Cu-Chitosan Nanoparticle Mediated Sustainable Approach To

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Cu-Chitosan Nanoparticles Mediated Sustainable Approach to Enhance Seedling Growth in Maize by Mobilizing Reserved Food Vinod Saharan, Kumaraswamy R.V., Ram Chandra Choudhary, Sarita Kumari, Ajay Pal, Ramesh Raliya, and Pratim Biswas J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02239 • Publication Date (Web): 27 Jul 2016 Downloaded from http://pubs.acs.org on July 28, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Journal of Agricultural and Food Chemistry

Cu-Chitosan Nanoparticles Mediated Sustainable Approach to Enhance Seedling Growth in Maize by Mobilizing Reserved Food Vinod Saharan*†, R. V. Kumaraswamy†, Ram Chandra Choudhary†, Sarita Kumari†, Ajay Pal‡, Ramesh Raliya§ and Pratim Biswas§

†

Department of Molecular Biology and Biotechnology, Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology, Udaipur, Rajasthan 313001, India ‡

Department of Chemistry and Biochemistry, College of Basic Sciences and Humanities, Chaudhary Charan Singh Haryana Agricultural University, Hisar, Haryana 125004, India

§

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, MO 63130, USA

*Corresponding author E-mail: [email protected] Phone: +91-9461180586; Fax: +91-294-2420447

ACS Paragon Plus Environment


1

ABSTRACT: Food crop seedlings often have susceptibility towards various abiotic and biotic

2

stresses. Therefore, in the present study, we studied the impact of Cu-chitosan nanoparticles

3

(NPs) on physiological and biochemical changes during maize seedling growth. Higher values of

4

percent germination, shoot, and root length, root number, seedling length, fresh and dry weight

5

and seed vigor index were obtained at 0.04 to 0.12% concentrations of Cu-chitosan NPs as

6

compared to water, CuSO4, and bulk chitosan treatments. Cu-chitosan NPs at the same

7

concentrations induced the activities of α-amylase and protease enzymes and also increased the

8

total protein content in germinating seeds. The increased activities of α-amylase and protease

9

enzymes corroborated with decreased content of starch and protein, respectively in the

10

germinating seeds. Cu-chitosan NPs at 0.16% and CuSO4 at 0.01% concentrations showed

11

inhibitory effect on seedling growth. The observed results on seedling growth could be explained

12

by the toxicity of excess Cu and growth promotory effect of Cu-chitosan NPs. Physiological and

13

biochemical studies suggest that Cu-chitosan NPs enhance the seedling growth of maize by

14

mobilizing the reserved food, primarily starch through the higher activity of α-amylase.

15

Keywords: Chitosan. Cu-chitosan nanoparticles. Seedling growth.


Page 2 of 30

Page 3 of 30


16

INTRODUCTION

17

Application of nanomaterials in agriculture is the subject of intense research and development.

18

The positive results of application of various nanomaterials in agriculture have encouraged the

19

further utilization of this technology. The majority of the nanomaterials used in agriculture for

20

plant growth and protection are metal based and have received a lot of attention and concern

21

related to toxicity.1-8 More vigilance is, therefore, needed during application of highly active and

22

non-degradable metal nanomaterials.9-11 The situation has galvanized the search for more

23

efficient and eco-friendly alternatives. Biopolymer based nanomaterials having certain exclusive

24

characteristics like biodegradability and biocompatibility could be utilized in agriculture.

25

Chitosan, a chitin derived amino polysaccharide has been used in various fields including

26

agriculture.12 Further, the benefits of nanotechnology innovations have been initiated to explore

27

the synthesis of various chitosan-based nanoparticles.13-18 Earlier results of in-vitro and in-vivo

28

studies have indicated a substantial effect of chitosan-based nanomaterials on plant growth and

29

protection.16, 17, 19-21

30

Chitosan has high affinity towards Cu as compared to other metals and with this

31

distinctive ability, Cu-chitosan based nanomaterials have been synthesized and applied in various

32

fields.13, 14, 16, 22, 23 We have already reported a reproducible method for the synthesis of stable

33

and monodisperse Cu-chitosan NPs (mean size 374±8.2 nm) by an ionic gelation method.16 Our

34

studies have resulted in a highly porous network of chitosan nanomaterial wherein Cu gets

35

entrapped with 80% encapsulation efficacy.16 We have further explained the potential of Cu-

36

chitosan NPs as an antifungal agent against various phytopathogenic fungi. Subsequently, we

37

systematically studied the growth promotory effect of Cu-chitosan NPs on tomato seedlings by

38

measuring germination, shoot-root length, and seed vigor index. Our findings unraveled that Cu-



39

chitosan NPs had significant antifungal and seedling growth inducer activity.16, 20 Based on the

40

studies, we predicted that Cu-chitosan NPs might enhance the activities of enzymes involved in

41

the mobilization of stored food in the seed. Hence, further insight may only come through in-

42

depth studies of physiological and biochemical responses of Cu-chitosan NPs on the plant. This

43

could also lead to precise and safe application of Cu-chitosan NPs in the future as an antifungal

44

and growth promotory agent.

