Methods for Determining Cell Wall-Bound ... - ACS Publications

Subscriber access provided by READING UNIV

Article

Methods for Determining Cell Wall Bound-Phenolics in Maize Stem Tissues Rogelio Santiago, Ana Lopez-Malvar, Carlos Souto, and Jaime Barros-Rios J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05752 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

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 23

Journal of Agricultural and Food Chemistry


Methods for Determining Cell Wall Bound-Phenolics in Maize Stem Tissues

Rogelio Santiago1*, Ana López-Malvar1, Carlos Souto2, Jaime Barros-Ríos3*

1

Universidad de Vigo, Dpto. Biología Vegetal y Ciencias del Suelo. Unidad Asociada BVE1-

UVIGO y Misión Biológica de Galicia (CSIC). Campus As Lagoas Marcosende. 36310, Vigo, Spain.

2

E.E. Forestales, Dpto. Ingeniería Recursos Naturales y Medio Ambiente, Pontevedra 36005,

Spain.

3

BioDiscovery Institute, University of North Texas, Denton, TX 76203, USA.

* Corresponding authors: Rogelio Santiago, (Tel: +34-986-854800; Fax: +34-986-841362; E-mail: [email protected]) Jaime Barros-Ríos, (Tel: 940-205-7636; Fax: 940-565-3821; E-mail: [email protected]) Santiago et al. Page 1

ACS Paragon Plus Environment


1

ABSTRACT

2

We compared two methods with different sample pretreatment, hydrolysis, and separation

3

procedures to extract cell wall-bound phenolics. The samples were pith and rind tissues from

4

six maize inbred lines reportedly containing different levels of cell wall-bound phenolics. In

5

method 1, pretreated samples were extracted with a C18 solid-phase extraction cartridge, and it

6

took 6 days to complete. In method 2, phenolics were extracted from crude samples with ethyl

7

acetate, it took 2 days to complete and the cost per sample was reduced more than 60%. Both

8

methods extracted more 4-coumarate than ferulate. Overall, method 1 yielded more 4-

9

coumarate while method 2 yielded more ferulate. The lack of a genotype × method interaction

10

and significant correlations between the results obtained using the two methods indicate that

11

both methods are reliable for use in large-scale plant breeding programs. Method 2, scaled, is

12

proposed for general plant biology research.

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

KEYWORDS: Zea mays, hydroxycinnamic acids, extraction, p-coumaric acid, ferulic acid Santiago et al. Page 2


Page 2 of 23

Page 3 of 23


31

INTRODUCTION

32

Ferulate (FA) and 4-coumarate (4CA) are hydroxycinnamic acids (HCAs) that are major

33

structural components of grass cell walls.1 Ferulate is deposited in both primary and

34

secondary walls, and is linked to arabinoxylans by ester bonds through its carboxylic acid

35

group with the C5-hydroxyl of α-L-arabinosyl side chains of xylans.2 During grass cell wall

36

lignification, glucuronoarabinoxylans (the major hemicellulosic polysaccharides in grasses)

37

are cross-linked by FA monomers into a complex array of dimers and trimers, and via ether

38

bonds to lignin.3,4 Overall, FA plays a significant role in cell wall development and affects

39

polysaccharide utilization in grasses. 4-Coumarate is mainly esterified to the γ–position of

40

phenylpropanoid sidechains of the syringyl (S)-rich units of lignin.5 Only small quantities of

41

4CA are esterified to glucuronoarabinoxylans in immature tissues. Most 4CA accretion occurs

42

alongside lignification, making 4CA a convenient indicator of lignin deposition.

43

Commelinid monocots and some dicots in the order Caryophyllales have significant

44

amounts of HCAs deposited in their cell walls,6 which are considered important natural

45

defense compounds against pests and diseases in plants,4,7. Industrially, HCAs are major

46

factors limiting forage grass digestibility, and therefore, animal productivity,8 and are

47

responsible for the recalcitrance of grass lignocellulose to enzymatic hydrolysis.9 They are

48

also potential antimicrobial molecules10 and important bioactive substances in the food

49

industry.11 These multiple applications require fast, reliable, and cost-effective methods to

50

extract and quantify cell wall bound phenolics from different plant tissues.

