Interaction of Salinity and CaCO3 Affects the Physiology and Fatty

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Agricultural and Environmental Chemistry

Interaction of salinity and CaCO3 affects the physiology and fatty acid metabolism in Portulaca oleracea Mouna BESSROUR, Najla Chelbi, Diego A. Moreno, Farhat Chibani, Chedly Abdelly, and MICAELA CARVAJAL J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01456 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 12, 2018

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Journal of Agricultural and Food Chemistry

Interaction of salinity and CaCO3 affects the physiology and fatty acid metabolism in Portulaca oleracea. Mouna Bessrour§, Najla Chelbi§,†, Diego A. Moreno‡, Farhat Chibani∥, Chedly Abdelly§ and Micaela Carvajal†* §

Laboratory of Extremophile Plants, Center of Biotechnology of Borj-Cedria (LEP-CBBC), P. O. Box 901, 2050 Hammam-Lif, Tunisia.

†

Aquaporins Group. Department of Plant Nutrition, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Campus Universitario de Espinardo, Edificio 25, 30100 Murcia, Spain.

‡

Phytochemistry Lab. Food Science and Technology Department, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC). Campus Universitario de Espinardo - 25, 30100 Murcia, Spain. ∥Laboratory

of Plant Molecular Physiology, Biotechnology Centre of Borj Cedria, PO Box 901, 2050 Hammam-Lif, Tunisia.

*Corresponding author: Phone: +34 968 396310; FAX: +34 968 396213; E-mail: [email protected]

ACS Paragon Plus Environment


1

ABSTRACT

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Due to the extreme conditions that usually occur in Mediterranean climates, the

3

objective of this work is to study the combined and/or separate effects of saline and alkaline

4

stresses in Portulaca oleracea. The study was carried out to determine the nutritional food

5

potential in relation to plant physiological parameters. The results show that alkaline media in

6

which CaCO3 was present did not affect growth, but exposure to 100 mM NaCl decreased it

7

greatly. Fatty acid content increased under all stress conditions, but to a higher extent with

8

salinity; however, the protein content was increased only by alkaline media. The beneficial

9

effect of each stress on P. oleracea is discussed in the light of the physiological response,

10

pointing out the suitability of this plant for human nutrition.

11 12

Keywords: Portulaca oleracea, Salinity, CaCO3, Fatty acids, Proteins, Minerals.

13 14 15 16 17 18 19 20 21 22 23 24 25


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INTRODUCTION

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Soil salinity is a problem for agriculture, as it affects the growth and yield of plants in arid

28

and semi-arid areas 1. The concentrations of salts tend to increase in soils due to evaporation

29

of precipitation, and poor drainage combined with high salt concentration in the fertilizer

30

irrigation2. In Mediterranean areas, groundwater and deep aquifers used for irrigation usually

31

are highly salinized (the concentration can reach 6 g/L) 3. Salinity has three different effects

32

on plants: a) osmotic, due to a decrease in osmotic potential, which reduces water uptake 4, b)

33

toxic, due to accumulation of Na+ and Cl- 5, and c) nutritional, due to the limitation of nutrient

34

uptake and transport 4. The high concentrations of Na+ and Cl- also cause oxidative stress in

35

plant tissues 6, generated by reactive oxygen species (ROS). To cope with this, plants

36

synthesize protective molecules - such as different secondary metabolites of the

37

phenylpropanoid pathway, including phenolic compounds - to eliminate and/or reduce these

38

ROS 7. The antioxidant activity of the phenolic compounds makes it possible for them to act

39

as reducing agents, hydrogen donors, and quenchers of singlet oxygen, preventing lipid and

40

protein damage 8.

41

Soils in some saline areas are rich in limestone, particularly at depth (NF ISO 10693).

42

In a calcareous medium, the soil solution is characterized by a high pH and a high

43

concentration of bicarbonate. Increasing the bicarbonate concentration causes a decrease in

44

the rate of root elongation, a decrease in the rate of anion uptake, and an increase in that of

45

cations 9. Under these conditions, some species take up large amounts of calcium that

46

accumulate in their roots in the form of calcium carbonate. In nature, this phenomenon allows

47

the roots to dissolve the limestone of the soil, by the transformation of the calcium of their

48

rhizosphere in their tissues, and thus make it possible to penetrate the compact calcareous

49

soils

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soluble organic nitrogen, relative to those of plants grown in the absence of CaCO3. Under

10

. Thus, leaf tissues of plants cultured with CaCO3 show high levels of K+, Ca2+, and



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saline conditions, plants with high Ca2+ intake have reduced Na+ accumulation and enhanced

52

growth

53

adverse effects of NaCl, through a mechanism involving aquaporins. In further investigation,

54

extra calcium produced a positive effect on aquaporins, allowing higher water uptake under

55

saline conditions 14.

56

11, 12

. Cabañero et al.

