Phytochemistry 85 (2013) 51–61

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Phytochemistry

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Cell wall compositional modifications of Miscanthus ecotypes in responseto cold acclimation

Jean-Marc Domon a, Laëtitia Baldwin a, Sébastien Acket a, Elodie Caudeville a, Stéphanie Arnoult b,Hélène Zub b, Françoise Gillet a, Isabelle Lejeune-Hénaut b, Maryse Brancourt-Hulmel b, Jérôme Pelloux a,Catherine Rayon a,⇑a EA 3900-BIOPI, Université de Picardie Jules Verne, 80039 Amiens Cedex, Franceb INRA USTL UMR 1281, Estrées-Mons BP50136, 80203 Péronne, France

a r t i c l e i n f o

Article history:Received 6 June 2012Received in revised form 5 September 2012Available online 15 October 2012

Keywords:MiscanthusPoalesCell wallCold stressGlucuronoarabinoxylan(1 ? 3),(1 ? 4)-b-D-glucanCelluloseUronic acidLigninPALCAD

0031-9422/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.phytochem.2012.09.001

Abbreviations: Gig, M. x giganteus; Gol, M. sinenAugust Feder; NA, non-cold-acclimated; CA, cold-accammonia-lyase; CAD, cinnamyl alcohol dehydrogenglucuronoarabinoxylan; b-glucan, (1 ? 3),(1 ? 4)-b-D

⇑ Corresponding author. Address: Université de PiBIOPI, 33 rue Saint Leu, 80039 Amiens Cedex, France.

E-mail address: catherine.rayon@u-picardie.fr (C. R

a b s t r a c t

Miscanthus, a potential energy crop grass, can be damaged by late frost when shoots emerge too early inthe spring and during the first winter after planting. The effects of cold acclimation on cell wall compo-sition were investigated in a frost-sensitive clone of Miscanthus x giganteus compared to frost-tolerantclone, Miscanthus sinensis August Feder, and an intermediate frost-tolerant clone, M. sinensis Goliath.Cellulose and lignin contents were higher in M. x giganteus than in the M. sinensis genotypes. In ambienttemperature controls, each clone displayed different glucuronoarabinoxylan (GAX) contents and degreeof arabinose substitution on the xylan backbone. During cold acclimation, an increase in (1 ? 3),(1 ? 4)-b-D-glucan content was observed in all genotypes. Uronic acid level increased in the frost sensitive geno-type but decreased in the frost tolerant genotypes in response to cold. In all clones, major changes in cellwall composition were observed with modifications in phenylalanine ammonia-lyase (PAL) and cinnamylalcohol dehydrogenase (CAD) activities in both non- and cold-acclimated experiments. A large increase inCAD activity under cold stress was displayed in each clone, but it was largest in the frost-tolerant clone,M. sinensis August Feder. The marked increase in PAL activity observed in the frost-tolerant clones undercold acclimation, suggests a reorientation of the products towards the phenylpropanoid pathway or aro-matic synthesis. How changes in cell wall physical properties can impact frost tolerance is discussed.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Miscanthus is a tall perennial rhizomatous C4 grass used for pro-duction of biofuels in Europe and in the US (Clifton-Brown et al.,2004; Zub and Brancourt-Hulmel, 2010; Pyter et al., 2010). Thegenus Miscanthus comprises about 20 species and originates fromEastern Asia (Zub and Brancourt-Hulmel, 2010). Two Miscanthusspecies, Miscanthus sinensis and Miscanthus sacchariflorus have po-tential for biomass production, but Miscanthus x giganteus, a sterilehybrid between the diploid species M. sinensis and the tetraploidspecies M. sacchariflorus has high potential for biomass in temper-ate regions (Glowacka, 2011).

ll rights reserved.

sis Goliath; Aug, M. sinensislimated; PAL, phenylalaninease; UA, uronic acid; GAX,

-glucan.cardie Jules Verne, EA 3900-Tel.: +33 322 827 536.ayon).

A serious drawback for the use of M. x giganteus is its high sen-sitivity to frost damage in the first year of planting (Lewandowskiet al., 2000; Jezowski et al., 2011). Important factors in improvingthe survival of M. x giganteus in the first year of planting arerhizome size, depth of planting and the effect of cold storage onrhizome emergence (Pyter et al., 2010). In Northern Europe, M. xgiganteus is less frost-tolerant than M. sinensis during the first win-ter after planting, whereas M. sacchariflorus is more adapted to awarmer climate (Lewandowski et al., 2000; Farrell et al., 2006).Frost reduces growth to variable extent in different Miscanthusgenotypes (Farrell et al., 2006). Chilling and freezing damage mod-ify transcriptional and plant metabolism with consequential effectson many biological functions (Stitt and Hurry, 2002; Goulas et al.,2006; Chinnusamy et al., 2007; Chang et al., 2010). The response tocold involved the barrier properties of the plasma membrane andthe cell wall (Hoson, 1998; Fujikawa and Kuroda, 2000; Yamadaet al., 2002).

