Fruit Ripening

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Biochemistry and Cell Wall Changes Associated to Noni (Morinda citrifolia L.) Fruit Ripening Wendy Guadalupe Cárdenas-Coronel, Armando Carrillo-López, Rosabel Vélez-dela-Rocha, John M. Labavitch, Manuel A. Báez-Sañudo, José Basilio Heredia, José de Jesús Zazueta-Morales, Misael O. Vega-García, and JOSEFA ADRIANA SAÑUDO J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03681 • Publication Date (Web): 02 Dec 2015 Downloaded from http://pubs.acs.org on December 6, 2015

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Journal of Agricultural and Food Chemistry

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Biochemistry and Cell Wall Changes Associated to Noni

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(Morinda citrifolia L.) Fruit Ripening

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Wendy G. Cárdenas-Coronel 1, Armando Carrillo-López 1**, Rosabel Vélez de la Rocha 2, John M. Labavitch 3, Manuel A. Báez-Sañudo 2, José B. Heredia2, José J. Zazueta-Morales 1, Misael O. Vega-García 1, J. Adriana Sañudo-Barajas 2*

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1

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2

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3

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*Corresponding author ([email protected])

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**Additional corresponding author ([email protected])

Programa Regional de Posgrado en Biotecnología, Facultad de Ciencias Químico-Biológicas, Universidad Autónoma de Sinaloa, 80013, Culiacán, México. Centro de Investigación en Alimentación y Desarrollo, A.C. Unidad Culiacán. CP 32-A. 80110, Culiacán, Sinaloa, México. Plant Sciences Department, University of California, Davis, CA 95616, USA.

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ACS Paragon Plus Environment


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Abstract

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Noni fruit was harvested at five ripening stages, from stage dark-green to

31

translucent-grayish. Quality and compositional changes were determined at

32

different stages. The noni fruit ripening was accompanied by acidity and soluble

33

solids accumulation but pH diminution; whereas the softening profile presented

34

three differential steps named as early (no significant softening), intermediate

35

(significant softening) and final (dramatic softening). At the initial stage the

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extensive depolymerization of Water Soluble Fraction and significantly increase of

37

pectinases activities did not correlate with the slight reduction of firmness. The

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intermediate showed an increment of pectinases and hemicellulases activities. The

39

final stage was accompanied of the most significant reduction in the yield of AIS as

40

well of the composition of uronic acids and neutral sugars, pectinases increase

41

their activity and depolymerization of hemicellulosic fractions. The noni ripening is

42

a process conduced by the coordinated action of pectinases and hemicellulases

43

that promote the differential dissasembly of cell wall polymers.

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KEYWORDS: Noni, softening, cell wall degradation, cell wall degrading enzymes.

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Introduction

51

The noni (Morinda citrifolia L.) fruit is produced by a small leafy, perennial

52

tree or shrub originated from south-eastern Australia and Asia. The noni fruit has

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been recognized worldwide as a folk medicine due to its health benefits. The noni

54

fruit, technically known as a syncarp, is oval shaped (5-10 cm long, 3-4 cm wide)

55

and fleshy. Its, color ranges from green to pale yellow, turning almost white at the

56

time of harvest. When fully ripe, the flesh is juicy, bitter, pale yellow or white,

57

gelatinous, develops a foul smell. At the end of the ripening period the fruit softens

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rapidly, a transformation that is influenced by fruit biochemical changes1-3. Due to

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their nutraceutical properties noni fruit have increased in commercial importance

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and been subjected to multiple investigations. Noni has been documented as a

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polyphenol-rich fruit that may help to alleviate several chronic diseases such as

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arthritis and diabetes; effects that may reflect its content of anti-oxidant

63

compounds3-5. Noni juice is in high demand as an alternative medicine for the

64

treatment of diseases such as cancer, atherosclerosis, diabetes and ulcers.

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However there is little information regarding the biochemistry and postharvest

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behavior of noni fruit6,7,8.