51

Currently, two main methods are used to analyze wall-bound phenolics in plant tissues 12

52

(Table 1). Method 1 was developed by Jung and Shalita-Jones

53

Dietary Fiber Method devised by Theander and Aman.13 This method determines the

54

composition of the cell wall and includes an in vitro fermentation step to assess forage

55

digestibility (Supporting Information, A). More recent approaches, with a special focus on

56

HCAs quantification, use simpler liquid–liquid extractions with organic solvents such as ethyl Santiago et al. Page 3


to complete the Uppsala


57

acetate. These recent methods vary in the amounts of starting material, sample pretreatments,

58

and alkaline hydrolysis conditions for extraction, and the separation and chromatographic

59

techniques used for quantification.14-16

60

In this study, we compared two methods using the same array of samples to provide a

61

detailed description of the techniques, to determine the consistency between their results, and

62

to assess their suitability for specific research purposes.

Santiago et al. Page 4


Page 4 of 23

Page 5 of 23


63

MATERIALS AND METHODS

64

Plant materials and experimental design. Six maize inbred lines reported to have different

65

cell wall-bound phenolics levels were grown in two geographical locations in Spain in 2012.14

66

Pontevedra (42° 30′ N, 8°46′ W) is a coastal location, approximately at sea level, whereas

67

Zaragoza (41° 44′ N, 0° 47′ W) is inland at 250 m above sea level. The experimental design

68

was a randomized complete block design with three replicates. Each plot had two rows spaced

69

0.80 m apart, and each row consisted of 25 two-kernel hills spaced 0.21 m apart. After

70

thinning to one plant per hill, the plant density was approximately 60,000 plants per hectare.

71

Irrigation, crop management, fertilization, and weed control were carried out according to

72

local practices.

73

Samples were collected at 30 days after female flowering. The third internode above

74

ground level was harvested from five to eight plants. Pith samples were obtained by manually

75

removing the rind tissues. Pith and rind tissue samples were frozen, lyophilized, and ground

76

through a 0.75 mm screen using a rotating-blade Wiley mill (Arthur H. Thomas, Philadelphia,

77

PA, USA).

78 79

Extraction of esterified cell wall-bound phenolics. In method 1, samples were first

80

pretreated to obtain a starch-free cell wall residue according to the Uppsala dietary fiber

81

method.17 Acetate buffer (5 mL, 0.1 M, pH 5.0) and 0.1 mL heat-stable α-amylase at 10-50

82

mg/mL biuret concentration (Sigma Chemical Co., St. Louis, MO, USA) were added to 100

83

mg sample and heated at 90 °C for 60 min. After cooling the mixture to 50 °C, 0.2 mL

84

amyloglucosidase (Sigma) was added and the resultant mixture was heated for 3 h at 60 °C.

85

Sufficient 95% ethanol was added to achieve a final concentration of 80% ethanol and the

86

sample was kept at 4 °C overnight. The crude cell wall preparation was recovered by

87

centrifugation at 1000 g for 15 min, washed twice with 5 mL of 80% ethanol and once with 5

88

mL of acetone, and allowed to air dry under a hood. Subsequently, ester-linked FA and 4CA Santiago et al. Page 5



89

were extracted from the starch-free cell wall residue with 10 mL of 2 M NaOH at 39 °C for

90

24 h. The alkaline extracts were acidified to pH 1.5–1.6 with concentrated phosphoric acid.

91

The acidified extract was filtered through a Whatman filter (0.45 µm pore size) and loaded

92

onto a C18 solid-phase extraction column (Supelco Inc., Bellefonte, PA, USA). The column

93

was washed with 2 mL of 2M NaOH acidified to pH 1.5 with phosphoric acid, and the HCAs

94

were eluted with two 2.5-mL of 50% methanol washes. The eluted samples were brought to a

95

final volume of 10 mL and stored at −20 °C until analysis.