Miscanthus, rice and maize are members of commelinid mono-cots, which make a type II cell wall (Carpita and Gibeaut, 1993).Type II primary cell walls contain small amounts of pectic

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polysaccharides and xyloglucans, but glucuronoarabinoxylans(GAX) and mixed-linkage (1 ? 3),(1 ? 4)-b-D-glucan (b-glucan)as the principal matrix polymers that interlace cellulose microfi-brils (Carpita and McCann, 2008). Unlike type I walls, ferulic andp-coumaric acid arabinosyl esters cross-link GAX in the primarywall as well as the secondary wall into a stiff matrix (Carpita,1996; Grabber et al., 2004).

In dicotyledons, cold stress induced accumulation of cell wallcomponents including pectin and non-cellulosic components. Theincrease in pectin induces leaf tensile strength and wall thickness,thus protecting cells from freezing-induced damage in winter oil-seed rape leaves (Kubacka-Zebalska and Kacperska, 1999). Inwheat, a decrease in (1 ? 3),(1 ? 4)-b-D-glucan and the branchingof GAX are observed in the beginning of a cold treatment (Zabotinet al., 1998).

The acclimation to cold displays an increase in cell wall metab-olism gene expression or enzyme activities including pectinmethylesterase in Arabidopsis and oilseed rape leaves, polygalac-turonase in pear, and b-glycosidases in wheat (Zabotin et al.,1998; Fonseca et al., 2005; Qu et al., 2011), leading to cell wallloosening or cell wall stiffening. Cold induces the biosynthesis ofcell wall protein structure such as proline rich protein in rice(Gothandam et al., 2010).

Cold also induces phenolic metabolism. A proteomic study inrice, shows an increase in PAL in response to cold (Cui et al.,2005), and another study on winter wheat leaves reports adecrease in PAL activity associated with an increase in solublephenolic compounds without any changes in lignin content. Theopposite was observed in wheat tillering nodes (Olenichenko andZagoskina, 2005). Other studies show an increased amount of sol-uble phenolic compounds which is correlated with a PAL activityincrease in oilseed rape leaves (Solecka and Kacperska, 1995). Inbarley leaves, lignin gene expression including CAD was upregu-lated under cold stress (Janská et al., 2011). These authors sug-gested that monolignols rather than lignin were biosynthesizedin the leaf of barley since peroxidase genes involved in lignin bio-synthesis were down-regulated. In transgenic tobacco plants, aCAD gene from sweet potato was highly induced in response tocold (Kim et al., 2010).

For Miscanthus, cell wall composition varies with genotype andharvesting time. There is a trend for lignin and cellulose to increaseand non-cellulosic polymers to decrease between early and lateharvesting dates (Hodgson et al., 2010, 2011). Furthermore, M. xgiganteus and M. sacchariflorus genotypes are higher in ligninand cellulose and lower in non-cellulosic wall components thanM. sinensis (Hodgson et al., 2010, 2011). M. x giganteus shows dif-ferences in the cell wall composition in its aerial organs includinginternodes, leaves and sheath harvested in the late fall or winter(Le Ngoc Huyen et al., 2010). These authors report that celluloseis the main cell wall polysaccharide, especially in internodesand GAX the main non-cellulosic polymer which contains lessarabinose ramification in the internode compared to leaves andsheaths. Galactose is higher in leaves and sheaths than in theinternodes. The highest lignin content is in the basal internodessuggesting a thicker secondary wall than in leaves and sheaths.The S/G ratio of lignin increases at late harvest in the organs whileno change in cellulose is observed.

We describe here the effects of cold acclimation on cell wallcomposition in Miscanthus genotypes that vary in frost sensitivity.M. x giganteus (Gig) is particularly sensitive to frost at the juvenilestage, whereas two ecotypes of M. sinensis, Goliath (Gol) andAugust Feder (Aug), display intermediate to strong frost tolerance,respectively (Zub et al., 2012). We found a decrease in lignin con-tent in the frost tolerant genotypes during cold acclimationalthough no significant changes in cell wall composition andconcentration were observed. However, the b-glucan content

increased in all genotypes during cold acclimation while the pectinlevel was only increased in the frost sensitive genotype, Gig. Themain changes during cold acclimation occurred with PAL and CADenzyme activities, the enzymes involved in the phenylpropanoidpathway. A marked increase in PAL activity was observed in thefrost-tolerant clones under cold acclimation. A large increase inCAD activity under cold stress was displayed in each clone, butwas largest in the frost-tolerant clone, Aug.