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In other parenchymatous fruits, ripening is accompanied by a progressive

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softening probably caused by enzymatic modifications of primary cell wall

69

polysaccharides that parallel changes in the ripening fruit´s texture. These changes

70

generally

involve

solubilization

and

depolymerization


of

pectins

and


and

a

subsequent

disruption

of

cell

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hemicelluloses,

wall

interpolymer

72

associations9,11. However, the patterns of changes in cell walls of ripening noni fruit

73

and alterations of the fruit´s cell wall-modifying enzymes (CWMEs) involved in this

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disruptive process are not yet known. Therefore, this study was designed to get

75

basic information about the physical, chemical and biochemical changes that occur

76

during noni fruit ripening, with a major focus on changes in the cell wall polymer

77

composition modifications and activities of CWMEs associated with noni softening.

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Material and Methods

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Fruit selection

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Noni fruit were harvested from a commercial orchard in Tepic, Nayarit,

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México at five ripening stages defined by traditional classification of skin color

83

(subjective evaluation7) in association with the firmness 1 (dark-green, very hard),

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2 (green-yellow, very hard), 3 (pale yellow, very hard), 4 (pale yellow, fairly hard)

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and 5 (translucent-grayish, soft). The orchard received in average annual rainfall of

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1,121 mm and temperature of 21.1 °C while in general wind speeds average 8

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mph12. After washing and classification, fruit quality measurements were recorded,

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the peel and seeds were removed, and a section of fruit was frozen for chemical or

89

biochemical assays (-20 °C or liquid nitrogen and stored at -80 °C, respectively).

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Chemical composition

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The methods of AOAC (1998) were used to measure pH, titratable acidity

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and total soluble solids. For pH and titratable acidity (% citric acid), 10 g of sample

94

were homogenized in 50 mL of distilled water and then filtered, 20 mL of this

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extract was used and titrated with 0.1 N NaOH to a pH of 8.2. A digital pH meter

96

(Model 140, Corning, Woburn, MA USA) was used for the initial determination of

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pH. The total soluble solids content (TSS) was determined using an aliquot from

98

the homogenized and filtered slurry residue with a refractometer (Abee, Leica Mark

99

II, NY, USA) and results were expressed in ° Brix.

100

Fruit quality

101

Fuit firmness was determined as the maximum force required to penetrate

102

the pulp (without peel), to 1 cm at a speed of 5.2 mm s-1; at points around the fruit

103

equator. A penetrometer (Model DFIS-50, Chatillon, Greenwich, USA) was used,

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and the results were expressed in Newtons. The color of the peel was evaluated in

105

7 fruits with a color meter (CM-2600d, Minolta Co., LTD, Japan). The results were

106

reported as Hunter Lab scale and converted to chroma (C) and hue angle (ºHue).

107 108

Cell wall extraction

109

Equatorial sections of 3 fruit from each ripening stage were obtained; the

110

skin and seeds were removed under cold conditions and 5 g of flesh were finely

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chopped and boiled for 30 min in 250 ml of ethanol at 80%, homogenized for 40 s

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at 13,500 rpm (T25 digital Ultra-Turrax), filtered with glass fiber filter paper (GF/A)



113

and washed for 20 min under magnetic stirring (in consecutive order) with 20 mL of

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different solvents, 80 % ethanol (twice), methanol-chloroform (1:1) and acetone13.

115

The residue was oven-dried at 40°C, weighted, ground to 40 mesh (Model

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3383L10, Thomas Scientific Mill) and stored in a desiccator until use. The residue

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was considered the cell wall and identified as alcohol insoluble solids (AIS) for

118

further evaluations.

119

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Cell wall composition analysis

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Two mg samples of AIS in concentrated 1 mL H2SO4 were continuously

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stirred, at intervals of 10 min 1 mL H2SO4, 1 mL H2O, and 7 mL H2O were added

123

until complete hydrolysis, keeping the reaction under ice bath. Uronic acid (UA)

124

content was assayed as described by the spectrophotometric method of Ahmed

125

and Labavitch14. Galacturonic acid (GalA, Sigma) was used for the standard curve

126

and calculations. For total sugars (TS), three mg of AIS were hydrolyzed with 3 mL

127

of 67 % H2SO4, stirred for 4 h and aliquots were assayed by the anthrone reaction

128

according to Yemm and Willis15; TS concentrations were calculated from a glucose

129

(Sigma) standard curve. For non-cellulosic neutral sugar (NS) composition, two mg