96

In method 2, 1 g dried ground tissue was pretreated with 30 mL 80% methanol and

97

mixed with a Polytron mixer (Brinkman Instruments, Westbury, NY, USA). Samples were

98

incubated for 1 h and centrifuged for 10 min at 1000 g. The supernatant containing soluble

99

phenolics was discarded; the remaining pellet containing the cell wall-bound material was

100

incubated for 4 h with 20 mL 2 N NaOH under nitrogen atmosphere in the dark with shaking

101

at 130 rpm at room temperature. After centrifugation at 1000 g for 10 min, the supernatant

102

was collected and the pellet was washed twice with 10 mL distilled water. Then, 6 N HCl was

103

added to lower the pH to 2.0. Supernatants were pooled and extracted twice with ethyl acetate

104

(40 mL each). The collected organic fractions were combined and reduced to dryness using a

105

Speed Vac (Savant Instruments, Holbrook, NY, USA) for 3–4 h. The final extract was

106

dissolved in 1.5 mL high-performance liquid chromatography (HPLC) grade methanol,

107

filtered through a 0.45-µm syringe filter (Chromatographic Specialties, Brockville, ON,

108

USA), and stored at −20 °C until HPLC analysis. The detailed lab protocols for both methods

109

and a budget estimate by protocol are provided as Supporting Information. Results obtained

110

by both methods were adjusted to a dry matter basis for data presentation and comparison.

111 112

HPLC-analysis of esterified cell wall-bound phenolics. The extracts obtained using the two

113

methods were quantified by reverse-phase HPLC, comprising a C18 stationary-phase column

114

coupled with a diode array detector as previously described.18 Analyses were performed using Santiago et al. Page 6


Page 6 of 23

Page 7 of 23


115

a 2690 Waters separation module (Waters, Milford, MA, USA) equipped with a 996

116

photodiode array detector (Waters) with a narrow bore column (100 × 2 mm i.d.; 3 µM

117

particle size) of YMC ODS-AM (Waters), which is a high carbon load C18 packing material.

118

The solvent system consisted of acetonitrile (A) and trifluoroacetic acid (0.05%) in water (pH

119

3.4) (B) at a flow rate of 0.3 mL/min. The elution conditions were as follows: initial

120

conditions 10:90 (A:B), changing to 30:70 in 3.5 min, 32:68 in 6.5 min, 100:0 in 4 min, then

121

isocratic elution with 100:0 for 4.5 min, and finally returning to initial conditions in 3 min.

122

The sample injection volume was 4 µL, and the elution profiles were obtained by monitoring

123

UV absorbance at 325 nm. Retention times were compared with those of 4CA and FA

124

(Sigma) in freshly prepared standard solutions. In method 2, FA dimers were identified by

125

comparison with the retention time and UV spectra of the 5–5-diferulic acid standard, and

126

confirmed according to previous literature.19 Calibration curves for the standards were built

127

and used for external quantitation.

128 129

Statistical analyses. Combined analyses of variance (ANOVA) for the HCAs concentration

130

were computed with the PROC MIXED procedure of SAS.20 Location, genotype, and

131

replication were considered to be random because they were representative of the variability

132

in climate and genetic materials. Comparisons of means among methods were made by

133

Fisher’s protected least significant difference (LSD) method.21 Correlations were computed

134

using the whole dataset (N = 36; 2 locations × 3 replications × 6 genotypes). The significance

135

of the correlations was computed using the PROC CORR and PROC REG procedures of

136

SAS.20

137

RESULTS AND DISCUSSION

138

The main differences between method 1 and 2 are highlighted in Table 1 (See Supporting

139

Information for details). Method 1 was proposed by Jung and Shalita-Jones12 to complete the

140

Uppsala Dietary Fiber Method by Theander and Aman13. In this method, cell wall residues are Santiago et al. Page 7



141

used subsequently for additional analysis and characterization of dietary fibers (i.e.

142

determination of Klason lignin and non-starchy polysaccharides for ruminant feed evaluation)

143

(Supporting Information, A). Method 2, instead, was specifically used to evaluate plant cell

144

wall bound phenolics in seed flours.22 The pretreatment carried out in method 1 took 3 days

145

and cost US $2.68 per sample, while the extraction steps took an additional 3 days and cost

146

US $5.28 per sample. However, the sample preparation and extraction method performed in

147

method 2 took 2 days and cost US $2.88 per sample. This latter method is faster and cheaper,

148

but it would be less useful if a further characterization of the cell wall properties is required.

149

Even when the cell wall residue preparation is not taken into consideration, method 2 is still

150

cheaper than method 1, mainly because of the use of the C18 SPE cartridges in the last one.