2. Results and discussion

2.1. Growth

The weight of the harvested tissue per plant was recorded in thethree clones (Fig. 1A). The total aerial organ mass ranged between2 and 4.2 g in the three ecotypes up to 3.6 g in Gig to 4.2 g in Golwith 2.9 g in Aug. An increase of 45% in the biomass was observedat the end of the control conditions in Gol, the intermediate frosttolerant genotype while no significant variations were observedin Gig and Aug. At the end of cold acclimation, an increase in thebiomass of 21% in Gig, the frost sensitive genotype, was observedcompared to 10% in Gol and 8% in Aug. These data suggest thatthe plant continued growth in response to cold and the growth ishigher in the sensitive frost genotype. However, these results showhigh variations in mass among plants in both control and cold con-ditions. Variability could be explained by the rhizome size andplanting depth in M. x giganteus (Pyter et al., 2010). The highestbiomass level was observed for Gol during both conditions com-pared to Gig. Gig is the genotype that is known to give higher bio-mass yield compared with M. sinensis (Clifton-Brown et al., 2004).However, plant height and yield are higher in Gol compared to Gigduring the first year of planting (Zub et al., 2011). Further, Gol isone of the tallest plants among M. sinensis clone. Although thesedata come from non-controlled environmental conditions, theycould explain why Gol has higher biomass compared to Gig andAug at a juvenile stage.

We determined the dry cell wall mass by isolating it from freshfrozen tissue (Fig. 1B). The cell wall biomass was higher in Gig thanin Gol and Aug in both conditions. During ambient temperaturecontrols, a 2-fold increase in cell wall was observed in Gig whileno significant changes were observed in the frost tolerant geno-types. During cold acclimation, no significant variations have beenseen in the three clones. The cold treatment had no significant ef-fect for cell wall accumulation. Some similar results have beenshown in oilseed rape leaves under cold condition (Solecka et al.,1999).

2.2. Fundamental cell wall composition

Cellulose content was 40% of total mass at the onset of theexperiment in Gig compared to 34% in Gol and 33% in Aug. No sig-nificant variations were found in the cellulose content in any cloneas result of cold treatment (Fig. 2A and SupplementaryTable S1).Total lignin ranged between 21% and 24% of total mass in the threeecotypes up to 24% in Gig to 21% in Aug, with 22% in Gol (Fig. 2B).In ambient temperature controls, the lignin content remained at asteady level in Gig and Gol. A transient decrease was observed inAug. No changes in proportion of lignin content of Gig were foundduring cold treatment. In contrast, a transient decrease in lignincontent was observed in Gol and at the end of cold acclimationin Aug (Fig. 2B). The non-cellulosic polysaccharides were estimatedto be ranged between 36% and 45% of total mass for Gig comparedto 41–45% for Gol and 46–56% in Aug (Fig. 2C). In ambient temper-ature controls, the non-cellulosic content remained at the samesteady state level in Gol and Aug while a 20% increase was

Fig. 1. Mean growth rate (A) and dry cell wall mass (B) for Miscanthus plants developed in climate controlled chamber. Gray columns represent the experiment at day 0; blackcolumns represent the non-cold-acclimated conditions (NA) and white columns the cold-acclimated conditions (CA). Harvest dates: after 28 days of the nursery period (day 0,D0) and after 4 and 8 days of cold acclimation Values are expressed in gram of fresh material. Mean values ± SD from four samples.

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observed in Gig. No significant variations were found in the non-cellulosic content in any clone as result of cold treatment(Fig. 2C). Values are in general in agreement with other reportson Miscanthus species (Hodgson et al., 2010, 2011; Le Ngoc Huyenet al., 2010; Lygin et al., 2011) and other maize (Sindhu et al., 2007)except for hemicellulose values where the range is from 24% to 34%(Hodgson et al., 2010; Lygin et al., 2011). Such discrepancy couldbe due to the different analytical procedures to quantify non-cellulosic materials. In our case, our values are based only onorganic constituents. Finally, no significant changes occur in thefundamental cell wall composition during cold acclimation inthe three clones at a juvenile stage, except for transient decreasesin lignin for Gol and Aug.

2.3. Activities of enzymes in monolignol synthesis

Activity of two enzymes involved in the phenylpropanoid path-way to lignin were assayed to investigate their induction undercold stress. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activitywas assayed according to Hano et al. (2006). At the onset of theexperiment, there were no significant differences in PAL activitybetween Gig and Aug. Gol showed a lower enzyme activity levelwith 0.10 nkat/mg protein compared to 0.16 in Gig and 0.18 inAug (Fig. 3A). Under ambient growth, a 1.5-fold decrease in PAL

was measured in the frost sensitive clone, Gig, while a 2-fold in-crease was observed in Gol and no significant variations occurredin Aug.

During cold acclimation, there were no significant changes inPAL activity in Gig, but a 1.5-fold increase in enzyme activity wasobserved in Aug, the most frost-tolerant genotype, at the end ofcold acclimation. A similar trend occurred in the intermediate frosttolerant, Gol. These data suggest changes in secondary metabolismpathways in response to cold in the frost tolerant lines. PAL is thefirst step enzyme in the biosynthesis of hydroxycinnamic acids(ferulate, coumarate) in the phenylpropanoid pathway as well asmonolignol synthesis. During cold acclimation an increase in feru-late molecules can make cross linking with the arabinoxylan chainsand between the arabinoxylan and the lignin thus increasing thecell wall rigidity. Increases in PAL activity could also indicate in-creases in soluble phenolics in addition to monolignol in Aug. Thiscould also contribute to the protection of the cell from reactiveoxygen species (ROS) as it has been discussed in oilseed rape plants(Olenichenko and Zagoskina, 2005). However, as ROS is involved inlignification, the appearance of soluble phenolics along withhydroxycinnamates and lignin directed to the interface of the plas-ma membrane and cell wall could be to scavenge free-radicals gen-erated during lignification (Ros-Barcelo et al., 2006; Dunand et al.,2007; Gill et al., 2010). This could prevent any plasma membrane