130

aliquots of dried AIS were hydrolyzed with 2 mol L−1 trifluoroacetic acid (TFA,

131

121°C for 1 h). The TFA-soluble fractions were recovered by centrifugation (clinical

132

centrifuge, ca, 2000×g) and free sugars recovered in the supernatant and three

133

methanol washes of the TFA-insoluble pellet were combined into a single sample

134

for chromatographic analysis (below). The washed pellets were dissolved in 67 %


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H2SO4 and assayed for cellulose using the anthrone reagent; a 67 % H2SO4-

136

solution of cellulose powder (CF11, Whatman) was used as a standard curve for

137

calculations. The pooled TFA-soluble supernatants were dried and converted to

138

alditol acetates according to Blakeney et al.16; and were injected into a gas

139

chromatograph (Model 3800, Varian Inc., Walnut Creek, CA, USA) with a 30m ×

140

0.25mm i.d. capillary column (model DB-23, J & W Scientific) as described17,18.

141

Results were expressed as mg100 g- fresh weight of rhamnose (Rha), fucose

142

(Fuc), arabinose (Ara), xylose (Xyl), mannose (Man), galactose (Gal), glucose

143

(Glc); calibration was made with known concentrations of sugars and myo-inositol

144

as an internal standard (Sigma).

145

Cell wall fractionation

1

146

The AIS was fractionated by solubility in water, EDTA in 0.05 mol L−1

147

sodium acetate pH 6.5, 0.05 mol L−1 Na2CO3 containing 0.02 mol L−1 NaBH4, 4 and

148

24 % KOH as described in Sañudo-Barajas et al.18. The fractions were obtained by

149

suspending 300 mg of the AIS powder in 20 mL of the extraction solvent and

150

stirring for 12 h at room temperature. Solubilized materials were separated by

151

centrifugation at 8500 ×g until the remaining insoluble material was fully pelleted.

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The supernatant was recovered and the pellet re-extracted with 10 mL of the same

153

solution, stirred for 4 h, and spin-pelleted. Corresponding supernatants were

154

pooled and stored for further filtration, neutralization, desalting and freeze-drying

155

(Freezone 18, Labconco, KC, Missouri, USA). The water-soluble fraction was

156

designated WSF, the EDTA-soluble fraction as ESF, the Na2CO3 soluble fraction

157

as SCSF, and the 4% KOH- and 24% KOH-soluble fractions as 4KSF and 24KSF,



158

respectively. GF/A (Whatman glass fiber filter) was used to retain insoluble solids;

159

glacial acetic acid was used to neutralize solubilized wall materials. Membranes

160

(6,000 MWCO; Spectrum) were used for desalting (4 times against distilled water

161

and changes each 8 h) to remove salts from ESF, SCSF, 4KSF and 24KSF. An

162

aliquot of the fractions was assayed in triplicate for UA and TS or in duplicate for

163

NS composition, as described previously. Finally, the samples were freeze-dried

164

and stored at -20°C until use. The SCSF was discarded due to low recovery

165

(insufficient sample to obtain useful UA and TS values), thus adding an error

166

(relativity small) to our total wall composition description.

167 168

Size exclusion chromatography (SEC)

169

Samples were lyophilized in freeze-dryer and then were dissolved in their

170

respective running buffer and fractionated on a SEC using a column (1.5 cm × 80

171

cm) packed with Sepharose CL-6B (Amersham-Biosciences). Dialyzed samples of

172

the WSF and ESF were dissolved in and eluted with NH4-acetate buffer (0.2 mol

173

L−1, pH 5.2), filtered through GF/A and chromatographed. NaOH (0.1 mol L−1) was

174

used as the solvent and eluant for SEC of the 4KSF and 24KSF. The size

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distributions were profiled by collecting fractions of 1.8 mL at a flow rate of 48 mL

176

h−1 (Fraction Collector Model 2110, Bio-Rad, USA). Fractions were assayed for UA

177

and NS, as described in Sañudo-Barajas et al.18. The column´s void and total

178

included volumes (Vo and Vt, respectively) were resolved with 2000 KD dextrans

179

and Glc (Sigma) chromatographed as described.

