Decomposition dynamic of higher plant pigments by HPLC analysis

Jourmal of Enionmertal Sciences Vol. 16, No.5,pp.847- -850,2004155N 101-0742CNI1- 28629/XArticle ID: 101-0742(200)05-0847-04CLC number: X17Document code: ADecomposition dynamic of higher plant pigments by HPLC analysisLUO Yil , ZHANG Ting zhou'.3, ZHOU Qi-xing?"，MAO Da-qing'，WAN Dong mei'(1. Departnent of Environmental Science， Liaoning University, Shenyang 110036， China. E-mail: yiluo90 @ hotmail. com; 2. Key Laboratory ofTerrestrial Ecological Process, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 10016, China; 3. Department of Food Science,Biotechnology & Environmental Engineeing, Hangzhou University of Commere，Hangzhou 310035， China; 4. Faculty of Agriculural and AppliedBiological Sciences, Leuven University, Leuven B-3001 ，Belgium; 5. Department of Life Science ，Liaoning University, Shenyang 110036, China)Abstract: The fate of the itter of dominant vegetation( willows and reeds) is one of the aspects studied in the frameof the project “Onderzoek Mileu Effecten Sigmaplan". One of the questions to be considered is how long the ltterstays within the estuary. In this paper, the time the leaf ltter( Salix triandra and Phragmites australis) stayed in theSchelde estuary was studied by using plant pigment as biomarkers with HPLC application. After analyzing the originaldata from the incubation experiment described by Dubuison and Geers( 1999)，the decomposition dynamics patternsof pigments were analyzed and described, and these decomposition dynamics patterns were used as calibrationpatterns. By using Spearman Rank Order Correlation, the calibration patterns of the pigments which were significant(p< 0.05) were grouped. In this way, several groups of the calibration patterns of pigment decomposition wereachieved. The presence or absence of these groups of pigments ( whether they can be detected or not from HPLC)was shown to be useful in determining the time the ltter has stayed in the water. Combining data of DW and POC,more precise timing can be obtained.Keywords: decomposition dynamics patterm; Salix triandra; Phragmites australis; Speaman Rank OrderCorrelation; Schelde Estuary; HPLCenergy input into the river systems. As a result the litterIntroductiondecomposition has long been studied in previous researchesHigh- performance liquid chromatographic ( HPLC )(Hill， 1996; Whiles， 1997; Latter, 1998; Tam， 1998;systems were developed for higher plant pigments in the lateKuehn，1998). We then use the pignents decomposition1970' s and early 1980' s. Subsequently, more complexdynamics pattem cornbining the POC ( particulate organicHPLC systerns were developed for chlorophylls andcarbon) and DW( dry weight) data to infer the time which thecarotenoids from microalgae and natural phytoplanktonltter stayed in the water body. This can be acted a thepopultions( Gieskes, 1991; Hdgson, 1997; Bris, 1998).biomarker to investigate the fate of the leaf ltter in theBoth reversed phase and normal phase systems were used.aquatie ecosystem( Sun, 2001).The HPLC technique was used more recently for plantPrevious studies ( within OMES) have described thepigment analysis, and it has led to a significant increase indecomposition dynamics pattermn of mass loss of vegetationseparation and in the number of pigments that can bealong the Schelde Estuary for willow and reed whenidentified in water and sediment samples( Hodgson，1997).submerged in the water in terms of DW and POC( Dubuisson,None method was ideal to be used for analysis for the 50 or so1999).chloropyll, carotenoids and degradation products likely toThe double exponential model implies a quick decline ofbe important in aquatic systems (Zhou, 2000; 2001a). NotDW(μg/mm2 ) or P0C(g/mm2 ) during the first week and aonly were selective HPLC methods needed for accuratevery slow decline afterwards for both willow and reedquantiative analysis of chlorophylls free from degradation( Dubuisson，1999 ). Considering the speed in replicateproducts but also pigments unique to certain algal classes，measurements it would be difficult to deduct the time thetaxa or processes needed to be unequvocally separated. Theleaves have been decomposed ( in the water ) from thesevalue of such pigments in field oceanography was becomingcurves. Thus we have to find other methods which could giveapparent(Zhou, 2001b).us a better decomposition dynamics pattem. The possibility toOne of the