Fig. 2. Biomass cell wall in Miscanthus plants. (A) Cellulose content. Cellulose was determined using the acetic–nitric acid reagent (Updegraff, 1969) and modified by Fosteret al. (2010). (B) Lignin content. Lignin was determined using the acetyl bromide method (Hatfield et al., 1999). (C) Non-cellulosic polysaccharides. Non cellulosic wascalculated using the formula:%100 � (% cellulose + lignin). Gray columns represent the experiment at day 0; black columns represent the non-cold-acclimated conditions(NA) and white columns the cold-acclimated conditions (CA). Harvest dates: after 28 days of the nursery period (day 0, D0) and after 4 and 8 days of cold acclimation Valuesare expressed in % of dry cell wall. Mean values ± SD from 12 samples.

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degradation from ROS associated with phenylpropanoid synthesisassociated with cell wall stiffening. Some studies on the effect ofcold exposure on winter oilseed rape plants or wheat leavesshowed changes in soluble phenolic acid but not in lignin content(Solecka et al., 1999; Olenichenko and Zagoskina, 2005). In ourstudy the transient decrease in lignin and the PAL activity increase

in the frost tolerant clones, could suggest a reorientation of PALactivity into soluble phenolics as well as in lignin. The decreasein lignin content could also arise from a down regulation of perox-idase genes involved in lignin polymerization as it has been ob-served in wheat leaves in response to cold (Janská et al., 2011).Another study reported that PAL activity was induced in soybean

Fig. 3. PAL (A) and CAD (B) activity assays in Miscanthus plants. PAL activity was determined as described by Hano et al. (2006) and CAD activity according to Hawkins andBoudet (1994). Activity is expressed in nkat per mg of protein. Harvest dates: after 28 days of the nursery period (day 0, D0) and after 4 and 8 days of cold acclimation. Graycolumns represent the experiment at day 0, black columns represent the non-cold-acclimated conditions (NA) and white columns the cold-acclimated conditions (CA). Meanvalues ± SD from 12 samples.

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roots at low temperature and phenolic acid soluble forms in-creased significantly (Janas et al., 2000). It was also shown thatPAL transcripts and major soluble phenylpropanoids (anthocyaninsand flavonoids) increased at low temperatures in leaves ofArabidopsis thaliana (Leyva et al., 1995; Solecka et al., 1999).Proteomic analysis of cold stress on rice seedlings identified thecold-responsive proteins involved in the biosynthesis of cell wallproteins including PAL (Cui et al., 2005).

A more specific enzyme of the monolignol biosynthetic path-way, cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195), wasmeasured in the three clones. At the onset of the experiment,CAD activity in the cold-sensitive genotype Gig (0.02 nkat/mg pro-tein) was at least half that in Gol (0.04 nkat/mg protein) and Aug(0.05 nkat/mg protein) (Fig. 3B). In the ambient temperature con-ditions, a significant decrease in CAD activity was observed onday 8 in the three clones with a 2-fold decrease in Gig and Golbut up to a 5-fold decrease in Aug compared to day 0. A significantincrease in CAD enzyme activity was recorded in all clonesthroughout the cold acclimation. The activity level was the highestin Aug, the frost-tolerant clone, with a 17-fold increase on day 8(0.7 vs. 0.05 nkat/mg of protein (NA experiment, day 8)). A similarprofile was observed in Gig with a 13-fold increase and, to a certainextent, in Gol with a 6-fold increase on day 8. Finally, CAD showedan increased level in the three clones during cold acclimation andwas higher in the frost tolerant clone, Aug.

These data showed that cold acclimation induced CAD enzymeactivity in each clone with their diverse genetic backgrounds. It hasbeen shown that CAD gene expression is induced in sweet potatoin response to cold (Kim et al., 2010). Our data suggest that mono-lignol biosynthesis increased under cold stress and in associationwith the rapid accumulation of phenolics are predictive of damageyet to come. The discrepancy between the small changes in lignincontent and the increase in CAD enzyme activity during cold accli-mation in each clone could be explained by a lack of time for ligninto accumulate against the background already there, such that themonolignols accumulate but lignification slowed. Another expla-nation could be an inhibition of peroxidase activity involved inmonolignols polymerization under cold stress. It has been ob-served in the leaf of barley that peroxidase genes involved in ligninbiosynthesis were down-regulated under cold stress (Janská et al.,2011).

2.4. Non-cellulosic polysaccharides

The former results showed an increase in PAL activity in thefrost tolerant genotypes than could be related to an increase inester-bound phenolics. These compounds are known to make link-ages with arabinoxylan. Arabinoxylan and the other non-cellulosicsugars were then determined in the three Miscanthus clones.