180


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Cell wall modifying enzyme isolation and activity assays

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Fruit mesocarp without peel and seeds (3 fruits per ripening stage) were

183

used for enzyme extraction according to Sañudo-Barajas et al.18 at 4°C. Briefly, 10

184

g of flesh were homogenized in 20 mL of pre-extraction buffer [0.05 mol L−1 sodium

185

acetate (HAc-NaAc) pH 5, containing 10 g L−1 polyvinylpolypyrrolidone and 0.1%

186

Triton X-100], centrifuged at 10,000 ×g and 4°C for 20 min (Allegra 64R, Beckman

187

Coulter, Brea, CA, USA) and the supernatant was collected. The pre-extraction

188

step was replicated once. The pellet was then subjected to saline resuspension

189

with 20 mL of the extraction buffer under cold stirring for 1 h (0.05 mol L−1 HAc-

190

NaAc pH 5, containing 10 g L−1 polyvinylpolypyrrolidone and 1 mol L−1 NaCl). The

191

supernatant was collected, filtered twice through GF/A, dialyzed (12,000 MWCO,

192

Spectrum) exhaustively against 0.05 mol L−1 NaAc pH 5 and used for assays of

193

enzymatic activities. The β-galactosidase (β-gal) activity was assayed according to

194

Pressey19, incubating every 10 minutes and calculating the rate of p-nitrophenol

195

liberation.

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galactopyranoside (0.8 g L−1 in HAc-NaAc 0.025 mol L-1, pH 4.5) and 300 µL

197

enzyme extract; free p-nitrophenol was determined by adding 1 mL of 1 mol L−1

198

Na2CO3 to 200 µL aliquots of the reaction mix; calculations were obtained from the

199

absorbance at 420 nm (Spectrofotometer Cary 1E, Varian, USA) and estimation

200

from a standard curve of p-nitrophenol. One unit of β-Gal activity was defined as

201

the amount of enzyme that liberates 1 µmol of free p-nitrophenol per min.

The

reaction

mixtures

contained

1

mL

p-nitrophenyl-β-D-

202

Polygalacturonase (PG) and galactanase (Gase) activities were measured

203

in reaction mixtures containing 1 mL enzyme extract and 1 mL polygalacturonic



204

acid (5 g L−1, MP Biomedicals) or 1 mL potato galactan (5 g L−1, Megazyme), with

205

both substrates prepared in 0.05 mol L−1 HAc-NaAc buffer pH 5.0. Kinetic of

206

reducing ends liberation were determined on 200 µL aliquots sampled from the

207

reaction mixture at progressive incubation times, every 10 or 20 minutes for PG or

208

Gase respectively, as described by Gross20. Reducing sugars liberated were

209

measured at 272 nm and calculated using GalA (PG activity) or Gal (Gase activity)

210

standards; a unit of PG and Gase activity was defined as the amount of enzyme

211

that released 1 µmol of reducing groups per min. Azurine cross-linked substrates

212

(Megazyme) were used for xyloglucanase (XGase), xylanase (Xase) and

213

rhamnogalacturonase (RGase) activity assays (AZCL-xyloglucan, arabinoxylan,

214

and rhamnogalacturonan respectively). Two mL of suspensions containing 0.5 g

215

AZCL-substrate L−1 (xyloglucan, arabinoxylan or rhamnogalacturonan type I) in

216

HAc-NaAc buffer and 1.3 mL of enzyme extract were incubated at 37 °C. At

217

selected intervals, the reaction mixtures were centrifuged and the increment in

218

supernatant absorbance at 595 nm was determined. A unit of XGase, Xase and

219

RGase activity was defined as the amount of enzyme that produces ∆ 1mU of

220

absorbance at 595 nm per min.

221

222

Data analysis

223

The data obtained were statistically analyzed using analysis of variance (ANOVA)

224

and comparison of means at significance values P≤0.05 was tested with Tukey’s

225

multiple range with the statistical program Minitab version 15. The molecular size

226

was presented in elution profiles and compared by changes in distribution.