45

Maize is one of the important food crops of the world population. Along with other crops,

46

maize seedlings have susceptibility towards various abiotic and biotic stress factors. Therefore,

47

the early vigor of seedlings is also pivotal for yield improvement. Application of Cu-chitosan

48

NPs in maize to understand the physiological and biochemical responses is therefore very

49

crucial. In the present study, Cu-chitosan NPs synthesized with well controlled characteristics

50

were evaluated for Cu content in treated seeds, seedling growth, starch and protein content, α-

51

amylase and protease activity in germinating maize seeds.

52

53

MATERIALS AND METHODS

54

The experimental plan and methodology are summarized in Table 1 and the following sections

55

are outlined here.

56

Materials. Chitosan (low molecular weight and 80% N-deacetylation) was procured

57

from Sigma-Aldrich, St. Louis, USA. Sodium tripolyphosphate (TPP) was supplied by SRL,

58

Mumbai, India. PVDF (polyvinylidene diﬂuoride) syringe ﬁlters (pore size 0.22 µm) and

59

chemicals for enzyme analysis including dinitrosalicylic acid, anthrone reagent, folin and

60

ciocalteus phenol were procured from HiMedia, Mumbai, India. All the chemicals and


Page 4 of 30

Page 5 of 30


61

reagents were used as received. The seeds of maize cultivar Surya local were obtained from

62

the Department of Plant Breeding and Genetics, Rajasthan College of Agriculture, Maharana

63

Pratap University of Agriculture and Technology, Udaipur, India.

64

Preparation of Cu–chitosan NPs. Cu-chitosan NPs were prepared following the methods

65

developed in our laboratory based on the ionic gelation of chitosan with TPP anions.16

66

Synthesized NPs

67

Scattering (DLS), Fourier Transform Infrared, Transmission Electron Microscopy, Scanning

68

Electron Microscopy and Energy Dispersive X-ray Spectroscopy and double beam Atomic

69

Absorption Spectrophotometry. The characteristic details of synthesized NPs were same as we

70

reported in our earlier report.16

71

Seedling bioassay. Maize cultivar Surya local was used to study the efficacy of NPs on seedling

72

growth using standard methods with slight modifications.24 Briefly, maize seeds were surface

73

sterilized by immersing in 10% sodium hypochlorite solution for 10 min and then rinsed thrice

74

with deionised water. The seeds were treated for 4 h with deionized water (control), bulk

75

chitosan (0.01%), CuSO4 (0.01%) and Cu-chitosan NPs at different concentrations (0.01, 0.04,

76

0.08, 0.012 and 0.16 % w/v). The treated seeds were placed in Petri plates (90 × 15 mm,

77

HiMedia, Mumbai, India), having moistening filter paper with 5.0 ml of deionized water. Each

78

treatment was performed in triplicates with 10 seeds in each plate. Sealed Petri plates were

79

maintained at 28 ± 2ºC in a dark growth room. Deionized water was applied daily to the Petri

80

plates to maintain required moisture level. Percent germination was recorded when seed showed

81

at least 1.5-2.0 mm shoot. After 10 days of growth, percent germination, shoot-root length, and

82

root number, seedling length, fresh and dry weight were measured. Seed vigor index (SVI) was

83

calculated by the formula described elsewhere.25

were characterized for physicochemical analyses using


Dynamic Light


84

Seed vigor index = (Germination %) × (Seedling Length)

85

Copper content. To estimate Cu content, treated seeds were oven dried for ~96 h at 75ºC till

86

constant dry weight was achieved. The dried seed samples were ground to a fine powder and

87

digested with 65% HNO3. Cu content in digested samples was measured using atomic absorption

88

spectrophotometer (AAS 4141 model, Electronics Corporation of India Ltd., India) by following

89

a method described elsewhere.26

90

Measurement of enzyme activity

91

Alpha (α) amylase assay. α-amylase at different growth stages (0, 1, 3, 5, 7 and 9 days of

92

germination) was extracted from germinating seeds by homogenizing in sodium acetate buffer

93

(100 mM, pH 4.7) at 4ºC. To initiate the enzyme reaction, diluted enzyme extract (1 ml) was

94

mixed with 100 mM sodium acetate buffer (pH 4.7) and 1 ml of starch solution (1% w/v) and

95

incubated at 37ºC for 15 min. The reaction was stopped by adding 2 ml 3, 5-Dinitrosalicylic acid

96

(DNS) followed by incubation at 100ºC for 5 min in the pre-heated oven. Before cooling the

97

reaction mixture, 1 ml of potassium tartrate (40% w/v) was added, and mixed by quick

98

vortexing. The absorbance of the enzyme activity was recorded at 560 nm. The enzyme activity

99

was expressed as µ mole/min/g dry weight basis which corresponded to µ mole of glucose

100

equivalent released per minute under the assay conditions.27

101

Protease assay. At various germination stages (0, 1, 3, 5, 7 and 9 days of germination), protease

102

was extracted from germinating seed tissues by homogenizing in 100 mM phosphate buffer at

103

4ºC. The enzyme reaction was started by adding casein (1% w/v), a substrate for protease assay.