151

Although both methods produced samples that showed good peak resolution for both

152

wall-bound 4CA and FA in the HPLC analysis, the samples prepared using method 2 allowed

153

the quantification of FA dimers (diferulates) (Figure 1A). The concentration of these

154

compounds in the pith tissues make up about 6% of total ferulates ester linked to the cell wall,

155

but less than 1% in the rind tissues, probably because of higher degree of lignification and

156

presence of ether linkages not extracted with a mild saponification. Those results are

157

consistent with previously published data.23 Diferulates function as cross-linking molecules

158

between cell wall polymers.9 These compounds affect numerous cell wall properties such as

159

digestibility, accessibility, and extensibility, and confer resistance to pest and diseases.4

160

Diferulates are also potentially valuable chemicals as natural antifungal agents.10 Method 2

161

has already been used in large-scale maize breeding programs aiming to increase the levels of

162

these plant metabolites.24 Other simpler methods, without sample pretreatment, have also

163

been used to screen maize mutants for HCAs.25 If diferulates were the target molecules,

164

method 1 could be potentially improved by: i) increasing the amount of starting material used

165

for analysis, ii) concentrating the volume of the eluted samples, or iii) using higher

166

concentration of methanol during the elution step from the SPE cartridges. Further Santiago et al. Page 8


Page 8 of 23

Page 9 of 23


167

experiments would be required to identify the actual reason of this limitation.

168

The concentration of 4CA was higher than that of FA in both tissues and using both

169

methods (Figure 1B). Similar results have been reported for several other plant species.26-28

170

Interestingly, the 4CA concentration was higher in the rind than in the pith, while the FA

171

levels were similar in both tissues. This reflected greater deposition of lignin in the rind

172

tissues,27 and suggested that the accumulation of cell wall-bound FA is independent of the

173

levels of 4CA and the degree of lignin polymerization in the cell wall. Extensive work has led

174

over recent years to identify cell wall-bound phenolics.29 This literature underlines the

175

variation observed among different plant species and tissues analyzed. In maize, for example,

176

cell wall-bound FA content can vary from 6.3 mg/g of dry weight in the whole stems to 1.1

177

mg/g in the roots.

178

The ANOVA revealed significant differences between the two methods for 4CA in the

179

pith and FA in the rind (Table 2). These differences cannot be explained by the physiology or

180

architecture of the tissues as the same trend was observed in both tissues (Figure 1B). Overall,

181

method 1 yielded more 4CA, while method 2 yielded more FA. The inherent characteristics of

182

the analytical methods may explain these differences. The single methanol wash in the sample

183

pretreatment in method 2 may have been insufficient to remove all soluble phenolics from the

184

alcohol-insoluble residue. This should have affected both 4CA and FA, but it may have had a

185

stronger effect on the extraction of 4CA than on FA, because the soluble 4CA level was much

186

higher than the soluble FA level in these tissues.30 This observation suggests that other steps

187

in the analytical procedure were responsible for the differences described above. Samples can

188

be saponified by treatment with sodium hydroxide at various concentrations with different

189

reaction times and temperatures.29 Cell wall HCAs ester-linked to wall polymers can be

190

extracted from the samples by a dilute alkali at room temperature.31 A previous study found

191

that increasing the alkaline hydrolysis temperature from 18 °C to 30 °C released 15% more

192

4CA but only 9% more FA from mature maize stems,32 indicating that the incubation Santiago et al. Page 9



193

temperature during the alkaline treatment has a stronger effect on the release of wall-bound

194

4CA than on the release of FA. Another factor to consider is the differential recovery of 4CA

195

and FA from the SPE columns. Method 1 used a C18 cartridge; this type of cartridge has been

196

widely used in phenolic compound separation, although it shows low recovery for particular

197

phenols.33 However, differences in the recovery of low molecular weight polar compounds

198

(4CA and FA) from a C18 cartridge was not noted in a more recent study.34

199

A non-cross over significant location × method interaction was found for 4CA levels

200

in the rind tissues (Table 2). For the sample from Pontevedra, the 4CA level was 10,1 mg/g as

201

determined by method 1 and 17,4 mg/g as determined by method 2. For the sample from

202

Zaragoza, the 4CA level was 13,1 mg/g as determined by method 1 and 12,8 mg/g as

203

determined by method 2. This interaction was attributed to the high variation in 4CA levels in

