Application of chlorophyll fluorescence performance indices to assess the wheat photosynthetic functions influenced by nitrogen deficiency

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1 Vol. 6, 14, No. : 2 21 Plnt Soil Environ. Applition of hlorophyll fluoresene performne inies to ssess the whet photosyntheti funtions influene y nitrogen efiieny M. Živčák 1, K. Olšovská 1, P. Slmk 2, J. Glmošová 3, V. Rtj 3, H.B. Sho 4, M. Brestič 1 1 Deprtment of Plnt Physiology, Slovk University of Agriulture, Nitr, Slovk Repuli 2 Deprtment of Agrohemistry n Plnt Nutrition, Slovk University of Agriulture, Nitr, Slovk Repuli 3 Deprtment of Mhines n Proution Systems, Slovk University of Agriulture, Nitr, Slovk Repuli 4 Key Lortory of Costl Biology n Bioresoures Utiliztion, Ynti Institute of Costl Zone Reserh (YIC), Chinese Aemy of Sienes (CAS), Ynti, P.R. Chin ABSTRACT Nitrogen efiieny influenes importntly the plnt photosyntheti pity n rop proutivity. Here, we employe the rpi, non-invsive mesurements of hlorophyll fluoresene kinetis for lultion of the integrtive fluoresene prmeters relte to the lef photosyntheti performne. In pot experiments with winter whet (Tritium estivum L.) we ultivte plnts uring the whole growing perio in the soil sustrte supplie with four ifferent oses of nitrogen. The lef nitrogen n hlorophyll ontent s well s the plnt ry mss were nlyze fter hlorophyll fluoresene reors in three growth stges. Our results inite tht the ommonly use prmeter F v (the mximum quntum yiel of photohemistry) ws lmost insensitive to nitrogen tretment. In ontrry, the performne inex (PI s ) n totl performne inex (PI tot ) were muh more responsive n signifint ifferenes mong plnts of ifferent nitrogen tretments s well s etween the youngest n thir lef from the top were oserve. Prmeter PI tot ws shown to express only smll iurnl hnges, thus eing more relile n more useful for omprison of ifferent smples in fiel onitions thn more frequently use prmeter PI s. Keywors: performne inex; photosynthesis; plnt eophysiology; non-invsive tehniques; JIP-test It ws well oumente tht the nitrogen nutrition influenes the plnt photosyntheti pity (Tershim n Evns 1988). Thus, the memrne proesses must e lne to mintin high effiieny in the onversion of energy n to voi the over-reution of photosyntheti eletron hin in onitions with ifferent nitrogen supply (Lu et l. 1). To ssess quikly the photosyntheti funtion in high numer of fiel grown plnts, the non-estrutive nlysis of polyphsi fst hlorophyll fluoresene (ChlF) trnsient ws evelope (Strsser et l., reviewe in Stiret n Govinjee 11). The metho is se on high-frequeny reor of ChlF emitte y rk pte lef uring short (usully one seon lsting) pulse of strong tini light y fluorimeter. The ChlF rise uring the first seon of illumintion shows sequene of phses (lele s O, K, J, I, P) from the initil (F o ) to the mximl (F m ) fluoresene vlue. The Supporte y the Europen Community, Projet No : Constrution of the AgroBioTeh Reserh Centre. 2