branches of the OMES ( Onderzoek Milieulook into pigment as “ biomarkers”of the duration offfecten Sigmaplan) Project which was approved by the中国煤化Ithe leaves have beenBelgium Govemment was to study the pigments decormpositionsubmerg:YHCNMH(this study . Pigmentsof dominant species of willow and reed leaf ltter along thewere uscu as uromankens 1 us suuy， to determine theSchelde Estuary. The riparian trees could be an importantretention time of the leaf litter in the Schelde, to describe theFoundation item: The National Natural Science Foundation for Distinguished Young Scholars( No.20225722) supported by the National Natural Science Foundation ofChina; the Shenyang S&T Planned ltem( No. 2001228-05-01) by S&T Bureau of Shenyang; * Corresponding author.848LUO Yi et al.Val. 16pigments decomposition dynamics pattern.significantly correlated， p < 0.05) were grouped， anseveral groups of decomposition calibration patterms of1 Materials and methodspigments for willow and reed leaves were described .1.1 MaterialsFor group 7(Fig.1), we can see that before 57 d ofHigher plants species willow( Salix triandra) and reedincubation, the concentration of the pigments ( RT 19.87( Phragmites australis) were selected .min, 22.12 min, 22.26 min) is fairly low and remained1.2 Samplingconstantly( absorbance peak below 2000 AU/24.52 mm2 ),Leaves of standing shoots of willow and reed wereeven could not be detected when incubation time was from 24collected in October, 1998 from the Schelde Estuary neard onward. After 57 d, the concentration increased sharplyDendermonde. They were put into the nets whose mesh sizeuntil at the end of the incubation experiment .is 500 μm and submerged into the water. During 3 months ,When we use the calibration pattern to infer the“age"leaves were collected from the nets at certain periods ( Oct.of the leaf ltter, since we only consider the presence(“+”)12, 19, 26; Nov. 5, 18; Dec. 8 in 1998 and Jan. 14 inor absence(“-”) of the pigment from HPLC detection,1999).while, the calibration pattem for group 7(reed) did not give1.3 HPLC measurementus the informationof“-”within 57 d of incubation(Fig.1).1.3.1 Pigment extractionHowever, if we use the concentration(in terns of AU/24.5Pieces with an average surface of 24.52 mm2(土10%)mm' ), the patterm still could be used, as variability for thesewere cut out of the leaves with a perforator. For eachdata was limited .sampling date， 3 leaves were analyzed， and 3 replicate50000pieces were taken out of each leaf. Each piece of leaf was40000士198730000brought into a test tube with 1.8 ml of 90% acetone. The0000leaf was crushed with a glass rod, and 0.2 ml of filtered1000(0.45 mm)，double distlled water added. The sample was0371424375794then further ground. The test tube was cooled during theprocedure by puting it in ice. When cornpletelyhomogenized, the sample was transferred to a centrifuge tubeFig.1 Pigments decompsitin dynamics pallern of group 7( red)and centrifuged for 3 min. The supematants were decantedIf combining calibration patterm of group 7 anand suck up in a syringe covered with a 0.5 mm filterchlorophyI]- a decomposition pattem ( Luo, 2002) we then( Millipore) .have the ratio( %) of pigment concentration in group 7 to1.3.2 HPLC analysischlorophylk-a as shown in Fig. 2. The ratio is quite lovThe samples werenalysed by using a Waters HPLC,within 57 d of incubation comparing with going up quicklyequipped with a Waters 600 Controller, an autosampler andfrom57 d onwards until the end of the incubation. So, thean absorbance fluorometer detector. A reversed phase column(Spherisorb 0DS2) was used. The fllowing solvents wereratio can also be used as an indicator of the“age”of the ltterused in a gradient protocol: ( 1 ) methanol/0.5 mol/Lwithin 57 d or not.amnonium acetate(80:20); (2) actoitrile/water( 90: 10);14019.87 Chla12022.12Chla(3) ethyl acetate .? 