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227

228

229

Results and Discussion

230 231

Fruit quality and chemical composition of noni fruit

232

During fruit ripening flavor changes usually occur in part, because of the

233

conversion of storage polysaccharides into soluble sugars, loss of tannins and

234

other astringent compounds, and reduction of acidity due to degradation and

235

conversion of organic acids. In this study these changes in noni fruit flavor were

236

indirectly measured as titratable acidity (TA), pH and total soluble solids (TSS). As

237

shown in Figure 1, the acidification of fruit occurred as denoted by an increase in

238

TA and decrease in pH, with more acidic values at stages 3 and later and a slightly

239

greater accumulation of TSS at stage 5. This result was consistent with other

240

reports of noni fruit from Tepic, Mexico and Brazil21,22. Interestingly, noni fruit

241

accumulates acidity and concomitantly pH decreases during ripening. This is an

242

unusual behavior in fruit ripening only observed in a few fruits (e.g., banana and

243

pineapple23). During fruit ripening, usually pH increases, while TA declines as

244

reported for ripe fruit detached from plant (mango24 and papaya25) or fruit ripened

245

attached to plant (Prickly Pear26).

246

Rubio-Pino et al.21 and Silva et al.22 have reported an unusually high

247

buffering capacity of noni fruit and Hernandez et al.27 argued that this profile for

248

acidic tropical fruits results from the synthesis of organic acids in higher amounts to



249

achieve optimum maturity. In relation to the increment in TSS, Rubio-Pino et al.21

250

and Silva et al.22 suggested that sugar accumulation derived from degradation of

251

storage and structural polysaccharides. According to our results only traces of

252

starch were found in noni fruit (data not shown). Thus the TSS increment observed

253

in our work could be due sugars in the sap supplied from the plant via the phloem

254

and/or the conversion of polysaccharides into soluble sugars mainly at the last

255

ripeness stage. Here, we discuss the cell wall changes that occur in parallel with

256

the physical and chemical changes of ripening noni fruit.

257

The noni fruit ripening was accompanied by three softening stages which for

258

the purposes of this study, are classified as early, intermediate and final (Figure

259

1D). The early stage corresponds to the loss of 14% of firmness over the noni

260

fruit´s three maturation stages 1 to 3. Over this developmental time period,

261

firmness was reduced only slightly but the fruit skin color shifted from green to

262

yellow (Table 1). The intermediate softening stage (from stage 3 to 4) involved an

263

additional 16% firmness loss and also continued yellowing of the skin. Finally, the

264

fruit transition from stage 4 to 5, included a substantial reduction in firmness (66%)

265

and loss of yellow color. The softening profile shown in this study is similar to that

266

of Silva et al.22 who reported a firmness value of 128.4 and 104.6 N in the green

267

and mature stage respectively, thus decreasing just 18.5% the strength. Below, we

268

discuss the cell wall changes occurring in parallel with the firmness change of noni

269

fruit.

270

Cell wall composition


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Page 13 of 31


271

Significant and progressive changes in cell wall yield and composition were

272

observed during noni ripening. The yield decreased most obviously from the

273

transition of stage 4 to 5, concomitant with the maximum softening and significant

274

changes in cell wall composition and integrity (Table 2). Fruit UA and non-cellulosic

275

NS content significantly decrease; these representative compounds suggest

276

changes at the level of linear and branched pectins, as well as pectin side chains

277

and hemicellulosic matrix. In contrast, wall cellulose content remained relatively

278

stable in terms of its contribution to overall fruit mass. Over the full course of fruit

279

ripening, in the intermediate stages with only slight firmness decrease, fruit

280

softening was associated to the changes in cell wall pectin content. In contrast, the

281

dramatic final softening was represented by changes in both pectins and

282

hemicelluloses.

283

The NS composition of total cell wall and chemically extracted fractions of

284

pectins and hemicelluloses of noni fruit presented changes and suggest an active

285

metabolism during ripening (Table 3). In terms of net content, the general profile of

286

NS decreased; however, the loss of cell wall Rha and Gal was readily apparent

287

even in the early stage of softening (from stage 1 to 2). The dramatic softening at

288

the end of ripening was associated with a progressive loss of Rha, Xyl and Glc but

289

in general all other sugars in the AIS also decreased slightly as fruit

290

ripening/softening continued. Gal, an important and predominant sugar in several

291

actively studied fleshy fruits like papaya18 and tomato28, is the quantitatively most

292

important non-cellulosic NS in the AIS of unripe noni fruit and displayed the most

293

significant losses from noni cell walls as ripening proceeded, even before the



Page 14 of 31

294

transition from stages 4 to 5 when fruit firmness decrease was greatest. This wall

295

change and others are discussed next.