13

showed that Ca2+ uptake can protect membranes against the

Recent research has shown that the forage plant purslane (Portulaca oleracea L.) is a 15

57

source of omega-3 fatty acids

, which are very important in preventing heart attacks and

58

strengthening the immune system 16. As P. oleracea grows naturally in arid and saline soils, it

59

is a promising species for consumption as a vegetable and oilseed production

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listed by the World Health Organization as one of the most used medicinal plants and has

61

received the term "panacea world" 18.

17

. It is also

62

Due to the natural Mediterranean growth environment of P. oleracea, the purpose of

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this study was to determine the influence of not only salt stress, but also combined with

64

alkaline constraints (CaCO3) on the chemical composition of purslane crops. A novel

65

comprehensive and comparative analysis of the nutritional composition (proteins, lipids, fatty

66

acids, and phenolic compounds) has been related to yield and physiological parameters

67

(growth, osmotic potential, and chlorophylls). Also, the fatty acids concentration in

68

chloroplasts was determined in relation to their synthesis for membranes.

69 70

MATERIAL AND METHODS

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Plant material and growing conditions. Portulaca oleracea is an edible plant rich in omega-

72

3 15 and with halophytic behavior 19. This species was chosen for its different responses to salt

73

stress and alkaline stress, and for its economic benefit as a forage crop 20.

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The germination was carried out at a temperature of 27°C in vermiculite. After one week, the

75

plants were transferred to a controlled environmental chamber. The temperature was


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25°C/17°C (day/night), with a photoperiod of 16 h, a luminous intensity of 400 µmol m-2 s-1,

77

and a relative humidity of 70-75%, After 5 days seedlings were disposed in 15 L-trays

78

containing a Hoagland nutrient solution 21: 6 KNO3; 4 Ca (NO3)2, 1 KH2PO4, 1 MgSO4 (in

79

mM), and 25 H3BO3, 2 MnSO4, 2 ZnSO4, 0.5 CuSO4, 0.5 (NH4)6, MO7O24 and 20 Fe-

80

EDTA (in µM). The solution was replaced every week. The following treatments were used:

81

control (no added NaCl or CaCO3), 100 mM NaCl, 10 mM CaCO3, and 10 mM CaCO3+100

82

mM NaCl. The pH and EC were checked at each renewal of the solution. After 30 days of

83

treatment, the plants were harvested.

84 85

Leaf Osmotic potential (Ψπ). The osmotic potential of the sap (Ψπ) of leaves was measured

86

using an osmometer (Digital Osmometer. Roebling. Berlin). This was calibrated using a

87

standard solution of KNO3.

88

The osmotic potential of the sap was calculated calculated by: Ψπ= nRT

89 90

Where “n” is the osmotic concentration (mOsmol); “R” is the gas constant (0.083 L atm K−1

91

mol−1) and “T” is the ambient temperature (K).

92 93

Chlorophyll concentration. The photosynthetic pigments in young leaves were determined

94

following extraction from samples of 100 mg of fresh material in 5 ml of 80% acetone. The

95

chlorophyll a (Chla) and chlorophyll b (Chlb) concentrations were calculated according to

96

Lichlenthaler 22.

97 98

Determination of the protein concentration. The protein concentration was obtained from

99

fresh material (0.5 g of leaves). The tissue was homogenized at 4°C with 1 ml of phosphate

100

buffer (KPB, 50 mM; pH 7.8) containing Triton x-100 (0.2%). After centrifugation at 8000 g



101

for 15 min, at 4°C, the protein was determined by the method of 23, using the Bio-Rad reagent

102

with BSA as standard, and expressed in mg g FW-1.

103 104

Determination of lipids. Total lipids were extracted according to the method of 24, including

105

some modifications. The leaves (100 mg of fresh material) were fixed in boiling water for 5

106

minutes, to denature the phospholipases, and then homogenized in chloroform-methanol (2:1,

107

v/v). The homogenate was centrifuged at 3000 rpm for 15 minutes. The lower chloroform

108

phase, containing the lipids, was separated and evaporated under nitrogen gas.

109 110

Determination of fatty acids. The total lipid fatty acids were transmethylated with NaMeO

111

(0.5 N in anhydrous methanol). To ensure rapid saponification of the fatty acids, the tubes

112

were placed in a water bath at 65°C for 10 minutes. The resulting fatty acid methyl esters

113

were extracted with hexane (2 ml), evaporated under N2, dissolved in ethyl acetate (200 µl),

114

and analyzed by GC using a capillary column bound to an HP5 column (30 x 0.25 x 0.25 µm)

115

with FID and the H2 as carrier. Fatty acids were identified and quantified by comparing peak

116

areas with those of known standars (Sigma)

117 118

Chloroplast isolation. Leaves of P. oleracea (20 g) were homogenized with 80 ml of 50 mM

119

Tris/HCl solution (pH 8) containing 1 mM DTT, 1 mM EDTA, and 0.4 M sucrose. After

120

filtration and centrifugation at 2000 x g for 15 min, at 4 ºC, the pellet (dissolved in

121

homogenization buffer) was used for the next steps. To purify the intact chloroplasts, a

122

density gradient of sucrose was used. Three sucrose solutions (4 ml) were used, each with a

123

different concentration of sucrose: 1.45 mM, 0.84 mM, and 0.45 mM, in Tris buffer (pH 7.6),

124

adding them one by one to the tube. After centrifugation at 26000 x g for 45 min, at 4 ºC, the


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intact chloroplasts were concentrated between the second phase and the lower phase. Then,

126

the intactness of the chloroplasts was checked under a microscope.