204

the rind when extracted using both methodologies (Figures 1B and 2). Consistent with these

205

results, the standard deviations among genotypes were moderate for FA in both tissues and

206

methods, but much higher for 4CA, especially in the rind samples (Figure 2). These variations

207

explain why the genotypes showed no significant differences in 4CA levels, and clarify the

208

origin of the location × genotype × method interaction (Table 2). These variations can be

209

explained by the intrinsic properties of the rind cell walls. The outer rind tissue used for these

210

analyses was highly heterogeneous, as it included the epidermis, parenchyma, sclerenchyma,

211

xylem vessels, and phloem cells, while the inner pith comprised mainly parenchyma cells and

212

randomly distributed vascular bundles. Different levels of lignin deposited in the rind during

213

the plant response to biotic or abiotic stress factors in the field could lead to different levels of

214

wall-bound 4CA, even within replicates from the same field. Our results suggest that the

215

variations are due to the nature of the sample rather than the methods used, although further

216

studies are required to confirm this. To better understand these variations, it may be useful to

217

apply the DFRC (derivatization followed by reductive cleavage) method, which quantifies

218

4CA specifically bound to lignin by cleaving lignin ethers while retaining esters.35 Santiago et al. Page 10


Page 10 of 23

Page 11 of 23


219

Importantly, no genotype × method interaction was detected (Table 2). Thus, the

220

genotypes showed a similar ranking in terms of the levels of wall-bound phenolics in both

221

tissues measured by both methods. This result suggested that method 2, which is faster and

222

cheaper than method 1, could be used to analyze large numbers of samples. The wide genetic

223

variation in FA content indicates that there is potential to breed or genetically engineer plants

224

that produce high levels of these phenolic compounds in different plant tissues.24,25

225

When the whole data set was analyzed, there was a significant positive correlation

226

between the two methods (N = 36, Figure 3). As expected, the r-squared values were slightly

227

higher for the pith (r2 = 0.7) than for the rind (r2 = 0.6). The strength of the correlation

228

between the two methods could be improved by using multiple methanol washes or preparing

229

a protein-free cell wall fraction, which might be suitable when using smaller solvent

230

volumes.36 In fact, method 2 has been scaled down for analyses with reduced amount of

231

samples in several recent studies (see Supporting Information, E).15,16,37,38 Overall, these

232

results indicate that the two methods are comparable in their ability to quantify wall-bound

233

HCAs in different maize stem tissues.

234

In summary, methods 1 and 2 can be used to analyze esterified phenolics in maize

235

stem tissues, and produce reliable and reproducible results. Method 1 is recommended for

236

analyzing forage digestibility when samples are processed as part of a full cell wall

237

characterization. Method 2 is faster and cheaper than method 1, and is appropriate for

238

analyzing cell wall FAs (both monomers and dimers). Method 2 requires larger samples,

239

which can be a limitation in some research applications. For these reasons, method 2, scaled,

240

is more appropriate for diverse research purposes in plant biology.




241

ACKNOWLEDGMENTS

242

We thank Dr. Lana Reid of ECORC, Agriculture and Agri-Food Canada for supplying inbred

243

line CO441 used in this study. We acknowledge Rosa Ana Malvar and Hans Joachim G. Jung

244

for critical reading of the manuscript.

245 246

SUPPORTING INFORMATION DESCRIPTION

247

Supporting Information : A: Simplified diagram of the Uppsala Dietary Fiber Method by

248

Theander and Aman (1980) completed with the analysis of cell wall-bound phenolics

249

proposed by Jung and Shalita-Jones (1990), highlighting the steps tested in the current study:

250

(a) cell wall residue preparation, and (b) ester phenolics extraction. DO/OM, dry

251

matter/organic matter; IVTF, in vitro total fermentation; A.I.R. PREP, cell-wall residue

252

preparation; DIw, deionized water; CHO, carbohydrates; B: Lab Protocol: Cell-wall residue

253

preparation; C: Lab Protocol Method 1: 2N Alkaline Extraction with SPE column cleanup; D:

254

Lab Protocol Method 2: 2N Alkaline Extraction with Ethyl Acetate separation; E: Lab

255

Protocol Method 2-Scaled: 2N Alkaline Extraction with Ethyl Acetate separation; F: Rough

256

budget estimates for each lab protocol.