296

The WSF (Table 3), representing wall polymers that had lost their tight

297

binding associations with other wall structural components, was mainly composed

298

of Gal, Glc and Ara. Presumably, most of the polymers and smaller sugar-rich cell

299

wall components shift into the WSF as wall metabolism events break the bonds

300

that tie them to the rest of the wall. Thus, accumulation of a given polymer in the

301

WSF reflects changes in the wall that lead to wall weakening. Some components

302

in the wall may continue to accumulate over the course of ripening or other aspects

303

of cell development. However, some may be digested further and, ultimately, be

304

recycyled for cellular energy metabolism or conversion to other cellular molecules.

305

The Rha content of WSF, along with the dynamic changes in Ara and Gal,

306

suggested

307

Rhamnogalacturonan I domain, representing the hairy region of pectin with the

308

repeating disaccharide: (1→2)α-L-Rha-(1→4)α-D-GalA. The accumulation of these

309

sugars in the WSF fraction at stage 5, most likely suggests a digestion and

310

solubilization of wall-associated RGI that probably can be explained by the active

311

participation of cell wall degrading enzymes (below). The ESF fraction was rich in

312

Glc, being the major component and having a behavior of relative accumulation

313

until stage 3 and then decreasing significantly toward stages 4 and 5. The total

314

recovered fraction was low as reported; however, the molar ratio of Gal and Ara

315

suggest the presence of important branching components, perhaps corresponding

316

predominantly to RGI pectins from the middle lamella potentially involved in

that

important

and

progressive

changes


occurred

at

the

Page 15 of 31


317

calcium bridged complexes (Carpita and Gibeaut29). These results suggest the

318

structural modifications of the ESF fraction were primarily during early softening;

319

furthermore, its decreased presence in the cell wall occurred concomitantly to WSF

320

accumulation.

321

The net content of hemicelluloses recovered as the 4KSF and 24KSF was

322

low (Table 3). The 4KSF analysis showed that major sugars were Glc, Xyl and

323

Man. In the intermediate stage Glc and Man increased significantly, and then

324

decreased in the final stage. By other hand, the composition of the 24KSF fraction

325

was mainly Xyl, Ara, Man, Gal and Glc, all showing significant reductions during

326

the transition from stage 4 to 5 (completion of ripening).

327

The presence of water-soluble polysaccharides in noni juice, presumably

328

RGI-type pectins rich in arabinan and type I arabinogalactan, was previously

329

suggested by Bui et al.30. Furthermore, galactan side chains may also play an

330

important role in noni fruit cell wall adhesion and organization. A distinctive feature

331

of EDTA-soluble pectins from noni was the fraction´s high Glc content; this was

332

also reported in small fruit such as raspberry and boysenberry (Vicente et al.31,

333

Vicente et al32). However, the ratio 5:14:12 of Rha, Ara and Gal suggests the

334

presence of an RGI-rich domain. The amount of this wall fraction increased until

335

maturity stage 3 and then decreased as the fruit became fully ripe. The role of

336

calcium-mediated cell wall adhesion attributed to this fraction was significant

337

weakly down toward the final stage of noni ripening and softening, suggesting an

338

important role for RGI pectin metabolism in the final ripening of fruit.



On

339

the

other

hand,

the

possible

presence

Page 16 of 31

of

xyloglucan

and

340

xyloglucomannan in noni is consistent with the idea that these polysaccharides are

341

the major hemicellulosic polysaccharides found in tomato fruit28. The hemicellulose

342

content reduction has been reported in boysenberry at intermediate stage of

343

ripening, and the authors suggested a more substantial metabolism of the wall's

344

cross-linked xyloglucan32. The composition of 24 KSF of Xyl, Ara, Man, Gal and

345

Glc were comparable to those reported in pineapple33,34. Our results suggest the

346

presence of xylan and xyloglucans, with a relatively high amount of Ara and Gal

347

potentially belonged to arabinoxylans and/or branched protopectins, probably

348

comparable to xyloglucan-RGI conjugates previously reported in raspberry30,

349

however, further studies are needed to figure out the importance of Ara and Gal in

350

this fraction.