127

Determination of fatty acids in chloroplasts Chloroplastic fatty acids were transmethylated

128

with NaMeO (0.5 N in anhydrous methanol), followed by addition of 1 ml of hexane,

129

centrifugation, and evaporation with N2. After adding ethyl acetate (200 µl), the extracts were

130

analyzed by GC as described previously.

131 132

Phenolic compounds. The phenolic compounds were analyzed according to the procedure of

133

25

134

vortexing every 5 min to improve the extraction, followed by centrifugation (15 min, 13000

135

rpm, 4 °C). The supernatants were collected and the compounds within them were separated

136

using HPLC, in a Luna C18 column (25 cm x 0.46 cm, 5 µm particle size, Phenomenex,

137

Macclesfield, UK) with a C18-ODS cartridge safety agent (4 x 30 mm). Phenolic acids were

138

quantified as chlorogenic acid (5-caffeoylquinic acid; Sigma, St Louis, MO, USA), flavonols

139

as quercetin3-rutinoside (Sigma), and sinapic acid derivatives assinapic acid (Sigma).

. Powders of lyophilized plant material (50 mg) were extracted with 1.5 ml of methanol,

140 141

Analysis of mineral elements. The concentrations of macronutrients (Ca, K, Mg, Na, P, S)

142

and micronutrients (Cu, Fe, Mn, Zn) were analyzed in finely ground (particle sizes of 0.5-0.7

143

mm) lyophilized material (roots, stems, and leaf tissues). Samples were digested in a

144

microwave oven (CEM Mars Xpress, Mattheus, NC, USA) by acid digestion (HNO3-HClO4,

145

2:1). Elemental analysis was performed using an inductively coupled plasma spectrometry

146

(Thermo Fisher iCAP 6500 duo, USA). The concentrations were expressed in mM of DW for

147

macronutrients and µM of DW for micronutrients.

148



149

Data analysis. The data were analyzed using ANOVA procedures and the means were

150

separated by Duncan's multiple-range tests.

151 152

RESULTS

153

Plant growth. The FW, DW, and water concentration were measured 30 days after the

154

application of salinity (100 mM NaCl), calcium carbonate (10 mM CaCO3), or a combination

155

treatment (100 mM NaCl-10 mM CaCO3). The results (Figure 1) shown that leaf FW slightly

156

decreased after NaCl treatment but increased with CaCO3. Under the combined treatment

157

(NaCl + CaCO3) leaf FW decreased compared with control plants. The stems showed similar

158

results but the decrease in FW with the NaCl treatment was higher and the increase with

159

CaCO3 was slighter. Root growth was not altered by the treatments compared with control.

160

Calculation of total FW per plants produced 213.2 ± 10.2 g in controls, a decrease to 138.2 ±

161

7.1 g in NaCl treated plants, and to 77.1 ± 1.7 g in NaCl + CaCO3 treated plants but an

162

increase to 257.2 ± 4.11 g in CaCO3 treated plants.

163

Similar results than for FW were observed for DW in stem and roots, but for leaves,

164

the treatments containing NaCl and CaCO3, did not gave significant differences from the

165

control values. In the same way as in FW, calculation of total DW per plants produced 14.3 ±

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1.1 g in controls and 15.65 ± 1.3 g in CaCO3 treated plants, and decreases to 9.1 ± 0.3 g in

167

NaCl treated plants, and to 4.6 ± 0.2 g in NaCl + CaCO3 treated plants.

168 169

Osmotic Potential. The osmotic potential (Figure 2) was also altered by the treatments. Both

170

salinity treatments (NaCl and NaCl + CaCO3) reduced the osmotic potential, strongly and

171

significantly in leaves and slightly but also significantly in roots of plants grown under both

172

treatments with NaCl.

173


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Chlorophylls. Chlorophylls a and b were determined (Figure 3), with similar results for both.

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The application of salinity alone reduced slightly, but not significantly, both types of

176

chlorophyll. The application of CaCO3 did not have an effect, but treatment with

177

NaCl+CaCO3 produced a decrease for both types of chlorophylls.

178 179

Proteins. The protein concentrations of the different plant organs were measured after 30 d of

180

treatment (Figure 4). The influence of the treatments different in leaves, stems and roots.

