The Effects of Ultraviolet Radiation on the Contents of Chlorophyll, Flavonoid, Anthocyanin and Proline in Capsicum annuum L.

Transcription

1 Turk J Bot 32 (28) TÜB TAK Reserh Artile The Effets of Ultrviolet Rdition on the Contents of Chlorophyll, Flvonoid, Anthoynin nd Proline in Cpsium nnuum L. K. MAHDAVIAN 1, *, M. GHORBANLI 2, Kh. M. KALANTARI 3 1 Biology Deprtment, Pym Noor University of Tehrn, Tehrn, I. R. of IRAN 2 Azd Islmi University of Gorgn, Gorgn, I. R. of IRAN 3 Biology Dept., Fulty of Siene, Shhid Bhonr University of Kermn, I. R. of IRAN Reeived: Aepted: Astrt: Although ultrviolet rdition is potentilly hrmful, it is n importnt omponent of terrestril rdition to whih plnts hve een exposed sine invding lnd. Sine then, plnts hve evolved mehnisms to void nd repir UV rdition dmge. Therefore, it is not surprising tht photomorphogeni responses to UV-B nd UV-C re often ssumed to e dpttions to hrmful rdition. Most of the ompounds umulted re diretly involved in UV-B nd UV-C protetion: they re either effiient in filtering exess rdition or in svenging rdils. In this study plnts were grown for 5 weeks in ontrolled environment room. The plnts were grown in vermiulite medium using pots. Before UV tretments, plnts were irrigted with nutrient solution (Hoglnd solution) for 5 weeks. Then plnts were exposed to UV-A (32-39 nm), UV-B (312 nm) nd UV-C (254 nm) irrdition with density of 6.1 (Wm -2 ), 5.8 (Wm -2 ) nd 5.7 (Wm -2 ) for 2 weeks. Plnts were treted with UV in their light period for 27 min per dy for 14 dys. The influene of UV-A, UV-B nd UV-C rdition on hlorophyll, flvonoids, nthoynin, proline, memrne permeility, lipid peroxidtion nd UV-soring ompound ws exmined. Results showed tht the ontents of hlorophyll, nd rotenoids of pepper leves were redued signifintly in those plnts whih were exposed to UV-B nd UV-C rdition nd ompred with ontrol nd UV-A treted plnts. In ontrst, UV-B nd UV-C inresed (P <.5) proline, queretin, rutin nd nthoynin onentrtions in leves of Cpsium nnuum L. Ultrviolet rdition indued oxidtive stress in pepper y inresing lipid peroxidtion nd memrne permeility whih inditing tht limits of tolerne re muh less thn dmged used y UVrdition. Key Words: Ultrviolet rdition, proline, lipid peroxidtion, Cpsium nnuum L. Introdution The spetrum of UV rdition rehing the erth, s surfe hs een divided into lower energy (UV-A, 32-4 nm), higher energy (UV-B, nm) nd UV-C ( nm) regions. A given dose of rdition, of UV- A is less effetive thn UV-B nd UV-C for indution of plnt responses (Brt et l., 24). UV rdition lso produes oxidtive stress (Cost et l., 22), whih rises from the deleterious effets of tive oxygen speies (AOS), whih ret with lipids, pigment, proteins nd nulei id (Di et l., 1997). Under onditions of norml helthy growth, plnts possess numer of enzymti nd non-enzymti detoxifition mehnisms to effiiently svenge either the AOS themselves or their seondry retion produts (Brtling et l., 1993). Sine flvonoids nd phenolis sore UV-B nds they represent seletive UV-B filter whih protet plnt tissue ginst hrmful rys (Rozem et l., 22). This hs led to the long-stnding hypothesis tht primry dptive dvntge onferred y these ompounds is tht they sor potentilly hrmful UV-B rdition t the lef surfe nd protet underlying photosyntheti tissues. It hd een rgued tht pigments lolized in the epiderml ells (minly flvonoids nd nthoynnins) redue epiderml penetrtion of UV-B rdition seletivelly proteting internl tissues of the UV- B irrdine without interfering photosynthesis (Cldwell et l., 1983). * E-mil: kormhdvi@yhoo.om 25