351

The profile of solubilization based in UA as shown in table 3 presented a

352

trend toward WSF and ESF accumulation from the transition of stage 1 to 3, when

353

the fruit is still firm, while toward he stages 4 to 5 the fractions were recovered in

354

less abundance. The study of the apparent molecular size of pectins of noni fruit

355

showed that the WSF undergo a downshift with as ripening progresses (Figure 2A)

356

whereas pectins in the ESF (therefore, presumably associated with the cell wall

357

fabric via ionic linkages through Ca2+ ions) showed discrete depolimerization at

358

stage 3 with no more changes at stage 5 (Figure 2B). Additionally, the

359

hemicellulose rich fractions (KOH-extracted) had a significative size reduction at

360

final stage of softening suggestion its major role at late softening (Figure 2C and

361

D).


Page 17 of 31


362

Depolymerization and solubilization of cell wall polymers are among the

363

distinctive changes contributing to softening in many types of fruit (cite some

364

general review of fruit wall changes). These changes in cell wall composition and

365

strength are due mainly to the action of fruit gene-encoded CWDEs enzymes. We

366

do not have specific glycosidic linkage composition data to unequivocally identify

367

particular cell wall structures in our wall polysaccharide extracts. However, the

368

changes in the general sugar composition data of the AIS over the course of

369

ripening (Table 3) suggests the presence of pectins such as homogalacturonan,

370

RGI, arabinan, arabinogalactan and xyloglucan. These inferences about the types

371

of wall components being metabolized as noni fruit ripen, led us to analyze the total

372

activity of pectin-degrading enzymes likely to be responsible for the wall changes

373

that converted interconnected wall components into the WSF. Our protein extracts

374

were assayed for activty of several pectin-digesting anzymes (PG, RGasa, β-gal

375

and Gase) and enzymes targeting the polysaccharide backbones of xyloglucan

376

and xylan hemicelluloses (XGase and Xase, respectively).

377

As seen in Figure 3, all of the extracted CWDE activities assayed displayed

378

their highest activities in stages 4 or 5, the time in fruit ripening in which most of the

379

fruit softening occurred. PG activity increased early in ripening and then remained

380

relatively stable until it increased again as the fruit lost essentially all resistance to

381

external pressure (Fig. 3A). The magnitude of this two activity peaks at the initial

382

and final stage of ripeness, were ~ 2 and 1.4-fold from stage 1 to 2 and from stage

383

4 to 5, respectively. This ripening-associated pattern of enzyme activation has

384

been reported previously in mango fruit (with a more gradual increase in activity).



385

In banana and boysenberry the PG activity pattern indicates early PG presence,

386

with activity decreasing at the end of ripening32,35-37. RGase action increased

387

noticeably from stage 2 to 3, just after PG activity increase and consistent with a

388

role in the solubilization of noni cell wall pectins. RGase cleaves RG-I and has

389

been proposed as a collaborator with PG in pectin network degradation,

390

particularly those pectins that contain galactan, arabinan and type-I arabinoglactan

391

side chains38. Gase activity increased steadily during ripening, with peak activity

392

measured at stage 5 showed a steadily progressing increase in activation being

393

the greatest at the final ripeness stage (Fig. 3). The cell wall target of Gase would

394

be the galactan side chains of RG-I; thus, its activity should contribute to exposure

395

of RG-I backbones to RGase, and the ultimate breakdown of cell wall pectin

396

networks. This would contribute to the extensive softening at the final ripening

397

stages, as has been reported in tomato and star fruit34,38. Finally, the glycosidase

398

β-gal, also had a steady activation that was significant from the change of stage 2

399

to 3 and even greater from 4 to 5, coinciding with the main loss of non-cellulosic

400

neutral sugars. β-gal activity has been associated with softening of several fruits,

401

including banana where activity increased at the beginning of storage and

402

subsequently decreased. In boysenberry, β-gal activity was low and constant

403

constant early in ripening and increased almost 18-fold at the time of ripening from

404

stage 4 to 5, a high value if compared with guava and carambola (1.7- and 1.6-fold

405

increase over the course of ripening)

406

increased with ripening but decreased between stages 4 and 5. The softening of

407

papaya and carambola has been associated with XGase and Xase activity during

408

storage18,39.