Page 12 of 23

Page 13 of 23


257

REFERENCES

258

1. Hatfield, RD; Rancour, DM; Marita, JM. Grass cell walls: A story of cross-linking.

259

Front.Plant Sci.2017,7, 2056.

260

2. Grabber, JH; Ralph, J; Hatfield, RD. Model studies of ferulate−coniferyl alcohol cross-

261

product formation in primary maize walls: implications for lignification in grasses. J.

262

Agric. Food Chem. 2002, 50, 6008-6016.

263 264 265 266

3. Bunzel, M. Chemistry and occurrence of hydroxycinnamate oligomers. Phytochem. Rev. 2009, 9, 47-64. 4. Santiago, R; Barros-Rios, J; Malvar, RA. Impact of cell wall composition on maize resistance to pests and diseases. Int. J. Mol. Sci. 2013, 14, 6960-6980.

267

5. Grabber, JH; Ralph, J; Lapierre, C; Barrière, Y. Genetic and molecular basis of grass cell-

268

wall degradability. I. Lignin–cell wall matrix interactions. Comptes rendus biologies,

269

2004, 327, 455-65.

270 271 272 273 274 275

6. Hartley, RD; Harris, PJ. Phenolic constituents of the cell walls of dicotyledons. Biochemical Systematics and Ecology, 1981, 9,189-203. 7. Marcia, MD. Feruloylation in grasses: current and future perspectives. Molecular Plant, 2009, 2, 861-72. 8. Jung, HJ; Samac, DA; Sarath G. Modifying crops to increase cell wall digestibility. Plant Science, 2012, 185, 65-77.

276

9. Oliveira, DM; Finger‐Teixeira, A; Rodrigues Mota, T; Salvador, VH; Moreira‐Vilar, FC;

277

Correa Molinari, HB; Mitchell, C; Andrew, R; Marchiosi, R; Ferrarese‐Filho, O; Dantas

278

dos Santos, W. Ferulic acid: a key component in grass lignocellulose recalcitrance to

279

hydrolysis. Plant Biotech Journal, 2015, 13, 1224-32.

280

10. Piotrowski, JS; Okada, H; Lu, F; Li, SC; Hinchman, L; Ranjan, A; Smith, DL; Higbee,

281

AJ; Ulbrich, A; Coon, JJ; Deshpande, R. Plant-derived antifungal agent poacic acid targets

282

β-1, 3-glucan. Proceedings of the National Academy of Sciences, 2015, 112, E1490-7.

283 284 285 286 287 288

11. Kroon PA, Williamson G. Hydroxycinnamates in plants and food: current and future perspectives. J. Sci. Food Agric. 1999, 79, 355-61. 12. Jung, HJ; Shalita-Jones, SC. Variation in the extractability of esterified p-coumaric and ferulic acids from forage cell walls. J. Agric. Food Chem. 1990, 38, 397-402. 13. Theander, O; Aman, P. Chemical composition of some forages and various residues from feeding value determinations. J. Sci. Food Agric. 1980, 31, 31-7.




289

14. Santiago, R; Butrón, A; Arnason, JT; Reid, LM; Souto, XC; Malvar, RA. Putative role of

290

pith cell wall phenylpropanoids in Sesamia nonagrioides (Lepidoptera: Noctuidae)

291

resistance. J. Agric. Food Chem. 2006, 54, 2274–2279.

292

15. Bouvier d'Yvoire, M; Bouchabke‐Coussa, O; Voorend, W; Antelme, S; Cézard, L; Legée,

293

F; Lebris, P; Legay, S; Whitehead, C; McQueen‐Mason, SJ; Gomez, LD. Disrupting the

294

cinnamyl alcohol dehydrogenase 1 gene (BdCAD1) leads to altered lignification and

295

improved saccharification in Brachypodium distachyon. The Plant Journal 2013, 73, 496-

296

508.

297 298

16. Barros, J; Serrani-Yarce, JC; Chen, F; Baxter, D; Venables, BJ; Dixon, RA. Role of bifunctional ammonia-lyase in grass cell wall biosynthesis. Nature plants 2016, 2,16050.

299

17. Theander, O; Aman, P; Westerlund, E; Andersson, R; Pettersson, D. Total dietary fiber

300

determined as neutral sugar residues, uronic acid residues, and Klason lignin (the Uppsala

301

method): collaborative study. Journal of AOAC International 1995, 78, 1030-44.