104

The reaction mixture was incubated at 37º C for 20 min. of the enzyme reaction was terminated

105

by adding trichloroacetic acid (5% v/v), cause precepetation of protein. The resulted precipitate

106

was removed by centrifugation at 10000 rpm for 15 min. and the supernatant was allowed to


Page 6 of 30

Page 7 of 30


107

react with alkaline copper reagent at room temperature for 10 min. To the reaction mixture, Folin

108

Ciocalteau Reagent (FCR) was added and incubated in dark at room temperature for 30 min. The

109

absorbance was measured at 620 nm after the development of blue color using a UV-visible

110

spectrophotometer. The enzyme activity was expressed as µ mole/min/g dry weight basis which

111

corresponded to µ mole of tyrosine equivalent released per minute under the assay conditions.28

112

Starch and protein estimation. The starch content in the germinating seeds was determined

113

using anthrone reagent.29 The seeds were homogenized in ethanol (85% v/v) and boiled for 10

114

min. The samples were centrifuged at 5000 rpm for 10 min, and the supernatant was removed.

115

One additional extraction in 10 ml of hot ethanol (80% v/v) was also carried out. The pooled

116

supernatant was allowed to react with 0.2% anthrone reagent. The absorbance was measured at

117

630 nm and starch content was expressed as mg/g dry weight basis. The protein content of

118

germinating tissue was determined according to the Lowry method28 using bovine serum

119

albumin as standard and the results were expressed as mg/g dry weight.

120

Statistical analysis. Statistical analyses of the data were performed with JMP software version

121

12 using the Turkey-Kramer HSD test to determine significant differences among treatment

122

groups at p = 0.05. Each experiment was repeated twice, and each treatment consisted of three

123

replicates.

124

125

RESULTS AND DISCUSSION

126

Cu-chitosan NPs. Well characterized Cu-chitosan NPs, as reported in our previous research

127

article16 were used to study its effect on physiological and biochemical response in seedling

128

growth. The mean physical diameter of the NPs was 150 ± 12.4 nm and mean hydrodynamic

129

diameter was 374.3 ± 8.2 nm along with zeta potential + 22.6 mV. The striking feature which



130

could play an important role in the growth aspect of the plants is the Cu component of NPs

131

which is entrapped in the chitosan network. The present study was conducted to compare the

132

effect of bulk chitosan, CuSO4 and Cu-chitosan NPs on growth aspects of maize seedlings.

133

Cu content in seed. The aim of this experiment was to quantify Cu content in differentially

134

treated seeds and make a further comparison with the seedling growth. Laboratory synthesized

135

Cu-chitosan NPs, water (control), bulk chitosan and CuSO4 were used separately to treat maize

136

seeds for 4h. The treated seeds were dried and used for estimation of Cu content by AAS.

137

Increasing amount (0.01, 0.04, 0.08, 0.12 and 0.16%) of Cu-chitosan NPs showed increasing

138

contents (0.012, 0.022, 0.028, 0.035 and 0.041 mg/g) of Cu into the treated seeds (Table 2).

139

Maximum 0.041 mg/g Cu content was recorded in 0.16% Cu-chitosan NPs treated seeds. CuSO4

140

(0.01%) treated seeds had 0.027 mg/g dw Cu whereas in water and bulk chitosan treatment, the

141

almost same content of Cu was found (Table 2).

142

Effect of NPs on seedling growth. To study the effect of various NPs on seedling growth seeds

143

treated for 4h were grown for 10 days and data were recorded for % seed germination, root-shoot

144

length, root number, seedling length, fresh and dry weight and seed vigor index (SVI). The

145

statistical analysis showed that Cu-chitosan NPs exert a statistically significant difference in all

146

parameters used for seedling growth measurement (Figure 1). Significantly higher values of

147

shoot length, root lengths, root number, seedling length, fresh weight, and SVI were recorded in

148

0.04, 0.08 and 0.12% Cu-chitosan NPs treated seeds (Figure 2 b - f and h). However, %

149

germination and dry weight were not statistically different among the treatments but

150

considerably higher in NPs treated seeds (Fig 2a and g). Effect of bulk chitosan on various

151

growth parameters was higher as compared to control and CuSO4 except % germination and

152

SVI. However, bulk chitosan has a significantly lower growth promotion effect than Cu-chitosan


Page 8 of 30

Page 9 of 30


153

NPs. But, Cu-chitosan NPs at 0.16% concentration was found to be strong growth inhibitory for

154

germinating seedlings followed by CuSO4 treatment (Figure 2). Most dramatic effect of 0.16%

155

Cu-chitosan NPs was on root number and length which was significantly lower as compared to

156

all other treatments (Figure 2c and d). From these experiments, it can be concluded that 0.16%

157

Cu-chitosan NPs exert seedling growth inhibitory effect while its lower concentrations showed a

158

growth promotion. The observed results on seedling growth were also correlated with the Cu

159

content of treated seeds. In general, higher content of Cu in seeds showed growth inhibitory

160

effect on seedlings (Figure 2).

161

Effect of NPs on starch and protein content in germinating seeds. To determine the starch

162

and protein mobilization pattern during germination of maize seed, seed tissues were collected

163

on 0, 1, 3, 5, 7 and 9 days of seedlings for starch and protein estimation. From zero to first day, a

164

slower and similar pattern of starch mobilization was observed in all treatments. From third day

165

onward, starch started to mobilize rapidly and maximum mobilization was observed in Cu-

166

chitosan NPs treated seeds. In control, bulk chitosan and CuSO4 treated seeds, starch

167

mobilization was slower and its content remained higher on all days as compared to Cu-chitosan

168

NPs treated seeds (Figure 3). Among Cu-chitosan NPs, 0.01, 0.04, 0.08 and 0.12%, induced

169

maximum mobilization of starch over the germination period. After nine days, starch could not

170

be estimated in Cu-chitosan NPs treated seeds because the seed material got almost exhausted

171

(Figure 3a). Total protein content was also measured in germinating seeds which increased from

172

the first day and reached a maximum at the third day in all the treatments. After the third day,

173

protein content started to decline (Figure 3b). From these experiments, we can observe that seeds

174

treated with Cu-chitosan NPs showed higher starch mobilization and have enhanced de nova

175

synthesis of proteins.



