IMPACT OF WATER DEFICIT STRESS ON GROWTH AND ALKALOID CONTENT OF ORGANS OF SPIGELIA ANTHELMIA (L.) * Umebese, C. E., Okunade, K. I. and Orotope, O. M.

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1 Ife Journl of Science vol. 14, no. 2 (12) 357 IMPACT OF WATER DEFICIT STRESS ON GROWTH AND ALKALOID CONTENT OF ORGANS OF SPIGELIA ANTHELMIA (L.) * Umeese, C. E., Okunde, K. I. nd Orotope, O. M. Deprtment of Botny, University of Lgos, Akok-Y, Lgos, Nigeri. *E-mil: cumeese@yhoo.com (Received: August 12; Accepted: Novemer 12) ABSTRACT Experiments were conducted to study the effect of wter deficit stress on the growth nd lkloid content of different orgns of Spigeli nthelmi (L), medicinl plnt used loclly s n nthelminthic. Plnts were sujected to 6 dys drought t the erly (EV plnts) nd lte (LV plnts) vegettive stges (30-35 nd dys fter plnting respectively). Wter stress cused reduction in height, lef re, root iomss, whole plnt iomss, lef re rtio nd reltive growth rte of stressed plnts ut the impct ws more intense in EV plnts. Miniml -1 differences in lkloid content ( mg g ) occurred mong orgns (fruit, leves, stem, nd roots) of plnts sujected to stress t oth vegettive stges nd the control. Thus, wter deficit stress t the vegettive stge of Spigeli nthelmi cused reductions in growth ut did not ffect the concentrtion of lkloid in the plnt orgns. Keywords: Wter Deficit, Alkloids, Growth, Spigeli nthelmi INTRODUCTION Alkloids re secondry metolites synthesized nd ccumulted y numerous higher plnts. Aout 12,000 lkloids hve een found to occur in pproximtely % of flowering plnts, mostly herceous dicots. A few lkloids my e found in severl gener or even fmilies ut most species disply their own unique, geneticlly determined ptterns (Hopkins nd Hüner, 04). Furthermore, individul lkloids my e restricted to prticulr orgns, such s roots, leves, cortex, young fruits nd seeds, in prenchym tissue or in cells. The sme plnt my ccumulte oth similr nd different lkloids. During the vegettive period, lkloid concentrtion undergoes chnges, the pek coinciding with flowering. At the end of vegettive period, lkloids ccumulte in seeds nd roots (Hopkins nd Hüner, 04; Hondelmnn, 1984). Though the physiologicl roles of these metolites hve not een clerly demonstrted, lkloids ccumulted in the underground prts of plnt hve een shown to prticipte in metolic processes, induce root growth nd mke rrier to microorgnisms s lechtes (Penev, 06). Alkloids re sid to protect plnts from pests which re put off grzing y the cidic tste (Wink nd Hrtmnn, 1982). They hve wide use in medicine. By 1985, ten out of the twelve commercilly most importnt plnt-derived drugs were lkloids. Alkloids generte vrying degrees of physiologicl nd psychologicl responses in humns, often y interfering with neurotrnsmitters (Hopkins nd Hüner, 04). The environment of the soil influences lkloid concentrtion in plnts. Wter stress deficit reduces indole lkloid nd increses jmlicine content in Cthrnthus roseus (Jleel et l., 07, 08). Mercuric chloride, cdmium, mngnese, nickel, led nd vndium enhnce the totl lkloids in Cthrnthus roseus (Fthll et l., 11, Srivstv nd Srivstv, 10, Tllevi nd Cosm, 1988) while chnges in temperture do not ffect totl lkloids in this plnt. Insect dmge of the roots of tocco plnts lso induces n ccumultion of lkloids (Ktoh et l., 05). Spigeli nthelmi L. Ch. (pink root, wormweed) of the fmily Lognicee, is common weed of wstelnd, clered res nd rodsides. It is very toxic plnt with leves nd roots tht hve locl medicinl use s n nthelminthic (Oliver, 1960). It produces n lkloid, spigeline (Clus nd Tyler, 1965). Spigeli is known to grow very well in lomy soil nd vergely in other soil types (Olorode, 1979; Akoundu, 1987). It lso shows phenotypic plsticity specific to popultions, in time of germintion, formtion of vrious orgns (Umeese nd Omolokun, 1998). In Nigeri, the leves of Spigeli re picked from rodsides nd wstelnd nd nothing is known out the possile effect of environmentl vriles on the lkloid content. The min ojective of this study is to determine the chnges tht occur in growth nd orgn lkloid content of Spigeli sujected to wter deficit stress t the erly