2 Plnt Soil Environ. Vol. 6, 14, No. : 2 21 mthemtil moel of the polyphsi trnsient ws evelope n nme s JIP-test (Strsser n Strsser 199). It enles lultion of speifi iophysil prmeters, quntum yiels n proilities hrterizing struture n funtion of the photosyntheti eletron trnsport system s well s some integrtive prmeters relte to plnt photosyntheti performne. Strsser et l. () introue the performne inex (PI s ) s the prout of three inepenent prmeters omining struturl n funtionl riteri: ensity of retion enters, the quntum effiieny of primry photohemistry n onversion of exittion energy in eletron trnsport. This prmeter ws foun to e muh more sensitive to environmentl effets thn ommonly use prmeter F v (Brestič n Živčák 13) n it ws shown to e well orrelte with photosyntheti pity mesure s CO 2 ssimiltion (vn Heeren et l. 3, Ripley et l. 4). Lter, the totl performne inex (PI tot ) ws introue (Smit et l. 9). This prmeter is se on PI s ut it is extene y the quntum effiieny of reution of photosystem I (PS I) en eptors (Reills et l. 11). The nlysis of fst ChlF trnsient ws pplie in numerous stuies in rop plnts, e.g. to ssess the environmentl effets in whet, suh s rought (Živčák et l. 8), high temperture (Brestič et l. 12), light stress (Klji et l. 12, Živčák et l. 14). The JIP-test nlyses were pplie severl times lso in stuies eling with nitrogen efiieny in plnts n the effet of poor nitrogen supply on PS II is reently well esrie (Lu et l. 1, Reills et l. 11, Li et l. 12, et.). Although in mny of pulishe works the rpi ChlF is enote s useful tool for ssessing the physiologil effets of nitrogen efiieny on plnts, there is still lk of t on the usefulness of the metho in ssessment of plnt photosyntheti performne in rop trils with ifferent nitrogen supply. In our stuy we present the results otine in three growth stges of winter whet expose either to full or prtil nitrogen efiieny ompre to plnts grown in norml nitrogen supply s well s in the exess of nitrogen. We fouse on prtil spets n we trie to suggest the useful wy to utilize the rpi hlorophyll fluoresene metho for eteting nitrogen efiieny or monitoring effets of nitrogen nutrition on whet photosyntheti performne. MATERIAL AND METHODS Cultivtion of plnts. Pot experiment with winter whet (Tritium estivum L., v. Akteur) ws estlishe on the 22 n Otoer 12 in vegettion ge of the Slovk University of Agriulture, Nitr. Eh pot ontine kg of meium hevy Cmisol. Owing to the ft tht there ws goo ontent of N min (1 mg/kg), P (9 mg/kg) n K (224 mg/kg) in the soil utumn fertiliztion with minerl fertilizers ws not performe. Sowing of 32 grins per pot ws relize y hn. After wintering regenertive fertiliztion of winter whet with nitrogen ws one in spring (18 th April 13) s follows: tretment 1. g N per pot (ontrol); tretment 2. g N per pot; tretment 3 1. g N per pot; tretment 4 2. g N per pot. Nitrogen ws pplie in the form of liqui fertilizer DAM-39 (UAN-39) ilute with wter in rtio of 1: (1 prt of fertilizer in prts of wter). Eh tretment ws repete 6 times, i.e. eh tretment onsiste of 6 pots. During the growing perio soil moisture in pots ws mintine t the level of 6% of full soil wter pity. Aovegroun phytomss of plnts from one pot in eh tretment ws smple three times (3 r My, 21 st My, n 7 th June) uring the vegettion perio. Anlysis of totl nitrogen. Totl nitrogen per ry mss (DM) unit in whet leves ws nlyze in ry smples (fter rying t 6 C). The ontent of totl nitrogen (mg/g of DM) ws nlyze using the stnr Kjelhl metho. The men vlue of two prllel smples is presente. Determintion of hlorophyll ontent. The lef hlorophyll ontent ws mesure spetrophotometrilly from etone extrt, n lulte using the eqution of Lihtenthler (1987), s esrie elsewhere (Živčák et l. 14). Six leves of eh tretment/lef position were nlyze. Chlorophyll fluoresene mesurements. ChlF mesurements were performe using the Hny- PEA ontinuous exittion plnt effiieny nlyser (Hnsteh Instruments Lt, Kings Lynn, UK). The lef lips were ple on the leves min to prior the mesurements to provie rk pttion. The light pulse intensity use ws 3 µmol/ m 2 /s for 1 s. Lef segment mesurements were performe in the mile prt of lef le, wy from the min lef vein. ChlF trnsient t were use to lulte prmeters. The mximum quntum 211

