1

Changes in the ultrastructure of three soybean cultivars in response to manganese

supply

Jos Lavres JuniorI,*; Eurpedes MalavoltaI; Neusa de Lima NogueiraII; Milton Ferreira

de MoraesI; Andr Rodrigues dos ReisIII

ABSTRACT

The deleterious effects of manganese (Mn) stress in many species have been studied,

mainly, considering the biochemical and yield parameters of the shoots, particularly,

where the symptoms are shown. However, there are few studies in the literature relating

the anatomical and ultrastructural changes in response to Mn nutritional disorders. This

10

research was aimed to study the changes in ultrastructures using Mn-efficient and Mn-

11

inefficient soybean genotypes as related to three rates of Mn in the nutrient solution.

12

Symptoms of Mn deficiency developed twelve days after transplanting of IAC-15 and

13

Santa Rosa cultivars (inefficient), followed by IAC-Foscarin 31 (efficient) at the fiftieth

14

day. Only IAC-15 and Santa Rosa leaves showed symptoms of Mn toxicity. Mn

15

concentration in these tissues ranged from 8.6 (deficiency) to 886.3 mg kg-1 (toxicity).

16

There were no neither in stomata length nor stomata number. Cytoplasm disorganization

17

was observed in IAC-15 under Mn-excess. In this case, the cytoplasm was amorphous,

18

densely stained, extensively disorganized and with increased vacuolation. Mn effects

19

were not found in either mitochondria or nucleus of the genotypes. Under all Mn rates,

20

many lipid globules were observed in IAC-15. There was an increase in the quantity of

21

plastids as well as in starch size inside IAC-Foscarin 31 chloroplasts directly related to

22

the rates of Mn. Genotypes showed distinct degree of the ultrastructures organization, in

23

particular the chloroplasts either under deficiency or toxicity, being the IAC-15 most

24

affected by the nutritional stress.

25

Key words: chloroplast, deficiency, nutritive solution, toxicity.

USP/CENA - Laboratrio de Nutrio Mineral de Plantas. C.P. 96 - 13400-970 - Piracicaba, SP Brasil

*Corresponding author <jjlavres@yahoo.com.br> In memoriam
II
USP/CENA - Laboratrio Histopatologia Vegetal. C.P. 96 - 13400-970 - Piracicaba, SP - Brasil
III
Waseda University Department of Environmental Engineer. Shinjuku Ku, 3-4-1 Okubo, 169 8555
Tokyo - Japan

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Alteraes na ultra-estrutura de trs gentipos de soja em resposta ao fornecimento de

27

mangans

28

RESUMO

29

Os efeitos negativos provocados tanto pela deficincia quanto pela toxidez de mangans (Mn) no

30

desenvolvimento das plantas tm sido avaliados, considerando-se os aspectos bioqumicos e

31

produtivos da parte area, particularmente, onde os sintomas visuais so manifestados. Entretanto,

32

h poucas informaes na literatura abordando as alteraes anatmicas e de ultra-estrutura, em

33

relao ao suprimento de Mn. Os objetivos do presente estudo foram avaliar os efeitos do

34

fornecimento de trs doses de Mn, em soluo nutritiva, nas ultra-estruturas de folhas de cultivares

35

de soja: Santa Rosa, IAC-15 e IAC-Foscarin 31, contrastantes quanto ao uso do Mn. Os sintomas

36

visuais de deficincia foram observados primeiramente em Santa Rosa e IAC-15 (ineficientes), os

37

nicos a exibirem sintomas de toxidez. As concentraes de Mn nas folhas com sintomas variaram

38

de 8,6 (deficincia) a 886,3 mg kg-1 (toxidez). No houve alteraes no comprimento e no nmero

39

de estmatos nos limbos foliares. Em condio de toxidez, constatou-se no IAC-15, citoplasma

40

desorganizado, vacuolado em excesso e denso evidenciando alteraes nas membranas dos

41

tilacides. No ocorreram alteraes ultra-estruturais nas mitocndrias e no ncleo das clulas dos

42

trs gentipos. Constatou-se presena de glbulos de lipdios nos cloroplastos do cultivar IAC-15,

43

em todas as condies de fornecimento de Mn. Constatou-se aumento no nmero de plastdeos e

44

gros de amido, bem como no tamanho destes no IAC-Foscarin 31 com o suprimento de Mn. Os

45

gentipos, tanto na condio de deficincia quanto de excesso, exibiram distintos graus de

46

organizao das ultra-estruturas, notadamente, os cloroplastos. O IAC-15 exibiu maiores alteraes

47

das ultra-estruturas em funo das desordens nutricionais em mangans.