2 358 Umeese et l.: Impct of Wter Deficit Stress on Growth nd Alkloid nd lte vegettive stges of development. MATERIALS AND METHODS Fruits of Spigeli nthelmi L. Ch. were hrvested from mture plnts growing t the nery ushes of the University of Lgos. They were stored in polythene gs for two weeks to relese the seeds y explosive mechnism. Plnting Procedure Seeds were plnted in three tches of 12 plnting pots, ech contining lomy soil using Rndomized Complete Block Design. Wtering ws done dily nd plnts were thinned to 4 plnts per pot fter 3 weeks. The first tch of plnts ws sujected to 6 dys wter stress t the erly th th vegettive stge (30-35 dys fter plnting, DAP). A second tch ws sujected to wter nd th stress t the lte vegettive stge (52-57 DAP) while the third tch ws wtered dily, serving s the control. Pots were kept t the greenhouse of the Botnic grden t the University of Lgos. Growth Prmeters th Smples of plnts were hrvested on the 35 dy fter plnting (DAP) for the determintion of lef re nd dry iomss of whole plnt nd plnt prts (leves, stem, roots nd fruits). Lef re ws mesured s outlined y Eze (1965). Finl hrvest th ws done on the 85 DAP, when the fruits were fully mture nd the lef re nd dry iomss were gin determined nd the numer of leves nd plnt height were lso recorded. Lef re rtio (LAR), net ssimiltion rte (NAR) nd reltive growth rte (RGR) were computed from the lef re nd plnt dry iomss vlues, s outlined y Noggle nd Fritz (1976). Extrction nd Mesurement of Alkloid Content Alkloids were extrcted from dry powdered whole plnt nd plnt prts using the method of Hrone (1960) t the Interntionl Institute for Tropicl Agriculture (IITA), Idn. 0.5 mg of ech powdered smple ws mixed with ml 10% cetic cid in ethnol nd left to stnd for 5 hours. The lkloid ws precipitted y dropwise ddition of concentrted mmonium hydroxide (NH OH). 4 This ws further centrifuged nd wshed with 1% NH OH. The residue ws then dissolved in few 4 drops of chloroform. The extrct ws chromtogrphed using silic gel G pltes in methnol: concentrted mmonium hydroxide (0:3) nd the presence of lkloids on the plte ws detected y fluorescence in UV light. Quntittive determintion ws done on fresh lkloid extrct y scnning t mximl vlue rnge of 2-3 nm using UV spectrophotometer, Bechmn utomted scnner model 990 equipped with computerized grphic nlyzer. Sttisticl Anlysis Mens of three replictes were quoted with their stndrd error. The level of significnce etween mens t p<0.05 sed on Anlysis of Vrince (ANOVA), ws determined using the New Duncn's Multiple Rnge Test. RESULTS AND DISCUSSION Spigeli nthelmi plnts sujected to 6 dys wter deficit stress t the erly nd lte vegettive stges (EV nd LV plnts respectively) showed differences in growth prmeters ut not in lkloid concentrtion. Wter stress cused significnt reduction (p<0.05) in height nd lef re of EV plnts while the impct ws only slight in LV plnts (Fig. 1). Generlly, stress tretment cused reduction in fruit, root, shoot nd whole plnt iomss (Fig.2). Wter stress hs een shown to ffect the phenology, growth, yield nd qulity of plnts (Adejre nd Umeese, 1998; 08). The root iomss nd whole plnt iomss were significntly reduced in ll treted plnts ut the reduction in shoot iomss ws only significnt in LV plnts. Thus, EV plnts were more sensitive to wter stress thn LV plnts. This corroortes erlier reports tht the effect of wter deficit vries with the growth stge of the plnt; the vegettive stge eing more sensitive to wter deficit thn the reproductive stge, considering plnt iomss (Fores nd Wtson, 1992; Adejre nd Umeese, 07; Umeese et l., 09). Lef re rtio (LAR) is mesure of the proportion of the plnt which is engged in photosynthetic processes; net ssimiltion rte (NAR) is mesure of the mount of photosynthetic product going into plnt mteril while oth components contriute to the reltive growth rte (Noggle nd Fritz, 1976). Wter stress cused significnt decreses in LAR nd RGR of stressed plnts while the NAR ppered to e similr (Fig. 3). Wter stress cuses low lef wter potentil ccompnied y lef stomtl resistnce nd the resulting effect is reduction in cron

