

48.9

3

.3

45.1

6.1

6.7

1.0

19.0

4.4

Alanine

21.0

0.1

27.8

8.0

30.4

10.6

94.8

11.9

40.5

6.6

167.0

2

1.1

128.4

-16.2

11.3

1.6

21.7

2.8

c-Aminobutiricacid

20.4

0.1

48.5

16.1

33.1

8.9

112.2

8.2

35.6

7.9

173.8

6

.9

206.5

17.6

44.0

3.9

43.5

-6.2

Tyrosine

2.5

.5

4.8

1.4

5.4

0.7

23.8

2.5

11.3

1.8

26.3

1

.0

16.0

1.2

4.1

0.7

12.3

2.9

Tryptophan

0.7

0.1

2.6

0.9

1.2

0.2

3.6

0.4

2.0

0.5

7.2

0

.4

5.4

0.6

1.5

0.2

3.8

0.4

Methionine

0.0

0.0

0.2

0.0

0.5

0.1

1.4

0.2

12.2

0

.7

9.2

0.8

2.4

0.1

6.7

1.1

Valine

3.0

0.0

3.1

0.3

3.0

0.3

14.7

2.4

7.2

2.1

32.9

3

.1

26.0

3.2

5.7

0.4

16.4

3.0

Phenylalanine

1.1

0.1

4.0

0.3

4.2

0.3

16.3

2.8

6.1

1.7

25.3

1

.4

20.1

2.4

5.9

0.7

11.7

2.2

Isoleucine

1.7

0.2

2.3

0.4

2.5

0.4

11.7

2.1

4.8

-1.5

23.5

2

.2

26.2

2.9

6.0

0.7

11.8

2.6

Leucine

3.0

0.2

5.1

0.5

5.9

0.5

26.6

4.5

10.8

3.3

53.7

5

.2

39.0

5.1

11.0

0.5

21.1

4.4

Ornithine

2.3

0.1

1.4

0.0

1.7

0.0

2.2

0.5

1.8

0.1

2.0

-

0.2

3.2

0.2

1.7

0.2

1.9

0.0

Lysine

3.6

0.3

3.6

0.9

5.9

0.9

18.0

2.6

6.7

0.8

28.9

6

.0

25.2

2.0

7.3

1.4

19.1

-5.2

Totalaminoacid

353.0

2.3E

368.1

71.7E

369.7

91.4E

1,464.2

174.4C

823.4

196.3D

2,791.8

1

34.4A

2,285.8

329.0B

433.0

40.5E

592.0

70.1DE

Dataarepresentedasmean

stan

darddeviation(n=

3).Valuesfollowedbydifferentletters,insideofparametersindicatingsignificantdifferencesaccordingto

theSNKtest(P\

0.05)

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461 ratio was low during flowering. In the present work, the

462 proportion of PAs [Put.(Spd ? Spm)-1] was high during463 embryo development (30, 45 and 60 DAP), corresponding,

464 respectively, to the globular, heart and torpedo stages, the

465 highest being detected at the heart stage then decreasing

466 until the cotyledonary stage (Fig. 5b). These results are in

467 agreement with those reported in A. angustifolia (Astarita

468 et al. 2003b), P. taeda (Silveira et al. 2004b), and

469 O. catharinensis (Santa-Catarina et al. 2006), Thus, the PA

470 ratio could be considered as a reliable marker of embryonal

471 development in A. sellowiana.

472In the present work, at zero-time and at the time of

473fertilization and formation of the zygotic embryo (DAP47421), free Spd was predominant as compared to other PAs

475(Fig. 5c). It has been shown that high Spd levels are

476associated with induction and floral development (Aribaud

477and Martin-Tanguy 1994). Spd was the most abundant free

478PA in Brassica rapa, followed by Put and Spm (Puga-

479Hermida et al. 2003). These authors also pointed out

480that free PAs content did not change when the flower was

481still closed, changing the profile only after synthesis of

482carotenoids in petals.

a

a a

b b

b

b

bc

c

aa a

a

b

b

b

b

b

0

20

40

60

80

100

120

0 21 30 45 60 75 90 105 120

g/g

FW

free total PAs conjugated total PAs

a

b bb

bc

c

ddd

0,0

0,3

0,6

0,9

0 21 30 45 60 75 90 105 120

Put/(Spd+S

pm)

aa abab

bc bccd

dd

a

bbcbcd bcd

cdecde

de e

a

bbc bccd

dee e e

b

a

aa

b b b

bc

c

0

20

40

60

80

100

120

0 21 30 45 60 75 90 105 120


Concentration(g

/gFW)

PUT SPD SPM Total

a

b

c

Fig. 5 The mean concentration

a total polyamines (lg/g) of

fresh weight (FW), b ratio of

PAs: Put (Spd ? Spm)-1 and

c free PAs (lg/g) of fresh

weight (FW) during the

development and maturation of

the zygotic embryo of

A. sellowiana (n = 3). Values

followed by different letters,

inside of parameters indicating

significant differences

according to the SNK test

(P\ 0.05)

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483 In the present work, similar patterns of variation

484 between free PAs, mainly for Put at all stages, were noted

485 in subsequent developmental stages (Fig. 5c). This could

486 be attributed to Put conversion to Spd and Spm by means

487 of Spd and Spm synthases, as suggested by Puga-Hermida

488 et al. (2003). At 60 DAP the ratio was quite the same,

489 although levels were lower, ranging further (105 and 120

490 DAP) for Spd (Fig. 5c).491 It is important to note that arginine and ornithine are

492 direct precursors, whereas methionine is an indirect pre-

493 cursor, of PAs biosynthetic routes in plants (Antognoni

494 et al. 1998; Bouchereau et al. 1999). In the present study,

495 these amino acids were those that presented the highest

496 levels at 75 and 90 DAP, the later corresponding to the

497 cotyledonary stage. Accordingly, the accumulation of these

498 amino acids as detected in this developmental stage could

499 be due to the synthesis of PAs, as revealed by analysis.