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Salinity had a significant negative effect on the protein concentration, with decreases of

182

18.5% and 11.6% in the leaves and roots, respectively. The CaCO3 and CaCO3+NaCl

183

treatments had a significant positive influence on the protein concentration in leaves with an

184

increase of 47.7% compared to the control. Also, in roots, the increases were 55.4% of control

185

for CaCO3 + NaCl treated plants vs the control plants. In stems, CaCO3 and NaCl applied

186

separately had slight decreased and no effect, respectively, but an increase was observed when

187

they were supplied together (16.4%).

188 189

Fatty acids. The abundance of the different fatty acids of P. oleracea was analyzed (Table 1).

190

In leaves, an increase was observed for all treatments, especially the salinity treatments (3-

191

fold relative to the control leaves in NaCl-treated plants and nearly 4-fold for NaCl+CaCO3).

192

The total fatty acid concentration was also increased in stems by all treatments, but the

193

concentration was lower than in leaves. The increase was similar and high in both treatments

194

with salinity (7-8 fold). But in roots, a marked decrease was observed. In this case, both

195

treatments with salinity (NaCl± CaCO3) produced greater decreases.

196

Regarding the individual fatty acids in leaves, C16:1 and C18:1 showed an increase only

197

with the combined treatment of NaCl+CaCO3. The fatty acid C18:2 decreased in response to

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the NaCl treatment but showed a high increase with CaCO3, while C18:3 was increased by all



199

the treatments (to a very great extent by NaCl+CaCO3). A reduced concentration was found

200

with all treatments for C20:0 and with the NaCl and CaCO3 treatments for C20:4. But, the

201

NaCl+CaCO3 treatment increased the concentration of this latter fatty acid.

202

The fatty acid C16:1 did not appear in any of the treatments, neither in stems nor in roots. Also,

203

C20:0 occurred just with the CaCO3 treatment, in both roots and stems. In the latter organs,

204

C18:1, C18:2, and C18:3 were increased in response to treatments, compared to the control.

205

Greater increases were observed with NaCl and the combined treatment of NaCl+CaCO3 than

206

with CaCO3. The increases were 4, 4, and 3-fold in C18:1, C18:2, and C18:3, respectively, with

207

CaCO3, 7, 5, and 6-fold under salinity, and 10, 7, and 7-fold with the combined treatment.

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This significant increase in the concentrations was accompanied by a significant decline of

209

those fatty acids in roots, with all the treatments. While C20:4 was absent in the control stems,

210

it was found for the different treatments. A significant decrease in C20:4 occurred in roots with

211

all treatment, even disappearing under the NaCl+CaCO3 treatment.

212

In the chloroplasts (Table 2), all fatty acids showed an increase with all treatments,

213

compared with control values. The increase was greatest for C18:3; it’s concentration was 10-

214

times higher with the NaCl+CaCO3 treatment, 4.5-times higher with NaCl, and 6-times higher

215

with CaCO3 compared to plants produced under control conditions. All the increases in the

216

rest of the individual fatty acids were similar for the distinct treatments: they were highest for

217

the NaCl+CaCO3 treatment, lower for NaCl, and much lower for CaCO3. The highest

218

increases in the double bond index (DBI), 6 and 15-fold, respectively, were produced with the

219

NaCl and NaCl+CaCO3 treatments. Also, the ratio of unsaturated to saturated fatty acids

220

(UFA/SFA) was increased in all treatments, compared to the control, with the greatest

221

increases by DBI, 6 and 15 times, respectively, occurring with NaCl+CaCO3 followed by the

222

NaCl and CaCO3 treatments.

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Phenols. Figure 5 shows the abundances of the phenolic compounds, expressed in mg 100g-1

225

DW (sinapic acid and chlorogenic acid derivatives and flavonol glycosides), the UV-Vis

226

spectra, retention time, and comparison with external standards made possible the tentative

227

identification. The leaves were the organ richest in phenolic compounds. A negative effect of

228

NaCl, independently of its combination with CaCO3, was observed for sinapic acid and

229

chlorogenic acid in leaves, stems. However, there were no significant differences in these

230

organs between control and CaCO3-treated plants. In roots, the treatment did not alter the

231

concentrations of phenolic compounds except an increase in sinapic acid with CaCO3 and

232

NaCl+ CaCO3 plants. Also in roots, chlorogenic acid derivatives were decreased with NaCl+

233

CaCO3 treatments.

234

There were no significant differences in the flavonol glycosides concentration in

235

leaves and stems among the treatments except an increase in stems of NaCl+ CaCO3 plants. It

236

is noteworthy that there was no trace of flavonol glycosides in P. oleracea roots.

237 238

Mineral nutrients. The mineral nutrient analysis of the leaves, stems, and roots of P.

239

oleracea plants grown under the different treatments is shown in Figure 6.