302

18. Sen, A; Miller, S; Arnason, J; Fulcher, R. Quantitative determination by high-performance

303

liquid chromatography and microspectrofluorometry of phenolic acids in maize grain.

304

Phytochem. Anal. 1991, 2, 225–229.

305

19. Waldron, K; Parr, A; Ng, A; Ralph, J. Cell wall esterified phenolic dimers: identification

306

and quantification by reverse phase high performance liquid chromatography and diode

307

array detection. J. Sci. Food Agric. 1996, 7, 305–312.

308

20. SAS Institute Inc. SAS/STAT® 9.2 User’s Guide. SAS Institute Inc., Cary, NC. 2008.

309

21. Steel, RDG; Torrie, JH; Dickey, DA. Principles and procedures in statistics: a biometrical

310 311 312

approach, 3rd ed., McGraw-Hill Ed., 1997, New York. 22. Krygier, K; Sosulski, F; Hogge, L. Free, esterified, and insoluble-bound phenolic acids. 1. Extraction and purification procedure. J. Agric. Food Chem. 1982, 30, 330-4.

313

23. Barros-Rios, J., Santiago, R; Malvar, RA, Jung, HJ. Chemical Composition and Cell Wall

314

Polysaccharide Degradability of Pith and Rind Tissues from Mature Maize Internodes.

315

Anim. Feed Sci. Technol. 2012, 172, 226–236.

316

24. Barros-Rios, J; Santiago, R; Jung, HJ; Malvar RA. Covalent cross-linking of cell-wall

317

polysaccharides through esterified diferulates as a maize resistance mechanism against

318

corn borers. J. Agric. Food Chem. 2015, 63, 2206–14.

319

25. Jung, HG; Phillips, RL. Putative seedling ferulate ester (sfe) maize mutant: morphology,

320

biomass yield, and stover cell wall composition and rumen degradability. Crop Sci. 2010,

321

50. 403–18.



Page 14 of 23

Page 15 of 23


322

26. Sarath, G; Baird, LM; Vogel, KP; Mitchell, RB. Internode structure and cell wall

323

composition in maturing tillers of switchgrass (Panicum virgatum. L). Bioresource

324

technology 2007, 98, 2985-92.

325

27. Barros-Rios, J; Malvar, RA; Jung, HJ; Bunzel, M; Santiago, R. Divergent selection for

326

ester-linked diferulates in maize pith stalk tissues. Effects on cell wall composition and

327

degradability. Phytochem.2012,83, 43-50.

328

28. Anderson, NA; Bonawitz, ND; Nyffeler, K; Chapple, C. Loss of ferulate 5-hydroxylase

329

leads to mediator-dependent inhibition of soluble phenylpropanoid biosynthesis in

330

Arabidopsis. Plant Physiol. 2015, 169, 1557-67.

331

29. Barberousse, H; Roiseux, O; Robert, C; Paquot, M; Deroanne, C; Blecker, C. Analytical

332

methodologies for quantification of ferulic acid and its oligomers. J. Sci. Food Agric.

333

2008, 88, 1494–1511.

334

30. Santiago, R; Reid, LM; Arnason, JT; Zhu, XY; Martinez, N; Malvar, RA. Phenolics in

335

maize genotypes differing in susceptibility to Gibberella stalk rot (Fusarium graminearum

336

Schwabe). J. Agric. Food Chem. 2007, 55, 5186–5193.

337 338

31. Iiyama, K; Lam, TBT; Stone, BA. Phenolic acid bridges between polysaccharides and lignin in wheat internodes. Phytochem. 1990, 29, 733–737.

339

32. Sun, RC; Sun, XF; Zhang, SH. Quantitative determination of hydroxycinnamic acids in

340

wheat, rice, rye, and barley straws, maize stems, oil palm frond fiber, and fast-growing

341

poplar wood. J. Agric. Food Chem. 2001, 49, 5122-5129.

342

33. Malovaná, S; García-Montelongo, FJ; Pérez, JP; Rodríguez-Delgado, MA. Optimisation

343

of sample preparation for the determination of trans-resveratrol and other polyphenolic

344

compounds in wines by high performance liquid chromatography. Anal. Chim. Acta 2001,

345

428, 245–253.