Fig. 4. Major neutral sugar composition of non-cellulosic polysaccharides in Miscanthus plants analyzed by GC. (A) Xylose, (B) Arabinose, (C) Galactose, (D) Glucose. Graycolumns represent the experiment at day 0; black columns represent the non-cold-acclimated conditions (NA) and white columns the cold-acclimated conditions (CA).Harvest dates: after 28 days of the nursery period (day 0, D0) and after 4 and 8 days of cold acclimation. Relative content is expressed in % mol of sugar. Mean values ± SD from12 samples.

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2.4.1. GlucuronoarabinoxylanAt the onset of the experiments, the most abundant non-

cellulosic cell wall sugars were xylose (�65–67 mol%) andarabinose (�10–20 mol%), with smaller amounts of glucose andgalactose (Fig. 4). These findings are in accordance with thoseobtained for Miscanthus species (Le Ngoc Huyen et al., 2010; Lyginet al., 2011). In the ambient conditions (NA), xylose levelsincreased in Gig from 66 to 74 mol% but decreased markedly inGol from 71 to 58 mol%. No significant changes in xylose contentwere observed in Aug (Fig. 4A). The arabinose content significantlydiffered among the three clones at the onset of the experiment.Frost-sensitive Gig showed the highest amount of arabinose(20 mol%) whereas more frost-tolerant clones Gol and Aug con-tained 16 and 10 mol%, respectively. Under ambient conditions,the arabinose content decreased in Gig from 20 to 14 mol% andslightly increased in Aug from 10 to 12 mol% (Fig. 4B). A steadystate level in arabinose was observed for Gol.

GAX is quantitatively a major non-cellulosic polysaccharide inthe primary and secondary walls of grasses (O’Neill and York,2003; Scheller and Ulvskov, 2010). It is important for the develop-ment of the primary wall in grasses (Christensen et al., 2010; Tooleet al., 2010) due to its potential role in cell wall strength (Leucciet al., 2008).

As GAX is the major cross-linking glycan of the walls of grasses,we combined the mol% of Ara and Xyl to estimate the relative pro-portions of this polysaccharide and also estimated the relative de-gree of branching by the ratio of Ara/Xyl (Table 1). At the onset ofthe experiment, the GAX content was highest in the frost-sensitiveGig at 87 and 82 mol% in the intermediate frost tolerant Gol and76 mol% in the frost-tolerant Aug (Table 1). Under ambient condi-tions GAX remained relatively constant in Gig and Aug but de-creased in Gol to a level similar to Aug. Under ambientconditions, a significant decrease in Ara/Xyl ratio was observedin Gig on day 8 while an increase in the ratio was observed in bothGol and Aug, the frost-tolerant clones. Similar results have beenobserved where Gig has the lowest Ara/Xyl and M. sinensis clonesthe highest Ara/Xyl ratios (Lygin et al., 2011). Thus under ambienttemperature controls, arabinosyl residues would be progressivelyremoved in Gig leading GAX more tightly bound to the cellwall polysaccharides and then reducing ester-bound phenolicsinteraction. Unlike Gig, the increase in the Ara/Xyl ratio in theintermediate frost-tolerant clones (Gol) and the frost tolerant clone

Table 1Calculated arabinoxylan content (Ara+Xyl) and the ratio between arabinose and xylose (Araexpressed in % mol sugar. NA: non-cold-acclimated; CA: cold-acclimated. Harvest dates: aftvalues ± SD from 12 samples.

Genotype Culture condition Samples

M. giganteus NA 048

A 048

M. sin. Gol NA 048

A 048

M. sin. Aug. NA 048

A 048

Aug could make a loosely bound cell wall which could play asignificant role in protecting the cell during abiotic stress (Leucciet al., 2008). Thus, the increase in arabinose substitution on thexylan backbone in the frost tolerant clones might be importantin the hydration status of the cell wall i.e. cold tolerance. Theseresults suggest a genotype effect on cell wall composition at thejuvenile stage.

Under cold-acclimated conditions, a slight increase (7%) inxylose content was observed in Gol on day 8 (Fig. 4A) while no sig-nificant changes in that neutral sugar content were seen in Gig andAug (Fig. 4A). The GAX content remained the same throughout thecourse of the cold acclimation experiment in Gig and Aug. The Ara/Xyl ratio remained the same in Aug, the frost tolerant clone while,a slight increase in the Ara/Xyl ratio was seen at the end of coldacclimation in the frost-sensitive clone (Gig) suggesting an in-crease in arabinosyl side-chains on the xylose backbone (Table 1).The GAX content was significantly higher at the end of cold accli-mation in Gol with a decrease in the Ara/Xyl ratio compared toambient temperature controls, suggesting a decrease in arabinosylside-chains on the xylose backbone in Gol. These differences inarabinosyl side chains of xylans among the clones could affectthe cross-linkages between the hydroxynamic acids and the GAXresidues and other cell wall polymers in response to cold. However,the differences are small and in general, the overall profilesobserved in the GAX content and Ara/Xyl ratio during plantdevelopment were relatively similar between ambient and cold-acclimated experiments in Aug and Gig. Gol was the only clonewhere some changes in the GAX content and Ara/Xyl ratio occurredduring the cold acclimation.