48

Palavras-chave: cloroplastos, deficincia, soluo nutritiva, toxidez

49

INTRODUCTION

50

Although manganese (Mn) toxicity can be a common problem in the tropical

51

regions with acid soils, the deficiency on soybean grown in Brazilian Cerrado has been

52

recognized as a nutritional disorder frequently related to excessive liming (Tanaka et al.,

53

1992). The literature has presented different explanations. Mn toxicity has been

54

described in many plants. However, the variations of the concentrations of this

55

micronutrient in the plants have been attributed either to the genotypes differences or to

56

soil fertility conditions (Fageria, 2001). Plant species and genotypes within species may

57

differ widely in the tolerance to high Mn (Foy et al., 1988), as well as in the

58

susceptibility to the deficiency when growing under low availability (Graham, 1988),

59

being most of these hereditary differences (Pittman, 2005).

60

Mn plays important roles in plant metabolism such as its participation in

61

photosystem II and chlorophyll biosynthesis (Teichler-Zallen, 1969; Lerer & Bar-

62

Akiva, 1979). However, there are few studies in the literature relating the anatomical

63

and ultrastructural changes of soybean leaves in response to the Mn supply. The

64

deleterious effects of deficiency and the excess of Mn in many species have been

65

studied, mainly, considering the biochemical and yield parameters of the shoots,

66

particularly, where the symptoms are shown that is, the leaves.

67

Recently, a few of the genes responsible for transport of transition metal in plants

68

have been identified, and Mn2+ transport pathways began to be identified at the

69

molecular level. These include transporters responsible for Mn accumulation into the

70

cell and release from various organelles, and for active sequestration into

71

endomembrane compartments, particularly the vacuole and the endoplasmic reticulum.

72

These mechanisms could also be important for Mn leaf-tissue tolerance in

73

hyperaccumulator plants and, on the other hand, could increase the efficiency use in

74

crop species (Van Ho et al., 2002; Pittman, 2005; Martinoia et al., 2007). However,

75

there is a scarcity of papers dealing with soybean genotypic differences as related to

76

cellular structure under either low or high Mn levels (Weiland et al., 1975; Izaguirre-

77

Mayoral & Sinclair, 2005).

78

The present work was carried out to study the effect of Mn supply on changes in

79

the ultrastructure, in the leaf tissue organization as well as in the Mn concentration in

80

leaves of three soybean cultivars, which, in turn, could affect photosynthetic capacity,

81

and other aspects of metabolism.

82

MATERIAL AND METHODS

83

Plant material and cultivation of soybean plants

84

The experiment was carried out under greenhouse conditions, in Piracicaba, State

85

of Sao Paulo, during the period between 17 May and 27 June, 2006. Santa Rosa (Mn-

86

inefficient), IAC-15 (Mn-inefficient) and IAC-Foscarin 31 (Mn-efficient) cultivars of

87

the Glycine max (L.) Merrill were grown under three Mn rates (0.5; 2.0 and

88

200.0 mol L-1) in the nutrient solution. The solutions were prepared from that

89

recommended by Johnson et al. (1957) adapted by Epstein & Bloom (2005), diluted to

90

the 1/5 of the usual concentration, and with initial pH of 4.97 0.03. The experiment

91

was set up as a randomized complete block design with three replications.

92

Seeds were placed to germinate in a tray with vermiculite, moistened with calcium

93

sulphate (CaSO4, 10-4 mol L-1). Plants reaching about 5 cm of height (phenologic stage

94

V1, about five days post emergence), were transplanted to individual plastic pots, 20.0

95

cm in diameter and with a 2.5 L capacity. The solutions containing the desired Mn

96

concentrations were supplied the third day after transplanting, and were renewed every

97

seven days. The pots were rearranged within each block every three days.

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Plant Analysis

99

Plant samples were collected at the outset of the more visible symptoms of

100

deficiency (in the solution with 0.5 mol L-1 of Mn), and of toxicity (200.0 mol L-1)

101

and healthy plants (2.0 mol L-1) as well V3 and V4 phenologic stages. The remaining

102

plant parts were collected, washed and dried at 65C for 48 hours in a forced-air oven

103

before being ground in a stainless steel mill. The material was digested with 4 mL

104

HNO3 and 2 mL HClO4 of concentred acids on a digestion block heated gradually to

105

203C. Manganese was determined by atomic emission spectroscopy (Varian).