3 Vol. 6, 14, No. : 2 21 Plnt Soil Environ. yiel (effiieny) of PS II photohemistry (F v ), performne inex n totl performne inex were lulte oring to the equtions reviewe y Stiret n Govinjee (11). ChlF mesurements were one in 3 yles (in the growth stges of tillering, stem elongtion n efore nthesis). Mesurements were one lwys on the newest, fully evelope lef (lef 1) n on the thir lef from the top (lef 3). Moreover, fter nthesis, the test of prmeter stility uring hot n sunny y ws performe. Sttistil nlysis. The reporte t represent the men ± stnr error (SE). Sttistil nlysis ws one using the nlysis of vrine (ANOVA) followe y the Tukey s HSD test (α =.) y the Sttisti version 9. (Sttsoft In., Tuls, USA). RESULTS Different oses of nitrogen fertilizers le to ifferenes in the nitrogen ontent in plnts s well s to signifint ifferenes in proution of iomss n some relte prmeters, s shown in Tle 1. The highest ifferene ws etween tretment N n other three tretments in nitrogen ontent s well s in ll growth prmeters, the ifferenes mong ½N, 1N n 2N were lower ut in mny ses signifint, onfirming the positive reltionship etween nitrogen supply, the lef hlorophyll ontent n iomss proution. We hve exmine n effet of ifferent nitrogen nutrition on vlues of three ChlF prmeters in two lef positions (Figure 1). Our results inite, tht in the youngest, fully evelope lef there is no erese of mximum quntum yiel of PS II photohemistry (F v ) ue to nitrogen efiieny; moreover, the verge vlues of F v were the highest in nitrogen eplete plnts in erly growth stges. On the other hn, the oler leves (lef 3) h slightly lower F v in tretments with no or insuffiient nitrogen supply. The tren of F v in lef 1 neither in lef 3 i not follow the tren of plnt nitrogen ontent or nitrogen supply. Vlues of PI s prmeter erive from ChlF reors (Figures 1 f) onfirme muh higher responsiveness of PI s ompre to F v. The vlues in N tretment were signifintly lower in oth lef 1 n lef 3 in ll three growth stges. It orrespons with the lowest lef nitrogen n hlorophyll ontent s well s low proution of iomss (Tle 1). Moreover, the PI s vlues inite lower photosyntheti performne of lef 3 ompre to lef 1, espeilly in N n ½N tretments; the ifferene ws getting higher in lter growth stges. On the other hn, there were only insignifint ifferenes mong PI s vlues in lef 1 of tretments ½N, 1N n 2N in stges of tillering n stem elongtion (Figures 1,e). The results of PI tot prmeter (Figures 1g i) were similr to PI s (Figures 1 f); however, we oul oserve some ifferenes. Firstly, the ifferenes etween lef 1 n lef 3 were muh higher in PI tot. Moreover, in lter growth stges we oul oserve lso signifint ifferenes etween 1N n 2N tretments in top lef, whih ws not foun in PI s. We exmine the reliility of the mesure prmeters in fiel onitions expresse y the stility of the vlues uring hot n sunny y (Figure 2). We oul see tht the PI s vlues progressively erese ue to het stress n photoinhiition (similrly s shown y erese