Page 10 of 30

176

Effect of Cu-chitosan NPs on α-amylase and protease activity in germinating seeds. The

177

aim of this experiment was to correlate the mobilization of starch and protein with α-amylase and

178

protease activity. α-amylase activity was measured at 0, 1, 3, 5, 7 and 9 days of germinating

179

seeds (Figure 4a). At zero day, the negligible activity of α-amylase was observed in all

180

treatments. The activity increased from 1st to 5th day and declined rapidly in proceeding days in

181

all treatments. On 5th day α-amylase activity was maximum (2.32, 2.93, 2.14, 1.96 and 0.59

182

µmol/min/g dw) in Cu-chitosan NPs treatments (0.01, 0.04, 0.08, 0.12 and 0.16%) as compared

183

to all other the treatment groups. Among the treatments, lower α-amylase activity was recorded

184

in 0.16% Cu-chitosan NPs and CuSO4 treatments. Bulk chitosan induced comparatively higher

185

α-amylase activity as compared to control, CuSO4 and 0.16% Cu-chitosan NPs but considerably

186

lower to other concentrations of NPs (Figure 4a). Similar to α-amylase activity, protease activity

187

was also effected by Cu-chitosan NPs in the same manner (Figure 4b). Protease activity was

188

maximum at a 3rd day in all treatments of Cu-chitosan NPs except 0.16%. In 0.16% Cu-chitosan

189

NPs, lowest protease activity was recorded followed by CuSO4 treatment. After the 3rd day, the

190

enzyme activity got fall-off in all the treatments (Figure 4b). These data suggest that Cu-chitosan

191

NPs significantly induced starch and protein mobilization by enhancing the α-amylase and

192

protease activity in germinating seeds. As starch comprises about 80% of reserve food in maize

193

seed, it is, therefore, the main source of energy for germinating seeds.

194

In the present study, we have deciphered the physiological and biochemical effects of Cu-

195

chitosan NPs on seedling growth of maize. We ascertain that seed priming by Cu-chitosan NPs

196

significantly induces the seedling growth as compared to other treatment (Figs. 1 and 2). Further,

197

we observed that the elevated seedling growth is due to rapid mobilization of starch and protein

198

in germination process due to higher activity of α-amylase and protease in Cu-chitosan NPs


Page 11 of 30


199

treatments (Figs. 3 and 4). Our study also revealed that Cu-chitosan NPs significantly enhanced

200

the total protein content in the germinating seed which may be attributed to de nova synthesis of

201

proteins (Figure 3b). Few studies have been conducted on the applications of chitosan NPs in

202

enhancing the plant growth and protection with astonishing results.17,

203

studies we have established that in addition to antifungal activity, Cu-chitosan NPs can stimulate

204

the seedling growth in tomato16. But, further reports on the physiological and biochemical

205

response of seedlings are unavailable.

20, 21

. In our previous

206

Germination assay is a fundamental procedure to determine growth and toxicity of metals

207

and NPs on plants.9, 11 In the seed germination process, embryonic cells become metabolically

208

active through complex biochemical changes like protein biosynthesis including various

209

enzymes of the glycolytic pathway and carbon metabolism such as α-amylase.30 For vigor and

210

rapid seedling growth, the reserved food should be efficiently mobilized to the embryonic mass.

211

Chitosan and Cu, which are the core components of NPs could enhance the seedling growth by

212

inducing the higher activity of α-amylase and protease enzymes (Figs. 2 and 4). The two facts

213

could explain the physiological and biochemical responses of NPs in our experiments. First, in a

214

recent study, it has been found that chitosan biopolymer entails up-regulation of a number of

215

genes related to carbohydrate metabolism in the plant15 and thus application of chitosan/or its

216

NPs could persuade the growth of plants through up-regulation of various enzymes. Second, the

217

role of Cu is well established in plants as a micronutrient, acts as a key structural and catalytic

218

component of various enzymes of electron transfer and redox reactions and is imperative for

219

growth.31, 32 Thus, Cu component of NPs may contribute in accelerating the metabolic processes

220

in germinating seed. Alongside, it is interesting to reiterate that bulk chitosan (0.01%) treatment



221

did not show any significant effect on seedling growth, α-amylase and protease activity and total

222

protein content as compared to NPs treatment (Figs. 2, 3 and 4).

Page 12 of 30

223

We foresee that Cu-chitosan NPs has a weighty effect on plant cells as they easily pass

224

into the seeds along with encapsulated Cu and participate strongly in the metabolism of

225

germinating seeds. Whereas, large sized polymeric bulk chitosan with low surface area cannot

226

readily pass into the seeds and is not as effective as chitosan NPs towards plant cells (Figure 5).