240

Sodium was greatly and significantly increased in all organs by both treatments that included

241

NaCl (NaCl and NaCl+CaCO3). The concentration of K+ showed a slight decrease in leaves

242

stems and roots with all treatments, relative to the control, except the concentration in stems

243

with CaCO3 which was not altered. The concentration of Ca2+ in leaves only increased in

244

CaCO3-treated plants, relative to control plants. In stems, a decreased appeared in both

245

treatments with NaCl. In roots; a strong decreased only appeared with application of NaCl,

246

compared to control. The concentration of P did not change in leaves and stems, except for a

247

slight decrease in the latter with the NaCl and NaCl+CaCO3 treatments. In roots, an increase

248

with NaCl appeared together with a decreased with CaCO3. The concentrations of Mg2+ were



249

increased in the leaves with all treatments. In stems, an increase was only observed with

250

CaCO3 treatment but in roots, this treatment produced a decrease. The concentration of S in

251

leaves and stems were not altered by treatment except by NaCl+CaCO3 treatment that

252

produced an increase. However, in roots there was a decrease with all treatments compared

253

with control.

254

All the micronutrients were also analyzed, but only Fe2+, Zn2+, and Mn2+ were altered

255

by the treatments (Figure 7). The concentration of Fe2+ in leaves was only decreased

256

significantly by the NaCl+CaCO3 treatment. In stems there were no significant differences in

257

any of the treatments, but in roots, an increased appeared with NaCl treatment, but a decrease

258

was observed with CaCO3 and NaCl+CaCO3 treatments. The concentration of Zn2+ was

259

increased in leaves by both NaCl treatments. In stems and roots the results were similar than

260

for Fe2+. The Mn2+ concentration was significantly increased in leaves by all treatments.

261

However, it increased in stems by both NaCl treatments and decreased in roots with all

262

treatments.

263 264

DISCUSSION

265

The fresh and dry weights of CaCO3-treated plants were higher than those of control plants.

266

However, the growth of NaCl-treated plants was significantly reduced and the treatment

267

combining CaCO3 and NaCl produced a more negative effect than salinity alone. This effect

268

has been reported already

269

The reduction in fresh weight that is shown in our results for P. oleracea exposed to salinity is

270

typical glycophytic behavior. The reduction of plant tissue production by salinity has been

271

observed also in P. oleracea plants exposed to an irrigation solution with ECi greater than 6.8

272

dS m-1 27.

26

in tomato plants treated with 100 mM NaCl and 2 mM CaCO3.


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It has been reported that application of CaCO3 could have a negative effect on plants

274

due to the high pH (7) caused by the dissolved CO32-, which could affect nutrient uptake. The

275

limitation of growth by alkalinity

276

nutrient uptake. But in our plants, the fact that the pH of the nutrient solution was controlled

277

throughout the experiment could diminish that possibility. Furthermore, the application of

278

both treatments together did not have a positive effect on growth compared with the

279

application of NaCl alone, probably due to an antagonistic effect on one or more of the

280

physiological parameters.

28

has been reported to be related to interference with

281

The results for chlorophylls a and b (Figure 2) show that only when both treatments

282

(NaCl and CaCO3) were applied together did a significant decrease in their abundance appear.

283

Although a decrease in chlorophyll is usually related to growth, in the case of P. oleracea it

284

only occurred in NaCl+CaCO3-treated plants, not in those exposed to NaCl alone - for which

285

growth was also low. This could be related to the modification of CO2 fixation that P.

286

oleracea experiences under stress conditions. It has been reported that P. oleracea evolved

287

from an ancestor with the C4 photosynthetic metabolism, enabling it to be more tolerant of

288

drier habitats where CO2 is limited due to stomatal closure 29. Also, it has been observed that

289

any CO2 assimilated by the chloroplasts would be derived from re-fixation of CO2 produced

290

by respiration, rather than by intake of atmospheric CO2, inducing the plants to perform CAM

291

under water stress

292

indicate highly efficient photosynthesis in plants treated with NaCl+CaCO3 and high

293

synthesis of carbon-based molecules, to the detriment of growth. This could be related as well

294

with the fact that carbon is also taken up by roots in the form of carbonate. Although dark

295

fixation of inorganic carbon is not related to passive uptake of inorganic carbon as CO32- by

296

roots, the higher concentrations of organic compounds in cells must be a consequence of the

297

incorporation of carbon into the carbon skeletons that are necessary for conversion of NH4+

30

. Although this should be investigated more deeply, the results could



26

298

into amino acids

. For the CaCO3 and NaCl-CaCO3 treatments, the plant protein

299

concentration was significantly higher (particularly in the latter) than in the control and NaCl-

300

exposed plants. Therefore, although in control plants the protein concentration was slightly

301

lower than reported previously

302

increase in both treatments containing CaCO3 (46%) is an important feature.

27

, the reduction due to salinity was similar, but the high

303

However, the fact that the osmotic potential in leaves and roots decreased with all the

304

treatments, especially those containing NaCl, in comparison with control plants, shows that

305

osmotic adjustment is the predominant response and that the organic and inorganic

306

compounds involved are independent of the type of photosynthesis performed by the plants.