100-22.26Chla2 Results and discussions40202.1 Pigments decomposition dynamics patterns0571424175794The height of the peak of pigments at their specificretention time was shown by HPLC data. The data coveredthe whole time range of the incubation experiment from 12Fig.2 Ratio( % ) of pignent concentation in group 7 toCbla( re)October 1998 till 14 January 1999 .From the original HPLC ollcted data over the wholesenescence is accompanied by the metabolism oftime range of the incubation experiment, the concentration ofchlorophyll to nonfluorescent catabolites ( Hoertensteiner,different pigments versus the incubation time was considered.1999中国煤化工degradation comprisesIn this way, the dynamics pattern of pigment decompositionsever:C NMHG theoccurrence ofcan be described. This can be used as calibration patterms ofintermeauates." Arter removal or pnytol and the central Mgpigment decomposition. By using spearman rank non-chlorophyllase and Mg dechelataseparametric correlation statistics， the pigments which have arespectively, the porphyrin macrocycle of pheophorbide-a issimilar decomposition patterm ( equal of which thecleaved( Hoertensteiner, 1999; Zhou, 2003).concentration... changeswith the incubation time wereNo.5Decomposition dynamics of higher plant pigments by HPLC analysis849Although no pigment identification was done in thisalso make a table with combination with the pigmentstudy, we can still have some useful information from theinformnation, as shown in Table 1.calibration pattem of pigment decomposition. Combining theTable1 The “age" of leat ltter by pigmeat informuation and DW，POCcalibration pattemn of chlorophyl-a decomposition for reeddecomposition for willow(Luo, 2002) and the calibration patterm for group 7( reed ;DW ,4g/mm2POC,ug/mm2> 85<85> 39<39Fig.1), we found the sharp decomposition of chlorophyl-aCGroup1+<7 d7- -24d7- -24 dfrom 57 d onward( until under the limit of detection at the endGroup 7 +of the experiment) were accompanied by a sharp increase of7dthe concentration of the pigments in group 7 from 57 donward. This may imply that the pigments of group 7 may beNole: Group 1, 4, 7 are reviewed in Fig .1 (a, d, b) repecively( Luo, 2002)the degradation products ( chlorophyllides，pheophorbides ，In Table 1, for example, if calibration of group 1 and 7and pheophytins) of chlorophyll-a.are present, with DW > 85 μg/mm2 , the“age" of the ltter isCombining the calibration pattern of chlorophyll-a forless than 7 d. If calibration patterm of group 1 and 7 arereed( L.10, 2002) with the POC data( Dubuison, 1999), thepresent, with DW < 85 μg/mm2 ，the“age” of the liter isratio of POC/Chla was obtained as shown in Fig.3. It showsbetween 7 and 24 d. In this way, we can inferthe“age”ofclearly that within 57 d of incubation, the ratio is fairly lowthe ltter which is less than one week and 7- 24 d. If theand remained constant ( nearly zero)， the ratio increases“age”of the litter is longer than 24 d，we can use thesharply from 57 d onwards until the end of the incubationpigment information as we already described in the previousexperiment. This agrees with the sharp increase of pigmentstudy( Luo, 2002).concentration of group 7( probably the degradation products ofTable2 The “age" of the ltter combining DW loss and calibration patternchlorophyl-a ) , reflecting the converting of chlorophyIl in theof group 1I(RT 10.13) for reedliving tssues to non fluorescent catabolites in the detritus .Group 110.0025+POC/CblaDW, pug/mm20.00200.0015<85 .7-24d> 24d$ 0.0010Note; Group 11(RT 10. 13) decomposition calibration pattern are reviewed inFig. 2(Luo, 2002)05广424375794In Table 2, we can find that if group 11(RT 10.13)(Luo, 2002) is present, and DW > 85 μg/mm2，the“age"Fig.3 Ratio of P0C/Chla for reedof the ltter is less than 7d. If group 11 is present, and DW< 85 pug/mm', the“age”of the ltter is between 7 to 24 d.Furthermore，combining the calibration pattem ofIf group 11 is absent, and DW is < 85 μg/mm2，the“age”chlorophyl-a with the DW data( Dubuison, 1999)， the rationf the liter is longer than 24 d, if combining the ratio ofof DW/Chla shows us the similar trend over the incubationP0C/Chla ( or ratio of DW/Chla， ratio of pigmenttime as the ratio of POC/Chla, as shown in Fig .4.concentration in group 7 to Chla), we can get more precise, 0.006◆DWiChlatime the litter stayed(in the water) between 24 and 57 d ands 0.005里0.004 Ilonger than 57 d.A 0.0032.3 Advantages of the method and further suggestions, 0.0020.00(1) The advantage of the use of pigment analysis by0371424375794HPLC is that after sampling the leaves，we need not analysesthem immediately in HPLC. The leaves can be kept in thedeep freezer( - 85C).Fig.4 Ratio of DW/Chle for reed(2) Identification of the pigments and calibration2.2 Determination of the“age" of leaf litter combiningpattem of the pigments is not very necessary, since we canpigment information and DW，POC decompositiondefine the pigments in terms of their specific retention time.dynamicsTetention time only