No significant changes in galactose content were observed in Gigand Aug between the ambient temperature controls and cold-acclimated plantlets. In contrast, a 2-fold decrease was observedin Gol at the end of cold acclimation (6.52–3.35 mol%) (Fig. 4C).

In Gol, the decrease in galactose under cold stress could be re-lated to changes in the expression or activity of such enzymes.Galactose can arise from pectic side-chains, including galactan,which are very important in the hydration status of the cell wall(Willats et al., 2001). Galactose may also arise from xyloglucan.However, the xyloglucan structure found in grasses is differentfrom that in dicotyledons and can present some galactose substitu-tion but only to a very small degree (Shibuya et al., 1983; Gibeautet al., 2005).

/Xyl) based on cell neutral sugar composition of Miscanthus plants.Relative content iser 28 days of the nursery period (day 0), after 4 and 8 days of cold acclimation. Means

Ara / Xyl Ara + Xyl

Mean SD Mean SD

0.29 0.05 86.64 1.330.25 0.03 88.97 0.970.19 0.05 87.54 1.170.29 0.05 86.64 1.330.25 0.01 87.30 0.940.22 0.03 86.62 1.58

0.25 0.01 81.51 5.450.25 0.03 82.24 3.50.31 0.1 74.26 17.430.25 0.01 81.51 5.450.22 0.01 71.83 3.710.26 0.01 84.89 3.02

0.15 0.01 75.49 3.190.20 0.01 78.87 1.70.20 0.01 76.77 2.010.15 0.01 75.49 3.190.21 0.01 77.79 1.250.21 0.01 78.59 1.7

Fig. 5. (1 ? 3),(1 ? 4)-b-D-glucan content (A) and uronic acid (B) content in Miscanthus plants. NA: non-cold-acclimated; CA: cold-acclimated. Gray columns represent theexperiment at day 0, black columns represent the non-cold-acclimated conditions (NA) and white columns the cold-acclimated conditions (CA). Harvest dates: after 28 daysof the nursery period (day 0, D0) and after 4 and 8 days of cold acclimation. b-glucan was estimated using a standard assay based on the release of glucan oligomers producedby digestion of cell wall material with lichenase (Megazyme). Values are expressed in percentage of (1 ? 3),(1 ? 4)-b-D-glucan in dry cell wall. The uronic acid content wasdetermined colorimetrically by the Meta-hydroxydiphenyl assay according to van den Hoogen et al. (1998). Relative content is expressed as % of uronic acid in dry cell wall.Mean values ± SD from 12 samples.

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2.4.2. (1 ? 3),(1 ? 4)-b-D-glucanThe proportion of b-glucan was low, from 1.5% to 1.8% (W/W),

and similar in all clones at the onset of the experiment (Fig. 5A).It remained the same during the ambient temperature experimentin Gig and Gol and increased in Aug up to 1.5-fold. Under coldacclimation, an increase in b-glucan content was observed in allclones. That level was up to 54% in Gol to 36% in Gig and with35% in Aug compared to the ambient conditions.

Cold acclimation induced an increase in the amount of b-glucanin the three genotypes and is higher in the frost tolerant clones.The low amount could be explained by the development stage ofMiscanthus clones. For example, in Poaceae species, b-glucan isassociated with growing cells and declines when growth hasceased, being replaced by arabinoxylan (Carpita et al., 2001; Obelet al., 2002; Buckeridge et al., 2004). Temperature can affectthe b-glucan content in oats and barley and lower it in cold(Saastamoinen, 1995; Anker-Nilssen et al., 2008). However someother studies reports contradictory results or no correlation be-tween temperature and b-glucan content (Lim et al., 1992; Anders-son and Börjesdotter, 2011). Another study on drought stress inwheat showed an increase in b-glucan in the drought tolerant

wheat genotype (Leucci et al., 2008). The increase in b-glucan dur-ing cold acclimation could be more related to the inhibition of b-glucanase activity that may decrease the b-glucan degradation asit was observed in wheat under aluminum stress (Hossain et al.,2006). However, it could contribute to a lower level compared toGAX to the cell wall stiffness in association with phenolics/GAXcrosslinks formation.

2.4.3. Uronic acid contentThe uronic acid (UA) content which reflects the galacturonic

and glucuronic acid contents found in pectins and GAX, respec-tively, was low in the three clones suggesting not only a very smallamount of pectins but also low glucuronic acid substitution onarabinoxylan in these plants (Fig. 5B). Total UA ranged between0.2% and 0.3% of total mass in the three ecotypes up to 0.3% inGig and Aug, with 0.2% in Gol (Fig. 5B). In ambient conditions, a33% and 18% decrease in UA were observed in Gig and Gol respec-tively while a 30% increase in UA was displayed in Aug. Under coldacclimation, a 2-fold increase in UA was seen in Gig while a de-crease was found in the frost-tolerant clones at the end of coldacclimation. Cold acclimation induced an increased level in UA in