227

We assume that bulk chitosan might have developed a film on the seed surface which had

228

prevented water uptake by the seed. The reduced water uptake has subsequently affected the seed

229

germination and seed vigor index. Earlier results also concluded that chitosan NPs as compared

230

to bulk chitosan has better growth enhancing activity in plants as it can easily cross the cell

231

membrane and endows higher activity.10, 16, 19 However, a high concentration of Cu in plant cell

232

becomes toxic and induces metabolic disturbances.22, 33

233

In the AAS study, we estimated Cu content in the treated seeds and correlated it with

234

seedling growth characters and α-amylase and a protease activity. We observed that the pattern

235

of the seedling growth was correlated with the Cu content. As Cu content increases in the seeds,

236

the seedling growth abates and goes to a minimum at high Cu content (Table 2 and Figure 2). In

237

aerial parts of the plant, the upper critical concentration of Cu has been found from 15-30 mg/g

238

in various plant species.33. Seeds treated with 0.01, 0.04, 0.08, 0.12 and 0.16% Cu-chitosan NPs

239

showed 0.012; 0.022; 0.028; 0.035 and 0.041 mg Cu content per g of seeds, respectively (Table

240

2). The acute toxicity on seedling growth and enzymes activities at 0.16% of Cu-chitosan NPs

241

could be explained by the higher Cu content (0.041 mg/g) in germinating seeds (Table 2 and

242

Figure 2). Cu-chitosan NPs (0.01 to 0.12% ) showed higher vigor, seedling growth, enhanced

243

amylase and protease activity in germinating seeds compared to all other treatments (Figs. 2 and


Page 13 of 30


244

4). With respect to Cu toxicity in seedlings, the Cu content found in 0.08 and 0.12% NPs treated

245

seed was 0.028 and 0.035 mg/g which was higher than the seeds treated with CuSO4 (Table 2).

246

CuSO4 (0.01%) showed the more inhibitory effect on seedling growth, α-amylase and protease

247

activity as compared to treatment with 0.08 and 0.12% Cu-chitosan NPs (Figs. 2 and 4).

248

To understand the response of CuSO4 on seedling growth, we must understand the

249

physicochemical properties of Cu-chitosan NPs. Cu-chitosan NPs exhibits porous network

250

wherein Cu is entrapped in the pores.16 The porous network of chitosan NPs slowly releases Cu

251

from the nanostructures.13, 16 Therefore, we assume that after entering into seeds the Cu-chitosan

252

NPs releases Cu slowly and steadily, and the exposure of Cu to cellular system is squat. Thus,

253

chitosan entrapped Cu, and its slow release imposes a mild effect on the cell. Contrarily, in

254

CuSO4 treatment, a sudden and rapid exposure of Cu to cellular system exerts growth inhibitory

255

effect on seedlings (Figure 5). These results are in line with the previous results where PVP

256

entrapped Ag exhibited less toxicity to plants as compared to AgNO3.34

257

Cu-chitosan NPs at 0.12% concentration caused a little decline in growth but not as

258

cumbersome as it was at 0.16% concentration (Figure 1). Growth inhibitory effect of 0.16% Cu-

259

chitosan NPs could be because of excess Cu that has been reported to decrease the level35 and

260

activity of α-amylase36, and other metabolic enzymes in germinating seeds thereby inhibiting

261

seedling growth.22,

262

compared to other growth parameters (Figure 2). It appears that Cu was much more cytotoxic to

263

the root cells at higher concentration. In addition, roots have been found to have more affinity

264

towards Cu and as a result increase in lipid peroxidation and a striking decrease of the K content

265

has been reported.37 In the present study, the higher seedling growth of maize under Cu-chitosan

35, 36

Root number and length were drastically decreased at 0.16% NPs as



266

NPs treatment may be due to the involvement of chitosan and Cu in metabolic complex reactions

267

during germination which ultimately provides higher energy to growing seedlings.

Page 14 of 30

268

In summary, the present study shows that Cu-chitosan NPs can significantly enhance

269

maize seedling growth by up-regulating the enzymes responsible for mobilization of stored food.

270

It can also be concluded that micronutrient like Cu could be used in plants as Cu-chitosan NPs

271

for seed treatments for higher and rapid growth of seedlings. These findings will open the

272

possibility of using other micronutrients in the form of metal chitosan NPs to enhance seedling

273

growth. Our earlier and present study reveals that Cu-chitosan NPs shows promising antifungal

274

activity in plants. Therefore, we conclude that seed priming with Cu- chitosan NPs is beneficial

275

for seedling growth and may also help in disease protection especially for seed born and soil

276

born disease. Further investigations in this line are under progress.

277

Acknowledgments. The authors are indebted to the financial support from Rashtriya Kristi

278

Vikas Yojna (RKVY), Government of Rajasthan, India. The authors gratefully acknowledge the

279

PhD grants, IF140505 from DST and DBT/2015RCOA-2012/372 DBT, Government of India to

280

R.V. Kumaraswamy and Ram Chandra Choudhary. Authors are thankful to the Nano Research

281

Facility, Washington University in St. Louis for providing assistance in nanoparticle synthesis

282

and characterization.