of F v vlues). On the other hn, the vlues of PI tot (Figure 2) were muh more stle n there were only insignifint ifferenes etween mesurement in the morning n ny other mesurement uring the y. Tle 1. Nitrogen ontent n plnt growth prmeters in evlute growth stges Growth stge tretment Tillering (BBCH 2-29) Stem elongtion (BBCH 3-39) Pre-nthesis (BBCH -6) Nitrogen leves (mg/g) Dry mss totl (g/plnt) Chlorophyll, lef 1 (mg/m 2 ) Chlorophyll, lef 3 (mg/m 2 ) Smll letters inite sttistilly homogenous groups (Tukey HSD test, α =.) 212

4 Plnt Soil Environ. Vol. 6, 14, No. : 2 21 ().9 () () Lef Lef 3 F v () 3 (e) (f) 3 ef f 3 ef 3 2 e PI s (g) 4 (h) 4 (i) PI tot 3 3 g 3 3 f ef 2 2 e e e f e Figure 1. The lulte prmeters erive from fst fluoresene trnsients reore in the first, youngest fully evelope lef (lef 1) or the thir lef (lef 3) on the whet min stem, ounte from the top. The ChlF mesurements were one in three ifferent growth stges: in the stge of tillering (olumn left), uring stem elongtion (mile olumn) n efore nthesis (olumn right). Figures (), () n () represent the vlues of the mximum quntum yiel of PS II photohemistry (F v ), figures (), (e) n (f) the performne inex (PI s ) n figures (g), (h) n (i) the totl performne inex (PI tot ). Mens ± SE re presente; the smll letters inites sttistilly homogenous groups (Tukey HSD test, α =., n = ) DISCUSSION ChlF tehniques re frequently employe in physiologil nlyses of nitrogen efiieny in plnts; however, the most of ppers fous on F v prmeter s it is simple to mesure n interpret. However, F v is insensitive to mny ftors tht negtively ffet plnt photosynthesis, suh s rought (Živčák et l. 8). Unlike to results of some uthors (Guii et l. 1997, Jnušuskite n Feiziene 12), our results inite tht F v is insensitive even to nitrogen efiieny, exept the erese in ol leves initing initition of lef senesene. On the other hn, performne inies muh etter reflete expete hnges in lef photosyntheti performne, s it ws shown even previously (vn Heeren et l. 3, 4). This ws minly 213

5 Vol. 6, 14, No. : 2 21 Plnt Soil Environ. () () ().8 Lef 1 2 Lef F v.7.7 PI s 1 PI tot Hour of y Hour of Dy Figure 2. The ily tren of men vlues of the mximum quntum yiel of photohemistry (F v ) (); performne inex (PI s ) (), n totl performne inex (PI tot ) (). The reors were one uring the sunny n hot y (ir temperture rnge from 18 C t 7: to 32 C fternoon). Mesurements for ssessment of iurnl hnges were one in plnts with optimum nitrogen nutrition (1N) fter nthesis (BBCH 7 7). Mens ± SE from 1 mesurements re presente Hour of Dy Hour of y Hour of y Hour of y ue to the signifint effet of nitrogen efiieny on the ensity of retion enters (Lu et l. 1, Li et l. 12, et.). The ifferenes etween the PI trens foun in the youngest lef (lef 1) n ol lef (lef 3) represent nie exmple of the effets of nitrogen reistriution on photosyntheti performne, whih ws ominnt in plnts poorly supplie with nitrogen. After emergene of the spike, the nitrogen n e re-istriute lso from leves, s isssemly of photosyntheti pprtus uner stress onitions n e vlule soure of nitrogen for the grin (Vssilev et l. 12). Reently introue prmeter totl performne inex (Smit et l. 9) extene the reore effets lso to the pity of eletron trnsport etween photosystems (Shnsker et l. ), whih is not onsiere in PI s. In terms of the mehnism, the nitrogen efiieny ws proven to inrese the yli eletron trnsport n there