3 Umeese et l.: Impct of Wter Deficit Stress on Growth nd Alkloid 359 Plnt height (cm) Lef Are (cm 2 ) Numer of Leves per plnt Fig. 1: A B C Control Erly Vegettive Lte Vegettive Tretment stges Height (A), Lef re (B) nd Lef numer (C) of Spigeli plnts sujected to wter stress t the erly nd lte vegettive stges. Brs with similr letters re not significntly different t P < 0.05 using the New Duncn's multiple rnge test Whole Plnt Biomss (g plnt -1 ) Shoot Biomss (g) Root Biomss (mg plnt -1 ) Fruit Biomss (g plnt -1 ) C D A B Control Erly Vegettion Lte Vegettion Tretment stges c Fig. 2: Fruit (A), Root (B) Shoot (C) nd Whole plnt (D) iomss of Spigeli plnts sujected to wter stress t the erly nd lte vegettive stges. Brs with similr letters re not significntly different t P < 0.05 using the New Duncn's multiple rnge tests

4 360 Umeese et l.: Impct of Wter Deficit Stress on Growth nd Alkloid 0.00 B Net Assimiltion Rte (g cm -2 wk -1 ) Lef Are Rtio (cm 2 g -1 ) C c Control Erly Vegettion Lte Vegettion Tretment stges 24 Fig. 3: Reltive Growth rte (A), Net ssimiltion Rte 24 (B) nd Lef Are Rtio(C) of Spigeli plnts sujected to wter stress, t the erly nd lte vegettive stges. Brs with similr letters re not significntly different t P < 0.05 using the New Duncn's multiple rnge tests Lef 22 Root 24 Stem Fruit 22 Control Erly Vegettion Lte Vegettion Control Erly Vegettion Lte Vegettion Control Erly Vegettion Lte Vegettion Control Erly Vegettion Lte Vegettion Fig. 4: Alkloid concentrtion of vrious orgns of Spigeli plnts sujected to wter stress t the erly nd lte vegettive stges. Brs with similr letters re not significntly different t P < 0.05 using the Duncn's multiple rnge tests ssimiltion nd susequent iomss production (Adejre nd Umeese, 07). During environmentl stress such s drought, rective oxygen species (ROS) which include oxygen ions, free rdicls nd peroxides, increse drmticlly resulting in oxidtive dmge to proteins, DNA nd lipids (Apel nd Hrt, 04). This ws corroorted y the oserved reduction in root nd whole plnt iomss, LAR nd RGR in stressed plnts. Alkloid concentrtion ws not ffected y the stress tretments given t oth erly nd lte vegettive stges nd it ws lmost evenly distriuted mong ll plnt orgns: fruit, leves, stem, nd roots (Fig. 4). Thus, moderte wter stress t the vegettive stge cused reductions in plnt iomss ut it did not trnslte to reduction in concentrtion of lkloid. Mny plnt lkloids re ntioxidnts. Antioxidnts protect plnts exposed to environmentl stress from dmge.

5 Umeese et l.: Impct of Wter Deficit Stress on Growth nd Alkloid 361 The lck of impct of wter stress on lkloid concentrtion in Spigeli nthelmi plnts despite the significnt reduction in iomss nd reltive growth rte, my suggest tht spigeline, the lkloid present in Spigeli, does not hve ntioxidnt ctivity. Environmentl stress, such s wter stress, ffects the concentrtion of different lkloids in some medicinl plnts. The environment of the soil influences lkloid concentrtion in plnts. Wter stress deficit reduces indole lkloid nd increses jmlicine content in Cthrnthus roseus (Jleel et l., 07; 08). In the cse of Spigeli nthelmi wter stress during the vegettive period did not ffect the lkloid concentrtion. CONCLUSION While it is true tht environmentl stress, such s wter stress, ffects the concentrtion of lkloids in some medicinl plnts, when Spigeli nthelmi plnts were