Page 15 of 30

283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326


References 1. 2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

Ghormade, V.; Deshpande, M. V.; Paknikar, K. M. Perspectives for nano-biotechnology enabled protection and nutrition of plants. Biotechnology Advances 2011, 29, 792-803. Khot, L. R.; Sankaran, S.; Maja, J. M.; Ehsani, R.; Schuster, E. W. Applications of nanomaterials in agricultural production and crop protection: A review. Crop Protection 2012, 35, 64-70. Jayaseelan, C.; Ramkumar, R.; Rahuman, A. A.; Perumal, P. Green synthesis of gold nanoparticles using seed aqueous extract of Abelmoschus esculentus and its antifungal activity. Industrial Crops and Products 2013, 45, 423-429. Raliya, R.; Nair, R.; Chavalmane, S.; Wang, W.-N.; Biswas, P. Mechanistic evaluation of translocation and physiological impact of titanium dioxide and zinc oxide nanoparticles on the tomato (Solanum lycopersicum L.) plant. Metallomics 2015, 7, 1584-1594. Raliya, R.; Tarafdar, J.; Singh, S.; Gautam, R.; Choudhary, K.; Maurino, V. G.; Saharan, V. MgO nanoparticles biosynthesis and its effect on chlorophyll contents in the leaves of clusterbean (Cyamopsis tetragonoloba L.). Advanced Science, Engineering and Medicine 2014, 6, 538-545. Raliya, R.; Tarafdar, J. C.; Biswas, P. Enhancing the mobilization of native phosphorous in mung bean rhizosphere using ZnO nanoparticles synthesized by soil fungi. Journal of Agricultural and Food Chemistry 2016, 64, 3111-3118. Anderson, C. W. N.; Bhatti, S. M.; Gardea-Torresdey, J.; Parsons, J. In Vivo Effect of Copper and Silver on Synthesis of Gold Nanoparticles inside Living Plants. ACS Sustainable Chemistry & Engineering 2013, 1, 640-648. Hernandez-Viezcas, J. A.; Castillo-Michel, H.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Interactions between CeO2 nanoparticles and the desert plant mesquite: a spectroscopy approach. ACS Sustainable Chemistry & Engineering 2016, 4, 1187-1192. Feizi, H.; Moghaddam, P. R.; Shahtahmassebi, N.; Fotovat, A. Impact of bulk and nanosized titanium dioxide (TiO2) on wheat seed germination and seedling growth. Biological trace element research 2012, 146, 101-106. Shukla, S. K.; Mishra, A. K.; Arotiba, O. A.; Mamba, B. B. Chitosan-based nanomaterials: A state-of-the-art review. International Journal of Biological Macromolecules 2013, 59, 4658. Thuesombat, P.; Hannongbua, S.; Akasit, S.; Chadchawan, S. Effect of silver nanoparticles on rice (Oryza sativa L. cv. KDML 105) seed germination and seedling growth. Ecotoxicology and Environmental Safety 2014, 104, 302-309. Dzung, N. A.; Khanh, V. T. P.; Dzung, T. T. Research on impact of chitosan oligomers on biophysical characteristics, growth, development and drought resistance of coffee. Carbohydrate Polymers 2011, 84, 751-755. Brunel, F.; El Gueddari, N. E.; Moerschbacher, B. M. Complexation of copper (II) with chitosan nanogels: Toward control of microbial growth. Carbohydrate Polymers 2013, 92, 1348-1356. Jaiswal, M.; Chauhan, D.; Sankararamakrishnan, N. Copper chitosan nanocomposite: synthesis, characterization, and application in removal of organophosphorous pesticide from agricultural runoff. Environmental Science and Pollution Research 2012, 19, 2055-2062.