is less PS I ville for epting eletrons from PS II (Antl et l. ). Similr to our results, Nikiforou n Mnets (11) ttriute the positive orreltion etween lef nitrogen ontent n PI tot vlues to erese of PS I tivity in liner eletron trnsport use y nitrogen efiieny. In prllel, Reills et l. (11) oserve the erese of PI tot uring N-epletion s result of the erese in the reution of en eletron eptors on PS I. Another importnt issue is the reliility of the mesure prmeters. As the performne inies re very sensitive, they n lso respon to flututing environmentl ftors, suh s tul temperture or inient light intensity. High light, espeilly when ssoite with high temperture, n le to photoinhiition or slow relxtion of PS II photohemistry, whih uses erese of ll three omponents inlue in PI s, mostly of the retion enter numer (Klji et l. 12). It mkes this prmeter prtiulrly suitle to oserve iurnl hnges in the sttus of PS II photohemistry (e.g. in eophysiologil stuies), ut less relile to ompre the intrinsi properties of photosyntheti pprtus in ifferent smples. The vlues of PI tot were muh more stle uring the y, thnks to the ft tht the fewer eletrons relese y erese numer of retion enters were lne y higher proility tht these eletrons will e epte y photosystem I (s inite y iniviul omponents of PI tot, not shown here). Therefore, we n onsier PI tot to e more useful for omprison of plnt photosyntheti properties in fiel onitions, inluing photosyntheti performne of plnts grown uner ifferent nitrogen supply. In onlusion, our results inite tht prmeters erive from rpi n non-invsive mesurements of the ChlF kinetis n serve for ssessment of lef photosyntheti performne influene y ifferent nitrogen nutrition, useful for rop reserh n prtil pplitions. We suggest using the integrtive prmeter totl performne inex, whih ws shown to e suffiiently sensitive n relile. REFERENCES Antl T., Mttil H., Hkl-Ytkin M., Tyystjärvi T., Tyystjärvi E. (): Alimtion of photosynthesis to nitrogen efiieny in Phseolus vulgris. Plnt, 232:

6 Plnt Soil Environ. Vol. 6, 14, No. : 2 21 Brestič M., Živčák M., Klji H.M., Crpentier R., Allkhveriev S.I. (12): Photosystem II thermostility in situ: Environmentlly inue limtion n genotype-speifi retions in Tritium estivum L. Plnt Physiology n Biohemistry, 7: 93. Brestič M., Živčák M. (13): PSII fluoresene tehniques for mesurement of rought n high temperture stress signl in rop plnts: Protools n pplitions. In: Rout G.R., Ds A.B. (es.): Moleulr Stress Physiology of Plnts. Springer- Verlg, Berlin, Heielerg. Guii L., Lorefie G., Prossi A., Mlorgio F., Tognoni F., Soltini G.F. (1997): Growth n photosynthesis of Lyopersion esulentum (L.) plnts s ffete y nitrogen efiieny. Biologi Plntrum, 4: Jnušuskite D., Feiziene D. (12): Chlorophyll fluoresene hrteristis throughout spring tritile evelopment stges s ffete y fertiliztion. At Agriulture Sninvi, Setion B Soil n Plnt Siene, 62: 7 1. Klji H.M., Crpentier R., Allkhveriev S.I., Bos K. (12): Fluoresene prmeters s erly initors of light stress in rley. Journl of Photohemistry n Photoiology B: Biology, 112: 1 6. Li G., Zhng Z.S., Go H.Y., Liu P., Dong S.T., Zhng J.W., Zho B. (12): Effets of nitrogen on photosyntheti hrteristis of leves from two ifferent sty-green orn (Ze mys L.) vrieties t the grin-filling stge. Cnin Journl of Plnt Siene, 92: Lihtenthler H.K. (1987): Chlorophyll n rotenois Pigments of photosyntheti iomemrnes. Methos in