106

Scanning electron microscopy and transmission electron microscopy analysis

107

For the scanning electron microscopy, leaf samples were fixed and after post

108

fixation in 1% OsO4 the samples were dehydrated in a graded ethanol series (30-100%)

109

and processed in the critical point dryer through CO2. The dried samples were mounted

110

in metal stubs, sputter coated during 260 seconds with gold and examined under a Zeiss

111

LEO 435-VP scanning electron microscope at 20 kv and the images were then

112

digitalized.

113

For the transmission electron microscopy, small pieces of leaf tissues were fixed

114

for 2 hours in a modified solution (Karnovsky, 1965), composed of 2% glutaraldehyde,

115

2% formaldehyde in 0.05mol L-1 sodium cacodylate buffer at pH 7.2, followed by 1

116

hour post fixation in 1% OsO4, and then dehydrated gradually using acetone (25-100%).

117

Later, the segments were embedded in Epon 812 resin. Blocks were trimmed and the

118

ultrathin sections were cut in a MT2 ultramicrotome equipped with a diamond knife.

119

The sections were picked on formvar-coated cooper grids, and then stained with 2.5%

120

aqueous uranyl acetate for 15 minutes, followed by lead citrate solution for 8 minutes,

121

according to Reynolds (1963). Finally the sections were examined under a Zeiss EM-

122

900 transmission electron microscope at an accelerating voltage of 50 kv and the images

123

were then digitalized. The Morphometric assessments of leaves (cross section leaf

124

layer thickness) were obtained through optical microscopy, followed through processing

125

of images, regularly registered, using the PHOTOSHOP ADOBE version 6.0, and

126

calculated using the software SIARCS (System Integrated for Analysis and Covering of

127

Soil) version 3.0, as to the areas of chloroplast and starch grains measurements.

128

Statistical analysis

129

Data were subjected to analysis of variance using the statistical software SAS -

130

System for Windows 6.11 (SAS Institute, 1996), where the F-test showed significant

131

differences among means. In all analyses, the P = 0.05 level was required for

132

significance.

133

RESULTS AND DISCUSSION

134

Santa Rosa and IAC-15 cultivars showed visual symptoms of deficiency of Mn

135

twelve days after the beginning of the treatments at 0.5 mol L-1 Mn, whereas IAC-

136

Foscarin 31 showed it three days later. Symptoms of toxicity were observed only in

137

Santa Rosa and IAC-15 on the sixth day after Mn was supplied at the doses of

138

200.0 mol L-1 (Figure 1). Chlorosis of internerval areas first appeared in younger

139

leaves, whereas toxicity symptoms were observed both in the young (markedly) and old

140

leaves. The Mn average concentrations in to the sampled leaves are presented in Table

141

1. In the three genotypes, leaf Mn concentration increased with solution Mn. Higher Mn

142

concentrations were found in leaves of IAC-15 followed by Santa Rosa and IAC-

143

Foscarin 31 cultivars. Between the Mn concentrations in the upper leaves at the highest

144

Mn treatment, the shoot metal concentration increased by almost 19% from IAC-

145

Foscarin 31 to IAC-15. There were not, however, significant differences. In general, the

146

critical deficiency range in fully expanded leaves is quite narrow, varying between 10

147

and 20 mg kg1 dry mass. On the other hand, critical leaf concentration for toxicity can

148

vary within a very wide range, depending on plant species and genotypes within
6

149

species, and on environmental conditions, such as temperature and mineral nutritional

150

status (Fageria, 2001). Fageria (2001) attributed Mn concentrations in soybean leaves,

151

respectively, of 67 and 720 mg kg-1, as adequate and toxic contents. Lima et al. (2004)

152

pointed out concentrations of Mn of 1,800 mg kg-1 on shoots of soybean cultivar

153

Emgopa 316, cultivated in oxissols from Brazilian Cerrado.

154

Observations by scanning electron microscopy of sections from deficient leaves,

155

in the three genotypes, showed certain degree of tissue disorganization and minor

156

alteration in the epidermis (abaxial and adaxial face), and none stomata reduction in

157

relation of those observed in adequate Mn-supply. At higher Mn rate (200.0 mol L-1),

158

alterations in the epidermis and tissue agglomeration were verified, resulting in an

159

epidermical hypertrophy. Under all Mn doses, there were no neither in stomata length

160

nor stomata number both in the adaxial as well in the abaxial face (Figure 2), which it is

161

in accordance with the observations of Weiland et al. (1975) and of Baldisserotto et al.