307

It has been reported that P. oleracea is one of the most abundant sources of plant ω-3

308

fatty acids, which have a potential beneficial effect on human health. Furthermore, the

309

concentration of fatty acids and ω-3 can be altered by environmental stress conditions 31. In

310

our experiment, the levels of all individual unsaturated C18 fatty acids were increased in

311

leaves and stems by the NaCl, CaCO3, and NaCl+CaCO3 treatments, giving a high increment

312

in the abundance of total fatty acids. Teixeira et al.

313

desaturase (FAD7) gene under stress conditions, suggesting a role for this gene in the

314

response to abiotic stress, such as that of the present work.

32

reported activation of the fatty acid

315

Fatty acids form part of membranes, which are themselves an important source of

316

signaling molecules. Unsaturated fatty acids are essential components required for normal

317

cellular function, having roles ranging from control of membrane fluidity to acting as signal

318

molecules 33. This signaling derives from the fact that fatty acids are important regulators in

319

plants

320

jasmonate are a rich source of signaling molecules involved in the response of plants to stress.

321

The total fatty acid concentration in P. oleracea has been reported to be higher in

322

leaves than in stems 31. Also, the concentration of linolenic acid (C18:3) accounts for 60% of

34

. In fact, fatty acid-derived volatiles such as aldehydes, jasmonate, and acid methyl


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323

total fatty acids in leaves, and 10-25% in stems 32. In our experiment, for the control plants,

324

the concentration of total fatty acids in leaves was 10-times higher than in stems; this was

325

reduced to 3.5 to 4-times higher by the treatments, although both concentrations were much

326

higher. Furthermore, the proportion of linolenic acid was much higher than the values

327

reported in the literature, reaching 90-98%, independently of the treatment. The concentration

328

of fatty acids and proportion of linolenic acid in P oleracea have been reported to change

329

according to the cultivar and environmental factors 35. So, the fact that in our samples the total

330

fatty acids constituted 25-40% of the total lipids 27 and the high linonenic acid levels make P.

331

oleracea interesting for the pharmaceutical and nutraceutical industry. Even if we take into

332

account the lower rate of growth in NaCl and NaCl+CaCO3-treated plants, the production of

333

total fatty acids was 16.5 and 12.3 mg plant-1, respectively, while it was 8.3 mg plant-1 in

334

control plants. Also, the CaCO3-treated plants had a higher production of fatty acids, (14.3 mg

335

plant-1) than control.

336

In chloroplasts, photosynthesis provides an endogenous source of fixed carbon that is

337

utilized by acetyl-CoA for synthesis of fatty acids. This pathway occurs within the plastids

338

and the fatty acids are used for membrane formation and storage in plastids 36. Our fatty acids

339

results for the chloroplasts are consistent with the data obtained for leaves, as the treatments

340

with NaCl and NaCl+CaCO3 gave the highest levels. These results suggest a role for the

341

chloroplasts in the biosynthesis of fatty acid-derived signaling molecules 33.

342

Here, the natural antioxidants of P. oleracea were expressed as the concentration of

343

phenolic compounds (chlorogenic and sinapic acid derivatives and flavonoids) in leaves,

344

stems, and roots. A decreased abundance of phenolic compounds with salinity has been

345

reported previously in other plants like Brassica oleracea 37. However, induction of secondary

346

metabolic pathways by salinity, leading to an increase in phenolic compounds, has also been



347

reported 38. This indicates that polyphenols could be involved in the defense against stresses,

348

especially oxidative stress.

349

The concentrations of the different mineral nutrients changed depending on the plant

350

organ and the treatment. The concentration of Na+ was higher in shoots than in roots, a

351

normal compartmentation of potentially toxic ions

352

photosynthetic apparatus

353

reflecting the fact that, in general, under saline conditions, Na in the growth medium interacts

354

with Ca, lowering its uptake. The addition of extra Ca as CaCO3 compensates this effect 13.

37

, but this may not avoid damage to the

39

. The Ca concentration was lowest in NaCl-treated plants,

355

Considering the micronutrients, the high concentrations of Fe and Zn in roots indicate

356

a very low mobility of these elements, but there were no changes consistent with the

357

treatments, as reported previously with salinity 27. The Mn concentration was higher in leaves

358

than in roots, with no differences among treatments.

359

In conclusion, P. oleracea has the potential to be a crop in the Mediterranean area,

360

with high interest for human consumption in view of its nutritional qualities. Salinity affected

361

the growth and yield of P. oleracea plants, although the high increase in fatty acids suggests

362

that it is worth cultivating under severe stress conditions like those caused by saline or highly

363

calcareous (alkaline) soils. Also, its growth in a calcareous medium provides high nutritional

364

quality with no growth reduction. The high concentration of fatty acids obtained, together

365

with high levels of proteins and mineral nutrients, makes this plant very suitable for

366

cultivation under extreme environmental conditions.