327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372

Page 16 of 30

15. Chandra, S.; Chakraborty, N.; Dasgupta, A.; Sarkar, J.; Panda, K.; Acharya, K. Chitosan nanoparticles: A positive modulator of innate immune responses in plants. Scientific Reports 2015, 5. 16. Saharan, V.; Sharma, G.; Yadav, M.; Choudhary, M. K.; Sharma, S.; Pal, A.; Raliya, R.; Biswas, P. Synthesis and in vitro antifungal efficacy of Cu–chitosan nanoparticles against pathogenic fungi of tomato. International Journal of Biological Macromolecules 2015, 75, 346-353. 17. Xing, K.; Shen, X.; Zhu, X.; Ju, X.; Miao, X.; Tian, J.; Feng, Z.; Peng, X.; Jiang, J.; Qin, S. Synthesis and in vitro antifungal efficacy of oleoyl-chitosan nanoparticles against plant pathogenic fungi. International Journal of Biological Macromolecules 2016, 82, 830-836. 18. Tan, C.; Xie, J.; Zhang, X.; Cai, J.; Xia, S. Polysaccharide-based nanoparticles by chitosan and gum arabic polyelectrolyte complexation as carriers for curcumin. Food Hydrocolloids 2016, 57, 236-245. 19. Van, S. N.; Minh, H. D.; Anh, D. N. Study on chitosan nanoparticles on biophysical characteristics and growth of Robusta coffee in green house. Biocatalysis and Agricultural Biotechnology 2013, 2, 289-294. 20. Saharan, V.; Mehrotra, A.; Khatik, R.; Rawal, P.; Sharma, S.; Pal, A. Synthesis of chitosan based nanoparticles and their in vitro evaluation against phytopathogenic fungi. International Journal of Biological Macromolecules 2013, 62, 677-683. 21. Manikandan, A.; Sathiyabama, M. Preparation of Chitosan nanoparticles and its effect on detached rice leaves infected with Pyricularia grisea. International Journal of Biological Macromolecules 2016, 84, 58-61. 22. Lou, L.-q.; Shen, Z.-g.; Li, X.-d. The copper tolerance mechanisms of Elsholtzia haichowensis, a plant from copper-enriched soils. Environmental and Experimental Botany 2004, 51, 111-120. 23. Saharan, V.; Khatik, R.; Kumari, M.; Raliya, R.; Nallamuthu, I.; Pal, A. Nano-materials for plant protection with special reference to Nano-chitosan. International Conference on Advances in Biotechnology (BioTech). Proceedings 2014, 23. 24. Association, I. S. T. In International rules for seed testing: rules l996, 1996; International Seed Testing Association: 1996. 25. Abdul-Baki, A. A.; Anderson, J. D. Vigor determination in soybean seed by multiple criteria. Crop Science 1973, 13, 630-633. 26. Smiri, M.; Chaoui, A.; El Ferjani, E. Respiratory metabolism in the embryonic axis of germinating pea seed exposed to cadmium. Journal of plant physiology 2009, 166, 259-269. 27. Bernfeld, P.; Colowick, S.; Kaplan, N. Methods in enzymology. by SP Colowick and NO Kaplan, Academic Press Inc., New York 1955, 149. 28. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry 1951, 193, 265-275. 29. Yemm, E.; Willis, A. The estimation of carbohydrates in plant extracts by anthrone. Biochemical Journal 1954, 57, 508. 30. Zhang, H.; Lian, C.; Shen, Z. Proteomic identification of small, copper-responsive proteins in germinating embryos of Oryza sativa. Annals of Botany 2009, 103, 923-930. 31. Ahmad, P. Plant Metal Interaction: Emerging Remediation Techniques. Elsevier: 2015. 32. Rajasekaran, P.; Santra, S. Hydrothermally treated chitosan hydrogel loaded with copper and zinc particles as a potential micronutrient-based antimicrobial feed additive. Frontiers in Veterinary Science 2015, 2, doi: 10.3389/fvets.2015.00062.


Page 17 of 30

373 374 375 376 377 378 379 380 381 382 383 384 385 386 387


33. Adrees, M.; Ali, S.; Rizwan, M.; Ibrahim, M.; Abbas, F.; Farid, M.; Zia-ur-Rehman, M.; Irshad, M. K.; Bharwana, S. A. The effect of excess copper on growth and physiology of important food crops: a review. Environmental Science and Pollution Research 2015, 22, 8148-8162. 34. Yasur, J.; Rani, P. U. Environmental effects of nanosilver: impact on castor seed germination, seedling growth, and plant physiology. Environmental Science and Pollution Research 2013, 20, 8636-8648. 35. Ahsan, N.; Lee, D.-G.; Lee, S.-H.; Kang, K. Y.; Lee, J. J.; Kim, P. J.; Yoon, H.-S.; Kim, J.S.; Lee, B.-H. Excess copper induced physiological and proteomic changes in germinating rice seeds. Chemosphere 2007, 67, 1182-1193. 36. Singh, D.; Nath, K.; Sharma, Y. K. Response of wheat seed germination and seedling growth under copper stress. Journal of Environmental Biology 2007, 28, 409. 37. Adhikari, T.; Sarkar, D.; Mashayekhi, H.; Xing, B. Growth and enzymatic activity of maize (zea mays l.) plant: solution culture test for copper dioxide nanoparticles. Journal of Plant Nutrition 2015, 99-115.

388 389



Page 18 of 30

390

List of Figures

391

Figure 1. Effect of Cu-chitosan NPs on seed germination and seedling growth of the maize.

392

Figure 2. Effect of Cu-chitosan NPs on (a) % germination; (b) shoot and (c) root length; (d) root

393

number; (e) seedling length; (f) fresh weight; (g) dry weight and (h) shoot vigor index

394

of maize seedling. Data were recorded after 10 days. Each value is mean of triplicate,

395

and each experiment consisted of 10 seedlings. The same letter in the graph of each

396

treatment is not significantly different at p=0.05 as determined by Tukey-Kramer

397

HSD, Control with water. BCH (Bulk chitosan, 0.01%) dissolved in 0.1% acetic acid.

398

CuSO4 (0.01%). Cu content was measured in 4h treated seed.

399 400

401 402

403 404

Figure 3. Effect of Cu-chitosan NPs on (a) starch and (b) total protein content in germinating seeds of maize. Each value is mean of triplicate Figure 4. Effect of Cu-chitosan NPs on (a) α-amylase and (b) protease activity in germinating seeds of maize. Each value is mean of triplicate Figure 5. A hypothetical presentation of the effect of CuSO4, bulk chitosan and Cu-chitosan NPs on seedling growth of maize


Page 19 of 30


405

Figures

406

Figure 1.

407 408 409

Control

BCH

CuSO4

0.01 % 0.04 % 0.08 % 0.12 % 0.16% I-------------------------------- Cu-chitosan NPs ---------------------------

----I



410

Page 20 of 30

Figure 2.

