Effect of Calcium Sprays on Mechanical Strength and Cell Wall Fractions of Herbaceous Peony (Paeonia Lactiflora Pall.) Inflorescence Stems

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1 Int. J. Mol. Sci. 212, 13, ; doi:1.339/ijms Article OPEN ACCESS Interntionl Journl of Moleculr Sciences ISSN Effect of Clcium Sprys on Mechnicl Strength nd Cell Wll Frctions of Herceous Peony (Peoni Lctiflor Pll.) Inflorescence Stems Chengzhong Li 1,2, Jun To 1, *, Dqiu Zho 1, Cho You 1 nd Jinto Ge 1 1 College of Horticulture nd Plnt Protection, Yngzhou University, Yngzhou 2259, Chin; E-Mils: lichengzhong8@yhoo.com.cn (C.L.); dqiuzho@126.com (D.Z.); kyzy518529@163.com (C.Y.); gjtwd678@126.com (J.G.) 2 Deprtment of Lndscpe Architecture nd Horticulture Yngzhou Voctionl College of Environment nd Resources, Yngzhou , Chin * Author to whom correspondence should e ddressed; E-Mil: tojun@yzu.edu.cn; Tel.: ; Fx: Received: 2 Mrch 212; in revised form: 15 Mrc 212 / Accepted: 19 Mrch 212 / Pulished: 13 April 212 Astrct: Clcium is n essentil element nd imprts significnt structurl rigidity to the plnt cell wlls, which provide the min mechnicl support to the entire plnt. In order to increse the mechnicl strength of the inflorescence stems of herceous peony, the stems re treted with clcium chloride. The results shows tht prehrvest sprys with 4% (w/v) clcium chloride three times fter ud emergence re the est t strengthening D Fugui peonies stems. Clcium sprys incresed the concentrtions of endogenous clcium, totl pectin content s well s cell wll frctions in herceous peonies stems, nd significntly incresed the contents of them in the top segment. Correltion nlysis showed tht the reking force of the top segment of peonies stems ws positively correlted with the rtio of wter insolule pectin to wter solule pectin (R =.673) s well s lignin contents (R =.926) fter clcium pplictions. Keywords: clcium; reking force; cell wll frctions; herceous peony; inflorescence stem

2 Int. J. Mol. Sci. 212, Introduction Herceous peonies (Peoni lctiflor Pll.) re populr nd well-known ornmentl plnts [1,2], which originted in Chin, where most of the cultivrs come from nd the culture history of which hs een known for more thn 2 yers [3 5]. Modern peonies cvs hve een used for commercil cut flower in some countries for mny yers, such s USA, New Zelnd, Isrel, nd so on [6,7]. In recent yers, peonies cut flower production hs een expnded in Chin [8], however, mny cultivrs of peonies in Chin hve flexile peduncle or inflorescence stem due to wek stem strength nd seriously impeded commercil cut flower production. Mechnicl support is provided to the entire plnt ody minly y the plnt cell wlls [5,9], especilly the secondry wll, which functions s the skeletl frmeworks of plnts [1]. The thickness of cell wlls in the sclerenchym nd the numer of vsculr undles re the importnt fctors tht ffect the stem mechnicl strength [11,12]. Clcium is n essentil element s well s crucil regultor of growth nd development in plnts [13], nd prticiptes in cross-linking negtive chrges, especilly on the croxylic residues of pectin, imprting significnt structurl rigidity to the wll [14]. Previous studies indicted tht clcium pplictions could retrd the ending of the gerers inflorescence stems [15,16], nd tht clcium could improve the stem qulity of herceous peonies, such s thickness nd height [17], ut the effect of clcium on the mechnicl strength of peonies stems is uncler. The cell wll of the herceous peony stem is highly orgnized composite structure tht contins cellulose, hemi-cellulose, lignin, polyscchrides, nd proteins [18], ut the effect of clcium pplictions on the cell wll frctions of peonies stems hs not yet een descried. The im of this study is to evlute the effects of clcium on these spects of herceous peonies. 2. Results nd Discussion 2.1. Breking Force The effects of clcium pplictions on the reking forces in inflorescence stems of herceous peonies re shown in Tle 1. Quntittive nlysis showed tht the force required to rek the stems which were treted with clcium ws significntly higher thn tht required for the control, it ws t lest 118.%, 112.5% nd 16.4% in the control stems for the top, middle nd ottom segment, respectively. For the top segment, Tretment 3 (4% clcium three times) ws the est, nd significntly different from other schedules, Tretment 2 (4% clcium twice) ws second est, nd the effect of Tretment 4 (2% clcium three times) ws the worst. For the middle segment nd the ottom one, Tretment 3 ws oth the est, however, other schedules showed no cler difference. So we could rech the conclusion tht prehrvest sprys with 4% (w/v) clcium chloride three times fter ud emergence were the est for the mechnicl strength of D Fugui peony stems. The effect on the reking force ws significntly reduced when incresing the concentrtion to 6%, which mye in excess of the clcium sorption cpcity of the plnt. In ddition, when decresing the concentrtion to 2%, insufficient clcium supply my hve occurred, regrdless of the times of tretment.