forIf the“age" of the ltter is shown to be less than 24 dcharacte中国煤化工、vantages. , since smallfrom the pigment information, then we can combine furthershifts ir:YHC N M H Gbetween diferent runswith the DW or POC decomposition dynamics. If the DW weanalysis can not be excluded. This may also createmeasured is higher than 85 pg/mm2，or POC is above 39 μg/problems in comparison of pattems observed by variousmm2，we can infer that the “age”of leaf ltter is less thancolumns and various HPLC instruments. In our application,one week. Otherwise， it is longer than one week. We canwe tried to cover this aspect by using the shift in retention50LUO Yi et al.Vol.16time of a well-known and defined pigment( chlorophyll~b) as[J]. Hydrobiod Bull, 25(1): 65- -72.Hill A R, Brooks A M, 1996. Coarse particulate organic matter inpuls to a head-the accepted range of shift in retention time . The problem iswater swamp stream[J]. Arch Hydrobiol, 137(1): 25also minimized because the method is not only based on theHodgsonD A. Wright s w, Davies N, 1997. Mass spectrometry and reversepresence or absent of any one component, but also consideredphase HPLC techniques for the inifiation of degraded fosil pigments inthe groups of the components.lake sediments and their aplication in palaclinology [J]. Journal of(4) When we infer the“age" of the ltter from theHoertensteiner s, 1999. ChlorophyIl breakdown in higher plants and algae[J].calibration patterm， we only considered the qualitative dataCellular and Molecular Life Sciences, 56(3- -4): 330- 347.whether the pigments concentration can be detected fromKuehn K A, Suberkropp K， 1998. Decmposition of standing liter of theHPLC or not (“+”or“-")，instead of considering thefreshwater emengent macrophyte[J]. Juncus Efusus Freshwat Biol, 40(4):quantitative data( the concentration of the pigments) .717-727.(5) Further identification of the pigments will be usefulLatterP M, Howson G, Howard D M et al. 1998. Lang-term study of literdecomposition on a Pennine peat bog: which regesion? [J]. Oecologia,to study the chlorophyll degradation pathway and degradation113: 94-103.products to help to investigate the fate of leaf ltter in theLuo Y, Tackx M, LiF Y, 2002. Leaf ltere eologcal fate in the Sthelde EstuarySchelde Estuary.in Belgjum[J]. Journal of Environmental Scienes, 14(4): 563-567.SunT H, ZhouQX. LiP J, 2001. Pllution cology[M]. Beiing: Sciene3 ConclusionsTamN F Y，Wong Y s, Lan C Y et al.. 1998. Ltter production andPigments degradation is well studied in phytoplankton,decomposition in a subtropical mangrove swamp receiving wastewater[J].few studies on pigments dynamics in decaying of higher plantsJourmal of Experinental Marine Biolgy and Ecoloy, 226: 1-18.are reported. The reason may be due to more difficult on theWhiles M R, WallaceJ B, 1997. Leaf liter decomposition and macroinvertebraleidentification of pigmentsof. higher plants thanonmmunities in headeater streams draining pine and hardwod calchments[J].Hydrobiologia, 353: 107-119.phytoplankton. Without identification of pigments in thisZhouQ x, Gibson C E, Foy R H, 2000. Long-temmexperiment，we still can get the pigments decompositionpbosphorus loadings to a large lake in north-west Ireland [J]. Waterdynamics by defining each of their retention time from HPLCResearch. 34(3): 922- -926.and get some valuable information on inferring the“ age”ofZhou Q x, 2001a. Chemnical pllution and lransport of oganic dyes in water- soilcrop systems of the Chinee coast [ J ]. Bulletin of Enirmentalleaf ltter( willw and reed) in the Schelde Estuary .Contamination and Toxicology, 66(6): 784- 793.References:Zhou Q X, Huang G H, 2001b. Environrnental biogochemistry and globalenvironmental changes[M]. Beiing: Science Pres.BrisSL, Cuny M RP, Vacelet E, 1998. Characterization o[ bacerial and algalpignents and breakdown products by HPLC in mixed freshwater planktonictrace metals zine, copper and cadmium in three populations of the polychaetepopulatios[J]. Arch Hydrobiol, 143(4): 409- -434.Nerris diversicolor[J]. Jourmal of the Marine Biologieal Asociaion of theDubuison J, GeersJ, 1999. Decomnpositie processen van de oevervegetatie in theUnited Kingdom, 83(1): 65- -72.Schelde[Z] . Thesis Ersmus Hogecool Bnusel.GieskesW W C, Engelkes M M，Kraay G W，1991. Degradation of diatom( Received for review Oetober 8, 2003. Accepted November 20, 2003)chlorophyll to colourles，non-uorecing comppunds during copepod graxing中国煤化工MYHCNMHG