411

Germinatin % 100

a

Germination %

ab b

80

Cu content a

a ab

ab

b

60 40 20 0 Control BCH

CuS04

0.01

0.04

0.08

0.12

0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0

Cu content (mg/g dw)

(a)

0.16

412 413

Shoot length (cm)

9 8 7 6 5 4 3 2 1 0

Cu content

a

abc

ab

ab

abc abc bc c

Control BCH

CuS04

0.01

0.04

0.08

414


0.12

0.16

0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0


Shoot length

(b)


(c)

Root length

Cu content

Root length (cm)

14 a

12 10 8 6

b

b

bc

b

b

b

4

c

2 0 Control

BCH

CuS04

0.01

0.04

0.08

0.12

0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0


Page 21 of 30

0.16

415

Number of roots

6

a

Number of roots

5

ab

4 3

Cu content

bc

ab

ab

abc bc

2 c

1 0 Control

BCH

CuS04

0.01

0.04

0.08

416


0.12

0.16

0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0


(d)


Seedling length

Cu content

Seedling length (cm)

25

0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0

a 20 b 15

cd

bcd

10

d

c

bcd

e

5 0 Control BCH CuSO4

0.01

0.04

0.08

0.12


(e)

Page 22 of 30

0.16

417

Fresh weight 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00

Cu content a

a

a

Control BCH

a

a

a

a

a

Copper

0.01

0.04

0.08

418


0.12

0.16

0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0

Cu content (mg/gm dw)

Fresh weight (g)

(f)


(g)

Dry weight

Cu content

0.25

Dry weight (g)

0.20

a

a

a a

a

a

a a

0.15 0.10 0.05 0.00 Control BCH Copper

0.01

0.04

0.08

0.12

0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0


Page 23 of 30

0.16

419

Shoot vigour index 2000 1800 1600 1400 1200 1000 800 600 400 200 0

Cu content 0.045

a

0.04

ab

0.035

bc cd

d

0.03

cd

0.025

d

0.02 0.015

e

0.01 0.005 0

Control BCH CuS04

0.01

0.04

0.08

420 421 422


0.12

0.16


Shoot vigour index

(h)


423

Page 24 of 30

Figure 3. Control (Water) CuSO4 (0.01%) Cu-chitosan NPs (0.04%) Cu-chitosan NPs (0.12%)

(a)

Bulk chitosan (0.01%) Cu-chitosan NPs (0.01%) Cu-chitosan NPs (0.08%) Cu-chitosan NPs (0.16%)

Starch (mg/g dw)

1000 800 600 400 200 0 0

1

424

Control (Water) CuSO4 (0.01%) Cu-chitosan NPs (0.04%) Cu-chitosan NPs (0.12%)

(b)

Total protein (mg/g dw)

3 5 Time (days)

7

9


4 3 2 1 0 0

425

1

3 5 Time (days)

426


7

9

Page 25 of 30

427


Figure 4. Control (Water) CuSO4 (0.01%) Cu-chitosan NPs (0.04%) Cu-chitosan NPs (0.12%)

Activity (µmole/min/g dw)

(a)

3 2.5 2 1.5 1 0.5 0 0

1

3

5

7

9

Time (days)

428

Control (Water) CuSO4 (0.01%) Cu-chitosan NPs (0.04%) Cu-chitosan NPs (0.12%)

Activity (µmole/min/g dw)

(b)


8 6 4 2 0 0

429


1

3

5

Time (days)

430


7

9


431

Figure 5.

432 433


Page 26 of 30

Page 27 of 30

434


Tables

435 436

Table 1 Experimental outlines

437 Experiment Laboratory synthesis of Cu-chitosan NPs

Analysis/method

Notes

Ionic gelation approach following the method of Saharan et al. (2015)

Cu–chitosan NPs were synthesized

Seedling bioassay

Blotter test method

To determine the efficacy of NPs on seedling growth

Cu content in seed

AAS (Atomic Absorption Spectrophotometer) DNS reagents method

To determine Cu content in seeds

α-Amylase enzyme assay

To estimate α-amylase activity

Protease assay

Folin Ciocalteau reagent (FCR) method

To estimate protease activity

Starch estimation

Anthrone reagent

To estimate the mobilization pattern of starch during germination

Statistical analysis

JMP software version 10 using Turkey-Kramer HSD test

438 439 440 441 442 443



444

Page 28 of 30

Table 2 Cu content in seeds treated for 4h with various treatments Treatment (%)

Cu content (mg/g)

Control (water)

0.012± 0.0016d

BCH (0.01)

0.014±0.0014d

CuSO4 (0.01)

0.027±0.0017c

Cu-Chitosan NPs

445 446 447

0.01

0.011±0.0008d

0.04

0.021±0.0013c

0.08

0.027±0.0012bc

0.12

0.034±0.0017ab

0.16

0.041±0.0012ab

Each value is mean of triplicate. The same letter in a column of each treatment is not significantly different at p=0.05 as determined by Tukey-Kramer HSD, Control with water. BCH (Bulk chitosan, 0.01%) dissolved in 0.1% acetic acid. CuSO4 (0.01%).

448


Page 29 of 30

449


TOC Figure

450 451

TOC Figure: Cu – Chitosan nanoparticles increase the activity of α – amylase and protease that

452

enhances the mobilization of reserve food of the seed, resulting in an increased germination rate.

453




Page 30 of 30

Cu-Chitosan Nanoparticle Mediated Sustainable Approach To

Recommend Documents