3 Int. J. Mol. Sci. 212, Tle 1. Breking force mesurement in herceous peony inflorescence stems with different clcium spry schedules. Spry schedules Breking Force (N) Top Middle Bottom Control (wter) 21.7 ±.8 e 53.6 ± 3.1 c 16.7 ± 4.5 c Tretment 1 (4% clcium once) 28.4 ± 1.4 c 6.3 ± ± 3. Tretment 2 (4% clcium twice) 29.6 ± ± ± 3.9 Tretment 3 (4% clcium three times) 32.9 ± ± ± 4.9 Tretment 4 (2% clcium three times) 25.6 ± 1.6 d 63. ± ± 2.8 Tretment 5 (6% clcium three times) 27. ± 2.1 cd 61.6 ± ± 2.8 Dt re men vlues ± SE for 6 stems. Different letters ehind the figures in ech column indicte significnt difference t P =.5 mong different clcium spry schedules. The stems were spryed three times with 4% (w/v) clcium chloride. Clcium concentrtions together with cell wll mterils (CWM) contents in the stems were nlyzed to investigte the reltionship etween the reking force nd these fctors Clcium Concentrtions Clcium sprys incresed clcium concentrtions in peony stems regrdless of the segment (Tle 2). The results were 111.1%, 16.9% nd 15.9% in the control stem for the top, middle nd ottom segment, respectively. There ws significnt difference etween tretment nd the control in the top segment, nd none of the clcium sprys led to significnt increse of clcium concentrtion in the middle or the ottom prt. Tle 2. Clcium concentrtions (unit: μg/g D.W.) in herceous peony inflorescence stems fter sprys with clcium chloride (4%, w/v). Tretments Clcium concentrtion Top Middle Bottom Control 14.4 ± ± ±.81 Clcium sprys 16.3 ± ± ±.15 Dt re men vlues ± SE for 2 stems. Different letters with the columns indicte significnt difference t P =.5 etween control nd clcium treted stems Cell Wll Frctions Pectin in Different Forms For different pectin forms, regrdless of the stem segment, clcium sprys reduced the contents of the wter solule pectin significntly (Figure 1A). Menwhile, it incresed the wter insolule pectin contents significntly (Figure 1B). In other words, clcium sprys incresed the rtio of wter insolule pectin to wter solule pectin. Correltion nlysis showed tht the rtio ws positively correlted with the reking force of the top segment peony stems (R =.673). Clcium pplictions

4 Int. J. Mol. Sci. 212, incresed the contents of totl pectin of the peony stems (Figure 1C), The contents indicted gret increse in the top segment, ut not in the middle or the ottom one. Figure 1. Contents of wter solule pectin (A), wter insolule content (B) s well s totl pectin (C) in different segments of herceous peony inflorescence stems fter sprys with clcium chloride (4%, w/v). Different letters ove the columns indicte significnt difference t P =.5 etween control nd clcium treted stems. 5 W te r s o lu le p e c tin c o n te n ts (μ g /m g ) C lc iu m tre tm e n t C o n tro l A W te r in s o lu le p e c tin c o n te n ts (μ g /m g ) B T o t l p e c tin c o n te n ts (μ g /m g ) C T o p M id d le B o tto m Cellulose, Hemi-Cellulose nd Lignin Clicum sprys significntly incresed cellulose contents in the top segment of the peony stems, it ws 128.5% in the control stems, ut for the middle nd the ottom segments, it ws 15.2% nd 12.9% in the control stems, respectively, nd there ws no significnt difference etween tretment nd control (Figure 2A). Clcium sprys led to significnt increse of hemi-cellulose nd lignin contents, regrdless of the segment of the stems (Figure 2B,C). Correltion nlysis showed tht the reking force of the top segment stems ws positively correlted with lignin content (R =.926) fter clcium pplictions.

5 Int. J. Mol. Sci. 212, Figure 2. Contents of cellulose (A), hemi-cellulose (B) nd lignin (C) in different segment of herceous peony inflorescence stems fter sprys with clcium chloride (4%, w/v). Different letters ove the columns indicte significnt difference t P =.5 etween control nd clcium treted stems. Cellulose content(μg/mgd.w.) Clcium tretment Control A Lignin content(μg/mgd.w.) Hemi-cellulose content(μg/mgd.w.) B C Top Middle Bottom 3. Experimentl Section 3.1. Plnt Mteril The experiments were crried out with 3-yer-old D Fugui, one of the populr herceous peony cultivrs in Chin, which were cultivted in the ornmentl plnts grden of Yngzhou university, Jingsu Province, Chin (32 3 N, E). The plnts grew well in the nturl environment with norml mngement.