367 368 369 370

ACKNOWLEDGEMENTS The authors thank Dr. D. Walker, for correction of the written English in the manuscript.

371


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Page 17 of 31

372


FUNDING SOURCES This work was funded by the Consejo Superior de Investigaciones Científicas

373 374

(COOPA20120).

375 376 377 378 379

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482 483 484 485 486 487 488 489 490 491 492 493 494 495 496


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497

FIGURE CAPTIONS

498 499 500 501 502

Figure 1: (A) Fresh weight (FW) and (B) dry weight (DW) of Portulaca oleracea grown under the effects of salt stress and alkaline stress. Means of four replicates ± SE. Means followed by different letters are significantly different (P ≤ 0.05), as determined by one-way ANOVA.

503 504 505 506 507 508 509 510 511 512 513 514

Figure 2: Osmotic potential in Portulaca oleracea grown under the effects of salt stress and alkaline stress. Means of four replicates ± SE. Means followed by different letters are significantly different (P ≤ 0.05), as determined by one-way ANOVA.

515 516 517 518 519

Figure 5: Phenolic compound (A) sinapic acid, (B) chlorogenic acid, (C) Flavonol, in Portulaca oleracea grown under the effects of salt stress and alkaline stress. Means of four replicates ± SE. Means followed by different letters are significantly different (P ≤ 0.05), as determined by one-way ANOVA.

520 521 522 523

Figure 6: Macronutrient concentrations (A) sodium, (B) potassium, (C) calcium, (D) phosphorus, (E) magnesium, (F) Sulphur, in Portulaca oleracea grown under the effects of salt stress and alkaline stress. Means of four replicates ± SE. Means followed by different letters are significantly different (P ≤ 0.05), as determined by one-way ANOVA.

524 525 526 527 528 529 530

Figure 3: Chlorophylls a (A) and b (B) in Portulaca oleracea grown under the effects of salt stress and alkaline stress. Means of four replicates ± SE. Means followed by different letters are significantly different (P ≤ 0.05), as determined by one-way ANOVA. Figure 4: Total proteins content in Portulaca oleracea grown under the effects of salt stress and alkaline stress. Means of four replicates ± SE. Means followed by different letters are significantly different (P ≤ 0.05), as determined by one-way ANOVA.

Figure 7: Micronutrient concentrations (A) iron, (B) zinc, (C) magnesium, in Portulaca oleracea grown under the effects of salt stress and alkaline stress. Means of four replicates ± SE. Means followed by different letters are significantly different (P ≤ 0.05), as determined by one-way ANOVA.



Page 22 of 31

TABLES Table 1: Fatty acid content in Portulaca oleracea grown under the effects of salt stress and alkaline stress. Means of four replicates ± SE. Means followed by different letters are significantly different (P ≤ 0.05), as determined by one-way ANOVA. Fatty acids (µg g-1 FW) Leaves

Stems

Roots

C16:1

C18:1

C18:2

C18:3

C20:0

C20:4

Control

0.009±0.001 a

0.021±0.005 a

0.095±0.005 a

NaCl CaCO3 NaCl+CaCO3 Control NaCl

0.006±0.000 a * 0.033±0.003 b * *

0.029±0.001 a 0.023±0.004 a 0.116±0.008 b 0.007±0.001 a 0.052±0.001 c

0.245±0.001 c 0.127±0.002 b 0.535±0.027 d 0.043±0.003 a 0.268±0.001 c

CaCO3 NaCl+CaCO3 Control

* * *

0.030±0.002 b 0.075±0.001 d 0.072±0.001 b

NaCl CaCO3

* *

NaCl+CaCO3

*

33.769±3.379 a

0.014±0.005 c

1.243±0.431 a

35.15±3.87 a

96.07±1.62 a

92.994±2.448 b 42.811±7.399 a 128.076±0.279 c 3.708±0.147 a 23.308±0.284 c

0.005±0.000 b * 0.002±0.000 a * *

0.985±0.127 a 0.441±0.050 a 2.376±0.485 b * 1.318±0.075 b

94.26±2.58 b 43.40±7.48 a 131.14±0.81 c 3.76±0.15 a 24.95±0.36 c

98.65±0.12 b 98.64±0.14 b 97.66±0.47 b 98.71±0.10 b 93.43±0.45 a

0.177±0.001 b 0.302±0.009 c 0.478±0.018 c

11.526±0.711 b 25.582±0.423 c 10.519±1.240 c

0.028±0.002 a * *

0.480±0.139 a 2.186±0.243 c 1.976±0.120 c

12.24±0.85 b 28.14±0.68 d 13.05±1.38 c

94.16±1.05 ab 90.89±0.83 a 80.63±1.22 a

0.013±0.000 a 0.021±0.004 a

0.070±0.008 a 0.151±0.007 b

1.434±0.083 a 3.214±0.248 b

* 0.063±0.001 a

0.471±0.060 a 0.789±0.009 b

1.99±0.15 a 4.24±0.27 b

72.11±3.77 a 75.85±0.87 a

0.016±0.001 a

0.083±0.003 a

1.860±0.081 a

*

*

1.96±0.09 a

94.95±0.50 b


TOTAL

Linolenic acid (%)