32,36,39

. In noni fruit, Xase and XGase activity


Page 18 of 31

Page 19 of 31


409

In many commercially important fruits, fruit softening has been attributed to

410

the action of CWDEs encoded by genes that are expressed during the course of

411

ripening. In many cases, research has reported that there are several genes (i.e.,

412

gene families) encoding many of the most important CWDEs, and these genes are

413

expressed at particular points in the fruit ripening program10. Thus, the patterns of

414

activity change in parallel with noni fruit ripening (Figure 3) could represent

415

different patterns of the expression of several CWDE genes. Furthermore, several

416

CWDEs that are important for ripening-associated fruit softening (e.g., pectin

417

methylesterase, pectate lyase and expansin)10, 40 have not been examined in noni

418

but may contribute to fruit softening. Research on ripening tomato fruits over the

419

past few decades has identified factors other than fruit cell wall metabolism that

420

can contribute to fruit softening (e.g., changes in the turgor pressure of fruit cells,

421

changes in the chemistry and structure of fruit cuticles)40. Noni fruit ripening leads

422

to the complete loss of fruit firmness (i.e., resistance to pressure) and most of this

423

firmness loss occurs in a relatively short window of developmental time. The noni

424

fruit may represent a very useful and healthful food, therefore, understanding the

425

postharvest biology of the fruit, in particular the (1) factors that regulate (or can be

426

used to regulate) the fruit's ripening program and (2) the aspects of cell biology that

427

contribute to the fruits loss of firmness which are likely to limit the marketing of the

428

fruit will be important targets of research in the near future.

429

430



431

Conclusions

432

The ripening of noni fruit includes changes in chemical composition (pH, soluble

433

solids and titratable acidity) and decreased fruit firmness. The texture change is

434

particularly interesting because most of the fruit's softening occurs at the end of ripening

435

and the result is an almost complete loss of the fruit's resistance to pressure. Our

436

preliminary study describes changes in cell wall composition that indicate breakdown in

437

the fruit's pectin and hemicellulose polymers. Assays of several cell wall modifying

438

enzymes that were isolated from ripening fruits (e.g., polygalacturonase, β-galactosidase

439

and xyloglucanase) identified activities that may be responsible for the fruit's cell wall and

440

firmness changes. More detailed examination of some of the relationships suggested by

441

our data, as well as other ripening-asociated changes in noni fruits, will be needed to

442

understand the rapid loss of fruit structure that occurs at the end of noni fruit ripening.

443

444

Acknowledgment

445

We thank Israel Partida (FCQB-UAS) and Rosalba Contreras-Martínez (CIAD-

446

Culiacán) for technical assistance.

447 448 449

References

450

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451

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53, 51–68.

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2. Morton, J.F. The ocean-going Noni, or Indian mulberry (Morinda citrifolia, Rubiaceae) and some of its ‘‘colourful’’ relatives. Ecol. Botany. 1992. 46, 241–256


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562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588


Page 24 of 31

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589 590 591

A

Titratable acidity in %

pH in flesh

4.5

4.4

a

a

b

4.3

4.2

b

4.1

c

Total Soluble Solids in °Brix

4.0

12.0

1

2

3

4

11.5

b

a

b 11.0

b b

10.0

B

9.5

a

0.7

b a

0.6

c

c 0.5

0.4

0.3 300

5

Fruit ripeness stage

10.5

C

0.8

Firmness on flesh in N

4.6

2 Early

1

250

200

3

4

5

Intermediate Fruit ripeness stage

Final

a

a a

150

b 100

50

D

c

0

1

592 593 594 595 596

2

3

4


5

1

2

3

4

5


Fig. 1. Chemical composition and firmness of noni fruit at five different ripeness stages. A) pH; B)Total Soluble Solids; C) Titratable Acidity; and D) Firmness classified by softening rate. Mean ±S.E. Different letters indicate significant difference (P

Fruit Ripening

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