J.-M. Domon et al. / Phytochemistry 85 (2013) 51–61 59

the frost sensitive genotype, Gig. No significant differences in theamount of glucuronic acid in several Miscanthus genotypes havebeen reported (Lygin et al., 2011). UA has been shown under coldstress, to be induced in dicotyledons (Kubacka-Zebalska and Kac-perska, 1999). The cell wall of grasses, including rice, barley, maize,switchgrass and Brachypodium, is known to contain few pectins(Gibeaut et al., 2005; Dodd and Cann, 2009; Christensen et al.,2010; Konishi et al., 2011). Another study reports an increase inUA associated to pectin in wheat under abiotic stress but not inthe non-cellulosic components (Hossain et al., 2006). That resultcould indicate some differences more in pectin level betweengenotypes during cold acclimation than in GAX. Furthermore, anincrease in galactose in Gig suggests an increased synthesis in pec-tin, which may act as a gelling agent preventing from water dehy-dration. However, the influence of pectin in cell wall structure maynot be very important due to the low content of pectin in the cellwall of Miscanthus genotypes.

3. Concluding remarks

The three clones had similar but also some specific differencesin cell wall composition before cold acclimation. In response tocold, it appears that the most frost-tolerant clone makes phenyl-propanoid components to protect the cells upon freezing. Thesevariations indicate that plants have different genetic backgroundsleading to different cell wall compositions which could explainthe diverse responses to frost tolerance in these clones. Neverthe-less, our study was performed on only three clones so do not reflectthe genetic diversity of Miscanthus.

Further understanding of the phenolic metabolism is necessaryfor clarification of cold acclimation to frost tolerance mechanism inthese genotypes. Because a correlation exists between changes inphenolic metabolism and genotypes during cold acclimation, thehydroxycinnamate content has to be assessed in the different lines,to investigate to which extent arabinoxylan makes cross-linkagesin the cell wall. Alteration in the GAX phenylpropanoid networkcould have downstream consequence for biomass quality.

Drastic alterations in PAL and CAD activities underscore theneed for global assessment on cell wall and phenolic metabolismby RNA seq. Unbiased identification of genes involved with frosttolerance will require recombinant inbred lines of two M. sinensislines that vary widely for the trait.

4. Experimental

4.1. Plant material

Two experiments with ambient and cold acclimation were per-formed using climate-controlled chambers. Twelve plants of eachecotype (M. x giganteus, M. sinensis Goliath and M. sinensis AugustFeder) were grown in a growth chamber according to conditionsdescribed by Zub et al. (2012). Briefly, plants were grown by plant-ing rhizome in a growth chamber (24 �C day/21 �C night) with a14-h photoperiod and light intensity of about 100 lmol m�2 s�1

for 28 days. Plants were then cold acclimated (12 �C day/12 �Cnight) with a 12-h photoperiod for a period of 8 days. During thetime course of the experiments, aerial parts from 7-leaf stages offour plants from each clone were harvested at different samplingdates: after 28 days of the nursery period (day 0) and after 4 and8 days of cold acclimation (CA, 4, 8). For control conditions, sam-ples were harvested on the same developmental stage day as inCA conditions. Aerial parts of each plant were frozen in liquidnitrogen, ground to a fine powder in a ball mill and kept frozen(�80 �C) until further use.

4.2. Preparation of cell wall material

Plant cell wall material was prepared according to Carpita et al.(2001). Briefly, frozen plant powder (100 mg) was washed twice inabsolute ethanol at 70 �C. The extract was homogenized in 50 mMTris–HCl (pH 7.2) containing 1% (W/V) SDS detergent at 70 �C for30 min. Cell walls from the homogenate were collected on a nylonmesh filter disk (Millipore, 41 lm) positioned in a Millipore-typemanifold, washed sequentially with plenty of water, ethanol,acetone, and then put back into water. Finally, the material wasfreeze-dried. Dry cell wall material (DCW, 2 mg) was digested witha-amylase according to Fleischer et al. (1999). Briefly, the cell wallwas suspended in 100 mM potassium phosphate, pH 6.8 (500 ll),and digested for 24 h at room temperature with a-amylase (0.51U of a-amylase from Bacillus subtilis Type II A., Sigma mg�1

DCW). After digestion, the cell wall was centrifuged at13000 rpm for 5 min, washed three times with water and freeze-dried. All cell wall extractions were carried out in triplicate for eachstudied plant of each clone.

4.3. Neutral sugar composition of the non-cellulosic polysaccharides

Cell walls were hydrolyzed using 2 N trifluoroacetic acidcontaining 1 lmol of Myo-inositol for 90 min at 120 �C. The triflu-oroacetic acid was evaporated under a stream of nitrogen and thesugars were converted to alditol acetates (Gibeaut and Carpita,1991). Derivatives were separated by gas liquid chromatographyon a 0.25-mm � 30-m column of TR-FAME (Thermo Myo-inositol)according to the method described by Peña and Carpita (2004). Theflame ionization detector was integrated and the neutral sugaramount (mol of sugar per 100 mol of total identified sugars) wascalculated relative to the myo-inositol internal standard. Monosac-charide standards included L-rhamnose, L-fucose, L-arabinose,D-xylose, D-galactose, D-glucose and D-mannose. Sugar analysiswas performed in triplicate for each studied plant (4) of each clone(3) corresponding to 12 values.