Page 23 of 31


Table 2: Chloroplast fatty acid content in Portulaca oleracea grown under the effects of salt stress and alkaline stress. Calculation of double bond index (DBI) and the ratio of unsaturated to saturated fatty acids (UFA/SFA) is presented. Means of four replicates ± SE. Means followed by different letters are significantly different (P ≤ 0.05), as determined by one-way ANOVA.

Fatty acids (µg g-1 lipids) C16:1

C18:1

Control

0.17±0.06 a

0.04±0.02 a

NaCl

1.12±0.12 b

CaCO3 NaCl+CaCO3

C18:2

C18:3

C20:0

C20:4

0.65±0.45 a

52.28±18.21 a

0.01±0.00 a

0.24±0.00 a

53.3±18.7 a

0.16±0.02 b

1.13±0.15 ab

312.78±32.0 c

0.05±0.02 a

3.05±1.20 b

318.6±33.7 c

954.8±101.1 b

4328.1±820.3 b

1.07±0.22 b

0.21±0.05 c

1.15±0.26 ab

234.75±46.67 b

0.10±0.02 b

3.62±0.18 b

241.3±47.1 b

723.18±140.3 b

3130.2±212.6 a

1.39±0.13 c

0.26±0.04 c

1.58±0.14 b

507.67±68.81 d

0.09±0.01 b

4.40±0.63 c

515.7±68.4 d

1546.2±205.3 c

6510.9±126.4 c


Total

DBI

158.9±55.6 a

UFA/SFA

24447.7±0.90 d


FIGURES Figure 1:


Page 24 of 31

Page 25 of 31


Figure 2:



Figure 3:


Page 26 of 31

Page 27 of 31


Figure 4:



Page 28 of 31

Figure 5:

Sinapic acids (mg/100gDW)

120 100

Leaves d d

Stems

Roots

A cd

80 60

d c

b

b

40

c

b

ab

a

ab

20 0 100

Chlorogenic acids (mg/100gDW)

Leaves

Stems

Roots

B

80 60 40

d

d bc c

20

c

c

bc

a

ab b

a

a

0 100

Flavonols (mg/100gDW)

Leaves

Stems

Roots

C

80 60

d

cd

d

c

40

b

20 0

a

a

Controla Control

NaCla NaCl

a CaCO3a CaCO 3

Treatments


a NaCl+CaCO3 NaCl + CaCO 3

Page 29 of 31


Figure 6:

Leaves

Stems

3

Roots

A

h h

g

+

d

6

Roots

ab

Leaves

i g

f

e

d

c

de

b

c a b

B

h

1

3 0 3

Stems

i

2

e

9

Leaves

f

+

Na (mM)

12

K (mM)

15

ab b

a

a b

Stems

0 0,4 0.4

Roots

C

Leaves

Stems

Roots

D

d

d

PP(mM) (mM)

2

d

2+

c

0 0,4 0.4

c

Leaves

Stems

0.3 0,3

f

Roots

b

b b

b

e c d a

Stems

Roots

0.3 0,3 c

b

Leaves

c b d d c

c

F

d

0,2 0.2

0 0,4 0.4 E

e

e

0,1 0.1

b c c

b a

ab a ab

S (mM)

2+

Mg (mM)

e

1 b b

f

0.2 0,2

b b a

0,1 0.1

e

S (mM)

Ca (mM)

0.3 0,3

0.2 0,2 d

0.1 0,1

c b

b a

a

cd b

b

bc

ab

0 0,0

0,0 Control

Control

NaCl

NaCl

CaCO3

CaCO3

NaCl + CaCO3

NaCl + CaCO3

Control Control

Treatments

NaCl NaCl

CaCO3 CaCO 3

Treatments


NaCl + NaCl + CaCO 3 CaCO3


Page 30 of 31

Figure 7:

Leaves

Stems

Roots

e

A

20 d

2+

Fe (µM)

30

10

0 4

c b

b a

b a Leaves

Stems

c a a

a

Roots

B

3

d

2

c

Zn

2+

(µM)

e

1

b a

2+

Mn (µM)

0 2,0

a

a

Leaves

1,5

Stems

f

b

a a

b a

Roots

f

C

e

e d

1,0 0,5

c a

c

b

a

b

b

0,0 Control Control

NaCl NaCl

CaCO3 CaCO 3

Treatments


NaCl+ + CaCO3 NaCl CaCO3

Page 31 of 31


TOC Graphic 275x190mm (96 x 96 DPI)


Interaction of Salinity and CaCO3 Affects the Physiology and Fatty

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