6 Int. J. Mol. Sci. 212, Clcium Tretments Thirty-six plnts of peonies were chosen for tretments. The chosen stems together with the leves nd uds on them were spryed with clcium chloride (w/v) once week fter the ud emergence. The tretments were s follows: Control (wter), Tretment 1 (4% clcium once), Tretment 2 (4% clcium twice), Tretment 3 (4% clcium three times), Tretment 4 (2% clcium three times), Tretment 5 (6% clcium three times). All the sprys including the control contined surfctnt (.1% Tween-2). Aout 1 week fter ll tretments were complete, we hrvested the stems for experiments, the stge of peonies flower opening menwhile ws loose ud, the outer petls soft nd loosening, firm inside [8,19] Breking Strength Mesurement The inflorescence stems were cut nd seprted into three segments, 5 cm length from the upper pex or distl of the stem s the top segment, the sme length from the proximl nd the midst s the ottom nd the middle one, respectively. The reking strength of ech segment ws mesured y the method of Burk et l. [2] with universl digitl force testing device (Model NK-2; Huier, Hngzhou, Chin), nd force ws pplied mnully until the stems were roken. Stems from 6 plnts corresponding to ech tretment were tested for the reking force Clcium Concentrtion Determintion Clcium concentrtion in ech prt of the inflorescence stems ws determined y the method s follows. Ech prt of the inflorescence stems ws clened with deionized wter nd then dried to constnt weight t 7 C. The stems were then ground into powder nd sieved. The weighed powder ws digested with nitric cid nd wter (1:1, v/v) in the microwve digestion system (MARS 5, CEM, USA). Clcium concentrtion ws determined y tomic sorption spectrophotometer (Solr S4 + Grphite Furnce System 97, Thermo Elementl, USA). The experiment ws performed twice Cell Wll Mterils (CWM) Preprtion The cell wll mterils (CWM) were frctioned following the method of Rose et l. [21] with some modifictions. Briefly, the stems were ground into fine powder in liquid nitrogen nd extrcted with 95% lcohol, then wshed twice with oiling lcohol nd methyl lcohol: chloroform (1:1, v/v), respectively. Finlly, the cell wll residues were dried overnight t 5 C nd used for nlysis. The experiment ws performed in triplicte Uronic Acid, Hemi-Cellulose, Cellulose nd Lignin Contents Determintion Pectin content in different forms followed the methods of Mngnris et l. [22] for wter solule pectin nd Selvendrn & O Neill [23] for wter insolule pectin frction, nd the uronic cid ws determined y colorimetric ssy [24], using glcturonic cid s the stndrd. Totl pectin content ws the sum of pectin content in oth frctions. Hemi-cellulose content ws determined y the phenol colorimetric ssy [25] nd the cellulose content ws mesured y the nthrone colorimetric

7 Int. J. Mol. Sci. 212, ssy [26]. Lignin content ws determined following the method of Müse et l. [27]. Mesurements were done in triplicte Sttisticl Anlysis Sttisticl nlyses were conducted using SPSS 16.. Differences in reking strength nd the content of cell wll frctions mong tretments were nlyzed y LSD test t significnce level of.5, one-wy ANOVA. 4. Conclusions Our dt indicted tht prehrvest clcium sprys provided n increse in clcium contents in hercous peonies stems which corresponded to the increse of reking force to fford etter mechnicl support to the plnt ody. The results were in line with the reserch y Hui et l. [28] except for the concentrtions nd sprying times of clcium chloride, nd prtly inconsistent with the reserch y Yu et l. [17]. The minly reson ws the peony cultivrs were different. Clcium is one of the most importnt components to confer strength to cell wlls [29], nd over 6% of clcium compounds in plnts were ssocited with pectin [3,31]. Clcium is ccumulted in the middle lmell [32], ound inside nd etween pectin nd other components forming structure nmed egg ox [33,34]. Clcium mkes cell wlls rigid; if clcium ions were t high level, the pectin chins would e cross-linked nd ggregted, nd the wll mximlly rigidified [14]. Our dt indicted tht the rtio of wter insolule pectin to wter solule pectin rised fter clcium ppliction, which hd high positive correltion with the reking force of the peonies stems. Studies on the effectiveness of sprying clcium in pech storge lso found tht clcium ppliction could fortify the rigidity of cell wlls of the pech y high wter insolule pectin [35,36]. Our dt indicted tht clcium sprys were more effective in the top segment of herceous peonies stems regrding reking force, which hd high positive correltion with lignin contents. Previous studies on Aridopsis [2,37], crops [5,1,11,38] nd ornmentl plnts [39] indicted tht the mechnicl strength of the stems ws highly correlted with the content of the secondry cell wll components, such s cellulose, hemicelluloses, nd lignin, which prtly greed with our studies on herceous peony stems. Acknowledgments This work ws finncilly supported y Agriculturl Science & Technology Independent Innovtion Fund of Jingsu Province (CX[1]114, CX[11]117, CX[11]315), Agriculturl Science & Technology Support Project of Jingsu Province (BE211325), the Priority Acdemic Progrm Development from Jingsu Government nd 21 College Acdemic Science nd Technology Innovtion Fund of Yngzhou University for student (Bei Du & Qing-ping Geng). We thnk Bin Xu of Virgini Tech for his kind revision of the mnuscript.

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