162

(2004). However, it should not be discarded, also, the possibility of that in severe Mn

163

toxicity there are high disorganization of the tissue and then hidden the stomata through

164

the leaf area. Nevertheless, Lidon (2002) observed, in rice grown in nutrient solutions

165

containing 2.4; 145.0 and 582.0 mol L-1 of Mn, alteration in stomata length, which

166

decreased about 50% with the supply of the higher rates. This author concluded that the

167

reduction was associated with the physiological control of the rice plants, as responsible

168

mechanism for the low Mn transport from roots to shoots.

169

Malavolta (2006) stated, that external manifestation of abnormality caused by

170

toxicity of any element, essential or not, are the result of a chain of events that starts

171

with an alteration at the molecular level, continues with modification at subcellular

172

which, in turn, leads to a cellular alteration which, finally, results in modification in the

173

mesophyll, the visible symptom. The deficiency of any nutrient also can unchain this

174

series of events, until the visual symptom appears. Several studies, more specifically

175

dealing with the deficiency of Mn in plants, had demonstrated the role of this nutrient in

176

the maintenance of chloroplasts structure (Weiland et al., 1975), since, one of most

177

striking influences of Mn deficiency is shown in the chloroplast, leading to a reduction

178

in the rate of PS II electron transport (Abadia et al., 1986). On the other hand, it can be

179

also observed significant alterations in the Golgi apparatus and in the endoplasmatic

180

reticulum (Izaguirre-Mayoral & Sinclair, 2005), as well as in the mitochondria under

181

Mn toxicity condition (Santandrea et al., 1998), this, however was not been verified in

182

the present study.

183

Observations by transmission electron microscopy (Figure 3), showed

184

ultrastructural alterations in the mesophyll of the three genotypes. Since, the leaves were

185

sampled at the initial stage of the symptoms (deficiency and toxicity), the anomalies did

186

not progress into the complete disorganization of the epidermis (abaxial and adaxial

187

face), as the plants were not kept until the end of the cycle. Meanwhile, increased Mn

188

concentration in the nutrient solution caused a significant increase in leaf lamina

189

thickness, primarily due to increased length of palisade parenchyma cells (Table 2). The

190

spongy parenchyma thickness also increased with the increasing of Mn rates in the

191

nutrient solution, ranged from 63.1 to 285.7 m, from 12.4 to 115.3 m and from 42.7

192

to 191.9 m, respectively, for Santa Rosa, IAC-15 and IAC-Foscarin 31 cultivars.

193

Transmission electron microscopy of transversely cut leaves from all genotypes

194

grown at concentration of 0.5 mol L-1 revealed chloroplasts with round aspect

195

(markedly in Santa Rosa and IAC-Foscarin 31 cultivars), absence of plastids, scarce

196

starch granules inside chloroplasts, as well as a reduction in its length. Stacking

197

thylakoids (granum) around the estroma and a higher number of vesicles in the

198

cytoplasm were also observed. However, extensive cytoplasmic disorganization, and

199

increase in vacuolation, as well as amorphous cytoplasm were more evident in IAC-15.

200

In this case alterations in the thylakoid membranes were evident (Figure 3-2A). For all

201

genotypes, Mn-deficient chloroplasts are small than those normal and Mn-toxicity (2.0

202

and 200.0 mol L-1 of Mn, respectively) (Table 2), which occupy larger part of the cell

203

volume. Within the chloroplast, the discs are arranged parallel to the envelope. Each

204

disc was organized with two or three other discs into stack characterized by a close

205

association of adjacent disc surfaces. This effect was more evident in the Santa Rosa

206

genotype. Other cellular organelles, like mitochondria and nucleus, did not appear to be

207

altered. The symptom of Mn deficiency observed in soybean in this study are similar to

208

those described by Weiland et al. (1975).

209

On the other hand, chloroplasts at the highest Mn supply, especially with regard to

210

the IAC-15 leaves, had an elongated shape, with thylakoids piled in a disorderly

211

manner, underdeveloped grana, scarce starch granules in comparison with of those

212

cultivars, and hole-like folds in the thylakoid membrane. In short, there was a general

213

disorganization within the chloroplast. An incomplete structure of the plastid was

214

seldom observed. The cytoplasm presented amorphous and dense aspect (spotted),

215

widely disorganized and with great number of vesicles. In a few cells the protoplast was

216

separated from the wall towards the inner of the cell (Figure 3-2C).