500 These PAs could be important during maturation of zygotic

501 embryos.

502 IAA and ABA

503 The role of IAA in zygotic embryogenesis is well-known

504 (Ribnicky et al. 2002; Bassuner et al. 2007), and is mainly

505 associated with cell division and elongation, and differen-

506 tiation of the vascular system (Gaspar et al. 1996). Many of

507 its effects are dependent on transport across tissues and

508 organs (Fischer-Iglesias et al. 2001; Friml et al. 2003).

509 In the present work, IAA and ABA levels between the

510 heart and cotyledonary stages were inversely proportional

511 (Fig. 6). Dynamic changes in these hormones could be

512 associated with changes in the patterns of histodifferenti-

513 ation and the establishment of embryonal radial and axial

514 symmetry, as suggested by (Michalczuck et al. 1992). The

515 dynamics of variation at IAA levels follows the same

516 pattern observed in the development of seeds, in which

517 high IAA levels occur during embryo development, to then

518decrease in mature seeds (Bewley and Black 1994). In the

519present work, the highest IAA levels were observed in the

520torpedo stage (60 DAP), followed by a continuous decrease

521(Fig. 6). The same pattern of IAA accumulation was

522observed in seeds of O. catharinensis (Santa-Catarina et al.

5232006). High IAA levels were found in Quercus roburat the

524heart stage, with a decrease in more advanced develop-

525mental stages (Prewein et al. 2006). In A. angustifolia526seeds, high IAA levels occurred in tissues of the embryonic

527axis and in the early stages of development (Astarita et al.

5282003c).

529In P. glauca and P. taeda the high levels of IAA

530observed during initial seed development were linked with

531growth of the zygotic embryo (Kong et al. 1997; Silveira

532et al. 2004b). In orthodox seeds, as in the case of A. sel-

533lowiana, reduction in hormone levels in mature seeds is a

534common feature caused by the high degree of seed dehy-

535dration. These hormones can be conjugated for further

536release during seed germination (Kong et al. 1997).

537Tryptophan is considered the main precursor of IAA538biosynthesis (Bandurski et al. 1995). The biosynthesis of

539tryptophan is important for establishing embryo polarity in

540early stages of development (Astarita et al. 2003c). In the

541present work, a decrease in tryptophan levels in the torpedo

542stage (60 DAP) was observed (Table 1), this occurring

543concomitantly with the IAA peak, thus suggesting that

544tryptophan was consumed in IAA biosynthesis.

545ABA also plays a major role in embryonal development.

546Among other effects ABA prevents early germination

547(Kermode 1995). In the present study, the highest levels of

548ABA (Fig. 6) were observed at the cotyledonary stage

549(90, 105 and 120 DAP). The patterns of ABA accumulation

550in the present work are common in most angiosperms,

551which show an increase in ABA content during seed

552development and a decline in the mature seed (Bewley and

553Black1994). In embryos of Quercus robur, ABA peaked at

554the cotyledonary stage and then decreased progressively.

555Concomitant with ABA, the water content declined as the

556embryo reached the maximum size (Prewein et al. 2006).

557Rock and Quatrano (1995) showed that at the end of barley

558zygotic embryogenesis, a decrease in IAA levels and an

559increase in ABA levels was observed. This ran parallel

560with the accumulation of storage compounds. In the pres-

561ent case, similar features were observed regarding the

562dynamics of IAA and ABA accumulation.

563Conclusions

564In this study relevant information about the biochemical and

565physiological changes occurring during the formation and

566development of the zygotic embryo of A. sellowiana was

567acquired. Starch is the predominant storage compound

A

A

A

ccc

B B

Bc

b

a

0

4

8

12

16

20

24

45 60 75 90 105 120


Concentration(g/gFW)

AB A IA A

Fig. 6 Mean concentration of IAA and ABA (lg g-1) in fresh weight

(FW) for the development of the zygotic embryo ofA. sellowiana

(n = 3). Values followed by different letters, inside of parameters

indicating significant differences according to the SNK test (P\

0.05)

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568 during embryonal development. All amino acids are syn-

569 thesized during embryogenesis with an increased accumu-

570 lation in the heart and cotyledonary stages. Asparagine is

571 the major amino acid observed. Free PAs synthesis during

572 early developmental stages, as well as the accumulation of

573 PAs conjugates in the cotyledonary stage, may be consid-

574 ered as reliable biochemical markers of embryonal devel-

575 opment in A. sellowiana. IAA and ABA levels were

576 inversely proportional between the heart and cotyledonary

577 stages, suggesting their involvement with histodifferentia-

578 tion patterns, mainly the establishment of embryonal sym-579 metries. The information here obtained will help to explain

580 the biochemical and physiological changes that occur dur-

581 ing zygotic embryogenesis and may assist in the develop-

582 ment of improved somatic embryogenesis protocols in this

583 and other woody plant species (Fig. 7).584

585 References

586 Antognoni F, Fornale S, Grimmer C, Komor E, Bagni N (1998) Long-587 distance translocation of polyamines in phloem and xylem of588

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Fig. 7 Summary of

biochemical changes of

A. sellowiana zygotic

embryogenesis

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