4.4. Determination of (1 ? 3),(1 ? 4)-b-D-glucan

The amount of (1 ? 3),(1 ? 4)-b-D-glucan was determined by ab-glucan detection kit (Megazyme) which is based on the releaseof (1 ? 3),(1 ? 4)-b-D-glucan oligomers produced by digestion ofthe material with lichenase (McCleary and Glennie-Holmes, 1985).The procedure was performed as described in the manufacturer’sinstructions except that it was scaled down to 10 mg of dry cell wall.The amount of b-glucan was expressed as% of dry cell wall. Groundbarley was used as a positive control. b-glucan content was deter-mined in triplicate for each studied plant (4) of each clone (3).

4.5. Crystalline cellulose content

The TFA pellet was washed several times with water and dried.Its cellulose content was measured according to the method basedon material resistant to acetic nitric hydrolysis described byUpdegraff (1969) and modified by Foster et al. (2010). Cellulosecontent was determined in triplicate for each studied plant (4) ofeach clone (3).

4.6. Uronic acid assay

Uronic acid (UA) content was determined by the Meta-hydroxydiphenyl method according to Van den Hoogen et al.(1998). An amount of 36 lL of cell walls (from 0.5 to 1 mg/mL)was added to 180 lL of sodium tetraborate, 75 mM in sulfuric acid(72%). After incubation for 1 h at 80 �C and cooling, the backgroundabsorbance of the samples was measured at 540 nm (Powerwave,

60 J.-M. Domon et al. / Phytochemistry 85 (2013) 51–61

Biotek). Thirty-six microliters of Meta-hydroxydiphenyl reagent(M-hydroxydiphenyl 0.15% (W/V) in 5% (W/V) NaOH) were addedand absorbance was measured at 540 nm after 15 min. Uronic acidcontent was expressed as % of dry cell wall. UA analysis was per-formed in triplicate for each studied plant (4) of each clone (3).

4.7. Lignin content

Lignin content was determined using the acetyl bromide meth-od described by Hatfield et al. (1999). Cell wall was isolated from1 g of frozen powder and digested with a-amylase as previouslydescribed. Dry cell wall was incubated with 2.5 mL of acetylbromide reagent (25% (V/V) acetyl bromide in glacial acetic acid)for 4 h at 50 �C. After cooling, 1.5 mL of the reaction mixture wasspun at 12000 rpm for 3 min. To 500 lL of clarified supernatant,2 N NaOH (2 mL) and acetic acid (2.4 mL) were added. Hydroxyl-amine (350 lL) was added to each sample and diluted to 10 mLwith acetic acid. Absorbance was read at 280 nm. The amount oflignin was calculated using the extinction coefficient that was as-sumed to be 17.688 L g�1 cm�1. Lignin analysis was performed intriplicate for each studied plant (4) of each clone (3).

4.8. Enzyme assays

PAL activity was assayed according to Hano et al. (2006). Solu-ble proteins were extracted from 1 g of fresh frozen tissue byhomogenization in 3 ml of 0.1 M sodium borate buffer (SBB) pH8.8 containing 14 mM dithiotreitol and 0.2 M phenylalanine andkept on ice for 30 min. After centrifugation, the supernatant wascollected and used in the assay. Protein concentrations were mea-sured by Bradford method (1976). The reaction mixture contained50 lg of proteins and 50 mM of phenylalanine in 5 mL of SBB. PALactivity was monitored for 3 h at 40 �C by measuring the produc-tion of Trans-cinnamate at 290 nm every 15 min.

CAD specific activity was determined spectrophotometrically asdescribed by Hawkins and Boudet (1994). Soluble proteins wereextracted from 1 g of fresh frozen tissue in 1.5 ml of 0.1 M TrisHCl, pH 7.5, Ethylene Glycol 5% (W/V), PVP 2% (W/V) 0.1 M bmercaptoethanol. The crude extract was centrifuged (10 min,16000g) at 4 �C and the surpernatant was used in the assays. Pro-tein concentrations were measured by Bradford method (1976).The reaction mixture contained 50 lg of proteins in 100 mM KH2-

PO4/Na2HPO4 pH 6.25, 5–100 lM coniferaldehyde, and 100 lMNADPH. The oxidation of NADPH was followed at 340 nm. Enzymeactivity assays were performed in triplicate for each studied plant(4) of each clone (3).

4.9. Statistical analysis

Analyses of variance were performed to test the differences be-tween treatments (NA/CA) and genotypes at different samplingdates. They were all carried out using SAS 8.0 software (SAS Insti-tute 1999) and means were classified using the Newman–Keulsrank test at the 0.05 probability level.

Acknowledgments

This work was supported by a Grant from the Conseil Régionalde Picardie, France, within the framework of the AAP07-05 PAROI-FROID project (2007–2010).

We thank N.C. Carpita (Purdue University, USA) for his review ofthe manuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.phytochem.2012.09.001.

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