217

According to Santandrea et al. (1998), high levels of Mn result in damage to the

218

membrane structure and, probably, to their physiological functions. Furthermore,

219

separation of the membrane from the cell wall, and rupture with formation of many

220

cytoplasmatic vesicles in adjacent spaces can occur. Higher absorption of Mn, as of

221

other heavy metals, probably increases the formation of free radicals and then reduces

222

the content of ascorbic acid, causing the peroxidative damage of the membrane (Morita

223

et al., 2006).

224

Lipid globules were observed in the IAC-15 chloroplasts, under all Mn doses,

225

could indicate either alteration in the metabolic route in the starch synthesis, or still, a

226

characteristic of the genotype. The lipid globules were not evident in the IAC-Foscarin

227

31 at all Mn supplies. The role of the Mn in lipid synthesis process is not very well

228

known. However, the effect can be secondary, due to low photosynthetic rate which

229

restricts the carbon supply for the fatty acid synthesis. The reduction in the number of

230

chloroplasts and their membranes as well the amount of starch in Mn-deficient plants

231

are morphological evidences of the reduction of the dossel photosynthetic apparatus

232

(Weiland et al., 1975; Henriques, 2003, 2004).

233

In the mesophyll cells of Santa Rosa and IAC-Foscarin 31, chloroplasts showed

234

normal configuration with an abundant of well-organized inner membrane system with

235

usually three or four grains of starch (Figures 3-1C and 3-3C). As observed in IAC-

236

Foscarin 31, there was an increase in the size of starch grains which were swollen

237

(Table 2). Doncheva et al. (2005) observed chloroplasts with distorted thylakoids, as

238

well as increment both in size and in number of starch grains, presence of small vesicles

239

and darkened estroma in pea (Pisum sativum L.) grown at Mn rate of 3.000 mol L-1.

240

They concluded that the most evident structural alteration in the chloroplasts was the

241

increase of the starch grains, associate possibly due to the inhibition of the transport of

242

photosynthetic compounds from leaf for other organs. Papadakis et al. (2007) stated that

243

Mn affected the size and shape of chloroplasts in seedlings of Citrus volkamericana

244

(L.), which were shorter and thinner under 0 mol L-1 Mn compared to the treatments

245

with 2 to 686 mol L-1. Besides, the percentage of starch grains per chloroplast was

246

five-fold greater under highest dose than in the treatments with 0 to 98 mol L-1 Mn, as

247

it was also found for IAC-Foscarin 31 in the present study.

10

248

The effects of Mn deficiency and toxicity on the cellular ultrastructure, like

249

chloroplasts distribution (granum) and its starch grains and lipid globules, showed

250

distinct degrees for the genotypes, IAC-15 and Santa Rosa being more affected. In

251

accordance with the ultrastructural observations, of the present study, it was evident that

252

soybean genotypes showed distinct degree in the organization of ultrastructures, in

253

particular by in the chloroplasts both under deficiency and toxicity, being the IAC-15

254

most affected by the nutritional stress.

255

Final considerations

256

According to the ultrastructural alterations as well as the periods of the visual

257

symptoms of deficiency and toxicity, observed in this study, is able to be supposed the

258

existence in Mn-tolerant IAC-Foscarin 31 of several mechanisms that act jointly in the

259

maintenance of the structural and biochemical apparatus of the plants resulting in

260

different degree of cellular organization. These differences may result from the higher

261

levels of the antioxidant enzyme activities and lower oxidant stress level in Mn-efficient

262

IAC-Foscarin 31, like Mn-superoxide dismutase.

263

The existence of a cell adaptation mechanism to excessive Mn availability

264

(200.0 mol L-1 Mn) by increasing the size of chloroplasts as well as their number per

265

cellular area, as seem in Santa Rosa and markedly in IAC-Foscarin 31 cultivars (Table

266

2), is discussed. It should not be discarded, also, the possibility of that, like others heavy

267

metals as the Cd, Cu, Ni and Zn, there are the detoxification and chelation of the Mn-

268

excess by organic acids, such as citrate and malate, or phytochelatins. Beyond, root Mn

269

accumulation and compartmentalization in root apoplast of IAC-Foscarin 31 and then,

270

low Mn transport to shoots can occur as pointed out by Lavres Jr. et al. (2008).