2 The Effets of Ultrviolet Rdition on the Contents of Chlorophyll, Flvonoid, Anthoynin nd Proline in Cpsium nnuum L. The study on Sorghum vulgre showed tht UV-B irrdition indued derese in hlorophyll nd rotenoid onentrtions (Amsht & Agrwl, 1998). However, Strid nd Porr (1992) suggested tht UV-B irrdition in leves of Pisum stivum hs no speifi effets on enzymes of the hlorophyll iosyntheti pthwy ut rther, influenes the geneti regultion of hlorophyll inding protein, leding to destrution of hlorophyll. It hs een reported tht UV-B stimulted the iosynthesis of UV-B soring ompounds nd rotenoids, whih oth perform photoprotetive funtion (Cmpos et l., 1991). The rotenoids re implited in the diret protetion of the photosystems ginst UV-B rdition (Middleton & Termur, 1993). This is euse UV-B rdition is potentil oxidtive ftor nd rotenoids, s flvonoids hve een shown to quenh effetively tive oxygen speies (Lrson, 1998). By ontrst, drmti indution of synthesis nd umultion of flvonoids is often oserved in response to high (sun) light (Rey & Lnster, 21; Merzlyk & Solovhenko, 22). Experiments with trnsgeni plnts demonstrted n upregultion of the genes responsile for flvonoid (prtiulrly, kempferol nd queretin) iosynthesis under elevted UV-B onditions (Wng et l., 2; Ryn et l., 22). There lso reposed whih show tht upon exposure to UV, plnts inrese the prodution of lef flvonoids nd nthoynins (Krizek et l., 1998). Flvonoids hve mximum sorption in UV region of light (Termur & Sullivn, 1994; Kruss et l., 1997). Similr protetive effets hve lso een suggested for nthoynins (Burger & Edwrds, 1996; Coley & Kursr, 1996; Woodll & Stewrt, 1998). Numerous studies hve demonstrted tht the umultion of flvonoids nd nthoynins y plnt provide defene mehnism ginst UV-B rdition (Biez & Lois, 21). Rutin is highly ntioxidtive tive flvonoid. It n e found in mny plnts.there is some evidene tht UV rdition inreses the prodution of rutin in plnts (Umek et l., 1999). Studies hve shown tht UV rdition lters memrnes. This n e seen y the inrese in mlondildehyd onentrtion (MDA), redued monoglgtosyl diylglyerol (MGDG), s well s n inrese in ethylene nd ethne onentrtion (Di et l., 1997). The im of this study ws to study the importne of the umultion of flvonoids (speilly rutin nd queretin) nd non-enzymti detoxifition mehnisms whih develop during exposure of pepper plnts to UV-A, UV-B nd UV-C rdition. Additionlly, the ontent of lipid peroxidtion nd memrne permeility were exmined in pepper plnts in im to find if enhned flvonoids iosynthesis n protet plnts from the whih dmged used y UV rdition nd repir the dmge. Mteril nd Methods Plnt growth nd tretments Seeds of Cpsium nnuum L. were sown in plsti pots ontining 1 kg of orse snd nd vermiulite (2:1, V/V). Plnts were grown in the greenhouse t 25/2 o C (dy/night), with 16h light/8h drk photoperiod for 35 dys. After 35 dys, seleted plnts were sujeted to ultrviolet rdition with UV lmps. UV-A (32-39 nm), UV-B (312 nm) nd UV-C (254 nm) irrdition with density of 6.1 (Wm -2 ), 5.8 (Wm -2 ) nd 5.7 (Wm -2 ), respetively, (mesured with UV sensor model: LEYBOLD DIDACTIC). UV-A, UV-B nd UV-C lms were purhsed from the Philips Compny. Plnt were treted with UV in their light period for 27 min per dy for 14 dys. We hve four replites for eh tretment. Pigment nlysis To determine the sorption y flvonoids,.1 g of fresh lef tissue were tken from the distl ends of the lef nd were extrted in 15 ml glss entrifuge tues ontining 1 ml ethyl lohol: eti id (99:1 v:v). The smples were gently oiled for 1 minutes in