Enzymology, 148: Lu C., Zhng J., Zhng Q., Li L., Kung T. (1): Moifition of photosystem II photohemistry in nitrogen efiient mize n whet plnts. Journl of Plnt Physiology, 18: Nikiforou C., Mnets Y. (11): Inherent nitrogen efiieny in Pisti lentisus preferentilly ffets photosystem I: A sesonl fiel stuy. Funtionl Plnt Biology, 38: Reills M.C.F.R., Jeong J.S., Strsser R.J., Kim Y.S., Kim J.K. (11): JIP nlysis on rie (Oryz stiv v. Nipponre) grown uner limite nitrogen onitions. Journl of the Koren Soiety for Applie Biologil Chemistry, 4: Ripley B.S., Refern S.P., Dmes J.F. (4): Quntifition of the photosyntheti performne of phosphorus-efiient Sorghum y mens of hlorophyll- fluoresene kinetis. South Afrin Journl of Siene, : Shnsker G., Tóth S.Z., Strsser R.J. (): Methylviologen n iromothymoquinone tretments of pe leves revel the role of photosystem I in the Chl fluoresene rise OJIP. Biohimi et Biophysi At, 176: 261. Smit M.F., vn Heeren P.D., Pienr J.J., Weissflog L., Strsser R.J., Krüger G.H. (9): Effet of trifluoroette, persistent egrtion prout of fluorinte hyrorons, on Phseolus vulgris n Ze mys. Plnt Physiology n Biohemistry, 47: Stiret A., Govinjee (11): On the reltion etween the Kutsky effet (hlorophyll fluoresene inution) n photosystem II: Bsis n pplitions of the OJIP fluoresene trnsient. Journl of Photohemistry n Photoiology B, 4: Strsser B.J., Strsser R.J. (199): Mesuring fst fluoresene trnsients to ress environmentl questions: The JIP-test. In: Mthis P. (e.): Photosynthesis: from Light to Biosphere. Kluwer Aemi Pulishers, Dorreht. Strsser R.J., Srivstv A., Tsimilli-Mihel M. (): The Fluoresene Trnsient s Tool to Chrterize n Sreen Photosyntheti Smples. Proing Photosynthesis: Mehnisms, Regultion n Apttion. Tylor n Frnis, New York, Lonon. Tershim I., Evns J.R. (1988): Effets of light n nitrogen nutrition on the orgniztion of the photosyntheti pprtus in spinh. Plnt n Cell Physiology, 29: Vn Heeren P.D., Strsser R.J., Krüger G.H. (4): Reution of rk hilling stress in N-fixing soyen y nitrte s inite y hlorophyll fluoresene kinetis. Physiologi Plntrum, 121: Vn Heeren P.D., Tsimilli-Mihel M., Krüger G.H., Strsser R.J. (3): Drk hilling effets on soyen genotypes uring vegettive evelopment: Prllel stuies of CO 2 ssimiltion, hlorophyll fluoresene kinetis O-J-I-P n nitrogen fixtion. Physiologi Plntrum, 117: Vssilev V., Demirevsk K., Simov-Stoilov L., Petrov T., Tsenov N., Feller U. (12): Long-term fiel rought ffets lef protein pttern n hloroplst ultrstruture of winter whet in ultivr-speifi mnner. Journl of Agronomy n Crop Siene, 198: Živčák M., Brestič M., Olšovská K. (8): Physiologil prmeters useful in sreening for improve tolerne to rought in winter whet (Tritium estivum L.). Cerel Reserh Communition, 36: Živčák M., Brestič M., Olšovská K, Slmk P. (8): Performne inex s sensitive initor of wter stress in Tritium estivum L. Plnt, Soil n Environment, 4: Živčák M., Brestič M., Klji H.M., Govinjee (14): Photosyntheti responses of sun- n she-grown rley leves to high light: Is the lower PSII onnetivity in she leves ssoite with protetion ginst exess of light? Photosynthesis Reserh, 119: Reeive on Jnury 23, 14 Aepte on Mrh 2, 14 Corresponing uthor: Prof. Ing. Mrián Brestič, CS., Slovenská poľnohospoársk univerzit v Nitre, Fkult groiológie potrvinovýh zrojov, Kter fyziológie rstlín, Tr. A. Hlinku 2, Nitr , Slovenská repulik phone: , e-mil: mrin.resti@unig.sk 21