346

34. Nemes, SM; Orsat, V. Modeling the recovery patterns from solid phase extraction

347

purification of secoisolariciresinol diglucoside, p-coumaric acid glucoside, and ferulic acid

348

glucoside from microwave-assisted flaxseed extracts." Food and Bioproducts processing,

349

2012, 90, 453-465.

350

35. Lu, F; Ralph, J. Derivatization followed by reductive cleavage (DFRC method), a new

351

method for lignin analysis: protocol for analysis of DFRC monomers. J. Agric. Food

352

Chem. 1997, 45, 2590-2592.

353

36. Moreira-Vilar, FC; de Cássia Siqueira-Soares, R; Finger-Teixeira, A; de Oliveira, DM;

354

Ferro, AP; da Rocha, GJ; Maria de Lourdes, LF; dos Santos, WD; Ferrarese-Filho, O. The

355

acetyl bromide method is faster, simpler and presents best recovery of lignin in different




356

herbaceous tissues than klason and thioglycolic acid methods. PLoS One. 2014,

357

9:e110000.

358

37. Pellny, TK; Lovegrove, A; Freeman, J; Tosi, P; Love, CG; Knox, JP; Shewry, PR;

359

Mitchell, RA. Cell walls of developing wheat starchy endosperm: comparison of

360

composition and RNA-Seq transcriptome. Plant Physiol. 2012, 158, 612-27.

361

38. Petrik, DL; Karlen, SD; Cass, CL; Padmakshan, D; Lu, F; Liu, S; Bris, P; Antelme, S;

362

Santoro, N; Wilkerson, CG; Sibout, R. p‐Coumaroyl‐CoA: monolignol transferase (PMT)

363

acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon. The

364

Plant Journal 2014, 77, 713-26.

365

39. Dien, BS; Jung HJ; Vogel, KP; Casler, MD; Lamb, JF; Iten, L; Mitchell, RB; Sarath, G.

366

Chemical composition and response to dilute-acid pretreatment and enzymatic

367

saccharification of alfalfa, reed canarygrass, and switchgrass. Biomass and Bioenergy.

368

2006, 30, 880-91.

369 370

ACKNOWLEGMENT FOR FUNDING RESOURCES

371

This research was supported by the National Plan for Research and Development of Spain

372

(AGL2012-33415, AGL2015-67313-C2-1-R, AGL2015-67313-C2-2-R), the Autonomous

373

Goverment of Galicia (XUNTA: ED431F 2016/014) and co-financed by the European Social

374

Fund (FEDER). R. Santiago acknowledges postdoctoral contract “Ramón y Cajal” financed

375

by the Ministry of Economy and Competitiveness (Spain), Vigo University, and the European

376

Social Fund. J. Barros acknowledges a grant from the autonomous government of Galicia

377

(Spain).



Page 16 of 23

Page 17 of 23

378


FIGURE CAPTIONS

379 380

Figure 1. HPLC elution profile of 4-coumarate (A, 1) and ferulate (A, 2) using both analytical

381

methods tested in the current study. Panel A shows the HPLC elution profiles at 325nm of

382

maize inbred line CM151 extracted from rind tissues. Method 2 showed better resolution for

383

ferulate dimers (diferulates) (3). Panel B shows the average concentrations of 4-coumarate

384

(4CA) and ferulate (FA) in pith and rind tissues from mature internodes of six field grown

385

maize inbred lines estimated using both methodologies.

386 387

Figure 2. Levels of 4-Coumarate (4CA) and Ferulate (FA) in Pith and Rind Tissues from

388

Mature Internodes of Six Field Grown Maize Inbred Lines Estimated Using both Analytical

389

Methods tested in the Current Study. Capital and Lower Letters show Genotypes Comparison

390

by Method 1 and 2 Respectively. The Interaction Genotype x Method was not significant.

391 392

Figure 3. Scatter plot for concentrations of ferulate (FA, A) and 4-coumarate (4CA, B) in

393

µg/g of dry weight measured using the two analytical methods tested. The black-dashed line

394

shows how far methods differ from a one-to-one correspondence. An asterisk (*) denotes

395

significance at P

Methods for Determining Cell Wall-Bound ... - ACS Publications

Recommend Documents