271
272
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ACKNOWLEDGEMENTS

274

To FAPESP for financial support (04/09411-4). Milton Ferreira de Moraes have

275

FAPESP (04/15897-7) scholarship. Eurpedes Malavolta, Neusa de Lima Nogueira and

276

Jos Lavres Junior have a fellowship of CNPq. To Professor E. W. Kitajima

277

(NAP/MEPA, USP-ESALQ) for maintaining the electron microscope facilities. To

278

Monica Lanzoni Rossi and Cleusa Pereira Cabral (USP/CENA) for their skilful

279

assistance.

280

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362

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363

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364
365
366
367

15

368
369

Figure 1 - Soybean leaf showing deficiency symptoms (A), Mn-normal leaf (B) and high-Mn
concentration (C).

370

Figure 2 - Scanning electron micrographs showing stomata leaf of soybean Santa Rosa (1),

371

IAC-15 (2) and IAC-Foscarin 31 (3) cultivars, as related to Mn rates of 0.5

372

mol L-1 (A), 2.0 mol L-1 (B) and 200.0 mol L-1 (C) in the nutrient solution. ()

373

Detail of the stomata. The scale of each picture has a value of 20m.

374

Figure 3 - Transmition electron micrographs showing leaf cell structures of soybean Santa

375

Rosa (1), IAC-15 (2) and IAC-Foscarin 31 (3) cultivars, as related to Mn rates of

376

0.5 mol L-1 deficiency (A), 2.0 mol L-1 control (B) and 200.0 mol L-1

377

toxicity (C) in the nutrient solution. Abbreviations for all parts in alphabetical

378

order: c, chloroplasts; cw, cell wall; gr, grana; lp, lipid globules; m, mitochondria;

379

n, nuclei; sg, starch grains; v, vacuole. () Detail of separated protoplast from cell

380

wall. Scale bar: 1A = 1 m and other pictures = 2 m.

381
382
383
384

16

[ Fig. 1 ]

17

[ Fig. 2 ]
(A)

(1)

(C)

(B)

(2)

(3)

18

[ Fig. 3 ]
(A)

(C)

(B)

gr

(1)

gr

sg

sg

lg

(2)

m
c

cw

cw

gr
c

c
(3)

gr
sg

19

Table 1 Mn concentration (mg kg-1) in the deficient leaf (rate of 0.5 mol L-1 Mn), Mn-normal
and high Mn leaf (200.0 mol L-1) in three-soybean cultivars: Santa Rosa, IAC-15 and
IAC-Foscarin 31, as related to Mn rates of 0.5; 2.0 and 200.0 mol L-1 in the nutrient
solution.
Cultivars
Santa Rosa
IAC-15
IAC-Foscarin 31
MSD
CV (%)

Mn rates (mol L-1)

0.5
2.0
200.0
-1
----------------------------------- (mg kg ) ---------------------------------12.5a
30.6a
786.3a
8.6a
25.9a
886.3a
9.8a
24.0a
744.3a
4.8
24.0
254.9
16
30
11

Lower case letters on the same column do not differ significantly by the Tukey test (P < 0.05).

20

Table 2 Morphometric assessments of leaves (cross sections) and mesophyll chloroplasts of Santa
Rosa, IAC-15 and IAC-Foscarin 31, as related to Mn rates of 0.5; 2.0 and 200.0
mol L-1 in the nutrient solution.
Cultivars
Santa Rosa
IAC-15
IAC-Foscarin 31
CV%
Santa Rosa
IAC-15
IAC-Foscarin 31
CV%
Santa Rosa
IAC-15
IAC-Foscarin 31
CV%
Santa Rosa
IAC-15
IAC-Foscarin 31
CV%

Mn rates (mol L-1)

0.5
2.0
200.0
------------Leaf layer thickness (m) palisade parenchyma -----------103.8aC
124.8aB
416.7aA
101.8aB
72.0bC
318.8bA
77.2bB
77.1bB
332.2abA
9.0
13.7
12.9
------------ Leaf layer thickness (m) spongy parenchyma -----------63.1aC
97.0aB
285.7aA
12.4bC
21.9bB
115.3bA
42.7aC
63.4aB
191.9aA
16.7
10.5
20.1
----------------------- Area of chloroplast (m2) ----------------------7.8aB
9.3aA
9.9aA
7.2aB
9.0aA
5.4bC
6.9aC
8.7aB
11.5aA
9.9
14.3
10.2
----------------------- Area of starch grain (m2) ---------------------0.6aA
0.6bA
-0.5a
--0.4aB
4.2aA
-9.4
11.8

Lower case letters on the same column and upper case letters on the same row do not differ significantly by
Tukey test (P < 0.05).

21