sujected to wter stress t the erly nd lte vegettive stges of growth, the concentrtion of lkloid in ll orgns of the plnt remined the sme. The significnt reduction in growth in stressed plnts did not ffect the lkloid concentrtion in plnt prts. AKNOWLEDGEMENTS We re grteful to `the University of Lgos for reserch grnt nd the Interntionl Institute for Tropicl Agriculture (IITA), Idn for ench spce to execute this study. REFERENCES Adejre, F.B. nd Umeese, C. E Effect of wter stress on the phenology of two cultivrs of Glycine mx. L. Journl of Scientific Reserch nd Development 3, Adejre, F.B. nd Umeese C.E. 07. Stomtl resistnce to low wter potentil t different growth stges ffects plnt iomss in Glycine mx L. Americn Journl of Agriculturl nd Biologicl Science 2, Adejre, F.B. nd Umeese C.E. 08. Wter Stress induces cultivr dependent chnges in stomtl complex, yield nd osmotic djustments in Glycine mx L. Interntionl Journl of Agriculturl Reserch 3, Akoundu, I. O Weed Science in the Tropics: Principles. John Wiley nd Sons, New York. Apel, K. nd Hrt, H. 04. Rective oxygen species: metolism, oxidtive stress, signl trnsduction. Annul Review of Plnt Biology. 55, Clus, E. P. nd Tyler, V. E. Jnr Phrmcognosy. Kimpton, London. Eze, J. M. O Studies on Growth Regultion, Slt Uptke nd Trnsloction.PhD Thesis. University of Durhm, Englnd. Ftll, M. A., Ad-El Kwy, A. M. nd Th, H. S. 11. Effect of hevy metl (HgCl 2) on ccumultion nd production of totl indole lkloids, vinlstine, nd/or vincristine from Egyptin Cthrnthus roseus (L.) G. Don. clli cultures. Journl of Applied Sciences Reserch 7, Fores, J. C. nd Wtson, R. D Plnts in Agriculture.University Press, Cmridge. Hrorne, J. B Phytochemistry. Acdemic Press, London. Hondelmnn, W The lupin ncient nd modern crop plnt. Theoreticl nd Applied Genetics 68, 19. Hopkins, W. G. nd N. P. A. Hüner. 04. Introduction to Plnt Physiology. John Wiley nd Sons, Inc. U.S.A. Jleel, C. A., Mnivnnn, P., Snkr, B., Kishorekumr, A., Gopi, R., Somsundrm, R., Pnneerselvm, R. 07. Wter deficit stress mitigtion y clcium chloride in Cthrnthus roseus: effects on oxidtive stress, proline metolism nd indole lkloid ccumultion. Colloids nd Surf B Biointerfces 60, Jleel, C. A., Snkr, B., Murli, P. V. Gomthinygm, M. Lkshmnn, G. M. A. Pnneerselvm R. 08. Wter deficit stress effects on rective oxygen metolism in Cthrnthus roseus; impcts on jmlicine ccumultion. Colloids nd Surf B Biointerfces 62, Ktoh, A., Ohki, H., Ini, K., Hshimoto, T. 05. Moleculr regultion of nicotine synthesis. Plnt Biotechnology 22, Noggle, G. R. nd Fritz, G. J Introductory Plnt Physiology. Prentice Hll, Inc. New Jersey. Oliver, B Medicinl Plnts in Nigeri. The Nigerin College of Arts, Science nd Technology, Idn. Olorode, O Flowering Plnts of Nigeri. University of Ife, Ile Ife.

6 362 Umeese et l.: Impct of Wter Deficit Stress on Growth nd Alkloid Penev A, 06. Stimulting llelopthic effect of plnt extrcts on some crops s fctor for etter germintion nd growth. Proceedings of the 3th interntionl conference on non chemicl crop protection methods: Lille - Frnce, Srivstv, N. K. nd Srivstv, A. K. 10. Influence of some hevy metls on growth, lkloid content nd composition in Cthrnthus roseus L. Indin Journl of Phrmceuticl Sciences 72, Tllevi, S. G. nd Cosm, F. D Stimultion of indole lkloid content in vndiumtreted Cthrnthus roseus suspension cultures. Plnt Medic 2, Umeese C. E., Oltimilehin, T. O. nd Ogunsusi, T. A. 09. Slicylic Acid Protects Nitrte Reductse Activity, Growth nd Proline in Amrnth nd Tomto Plnts during Wter Deficit. Americn Journl of Agriculturl nd Biologicl Sciences 4, Umeese, C.E. nd E. Omolokun, Phenotypic plsticity in edphiclly different popultions of Spigeli nthelmi L. Nigerin Journl of Botny 11, Wink M. nd Hrtmnn, T Enzymtic synthesis of quinolizidine lkloid esters: tigloyl-coa: 13-hydroxy-lupnine O- tigloyltrnsferse from Lupinus lus L. Plnt 156,