wter th t 8 o C nd rought up to volume. Asorne ws mesured t three wvelengths: 27, 3 nd 33 nm with UV-VIS spetrophotometer WPA, (model: S21 Diod Arry) (Krizek et l., 1998). To determine the onentrtion of nthoynins,.1 g fresh leves were tken nd were extrted in 15 ml glss entrifuge tues ontining 1 ml of idified methnol (methnol: HCl, 99: 1, v:v) nd kept over night in the drk. The smples were rought up to volume, nd the sorne t 55 nm ws determined. Anthoynin onentrtion ws lulted using n extintion oeffiient of 33 mol -1 m -1 (Wnger, 1979) 26

3 K. MAHDAVIAN, M. GHORBANLI, Kh. M. KALANTARI Totl hlorophylls nd rotenoids were extrted from lef dises with 8% etone nd determined ording to Lihtenthler (1987). HPLC nlysis of flvonoids.2 g fresh lef tissue ws tken. The homogenized tissue ws inuted in 8% methnol, for 24 h t 4 o C. Methnoli (8%) lef extrts were sujeted to reversed phse HPLC (Agilent model: 11) nd monitored t 355nm with diod rry detetor. A 2 µl smple ws injeted into mm C-18 olumn (Zorx 3 s) with moile phse of mm phosphori id (ph = 3), nd etonitrile. A non-liner grdient of etonitrile ws then run to elute the flvonoids (3 min t 1%, 1.5 min t 11.5, 9 min t 14%, 2 min t 19%, 9 min t 22%, nd 6 min t 1% etonitrile) (Greenerg et l., 1996). Rutin eluted t 7.3 min nd the pek re ws ompred with the stndrd. Queretin eluted t 12.5 min nd the pek re ws ompred with the stndrd. Thiorituri id retive sustnes (TBARS).2 g of the lef tissue of plnts were homogenized in 1 ml of.1% (w/v) trihloroeti id (TCA), then entrifuged t 1 g for 15 minutes. 1 ml of superntnt ws then vortexed with 4 ml of 2% (w/v) TCA ontining.5% (w/v) 2-thiorituri id (TBA), nd the solution ws heted for 3 minutes t 95 C. The smples were ooled on ie for 5 min nd reentrifuged for 1 minutes t 1 g. The non-speifi sorne of superntnt t 6 nm ws sutrted from the mximum sorne t 532 nm for the MDA mesurement (Heth & Pker, 1969), nd t 455 nm for other ldehydes (Meirs et l., 1992). For the MDA nd ldehyds lultion, n extintion oeffiient (ε) of M -1 m -1 ws used t 532 nm for MDA nd n ε of M -1 m -1 ws used t 455 nm s the verge of the ε otined for five other ldehyds (propnl, utnl, hexnl, heptnl nd propnldimethyl etl). Proline nd memrne permeility determintion Free proline ws extrted, derivtized with id ninhydrin nd sorne red ording to Btes et l. (1973) method. Memrne permeility of leves ws mesured y eletrolyte lekge (Dhinds et l., 1981). Lef smples for ll experiments were olleted from the fully expnded third lef elow the tip of the stem. Sttistil nlysis Quntitive hnges of different prmeters were nlysed through nlysis of vrine (ANOVA), with Dunn, s multiple rnge test eing used to determine signifint differenes mong tretments. Results The experimentl results showed tht UV-B nd UV-C irrdine used the redution of the ontents of hl, hl nd (hl + hl) of pepper leves ut in UV-A treted plnts there ws no signifint derese in these pigments. Signifint derese in hlorophyll ontents ws oserved in UV-B y 8% nd y 15% UV-C treted plnts, ut in UV-A treted plnts there ws no signifint derese (1%) in omprison with the ontrol smples (Figure 1). Chlorophyll ontents ws signifintly deresed, in UV-B nd UV-C treted pepper plnts y 12 nd 21% respetively ut in UV-A treted plnts there ws no signifint derese (3%) in omprison with the ontrol (Figure 1). Totl hlorophyll ontents ws signifintly deresed, in UV-B nd UV-C treted pepper plnts y 9 nd 17% respetively ut in UV-A treted plnts there ws no signifint derese (2%) in omprison with the ontrol (Figure 1) Signifint derese in rotenoid ontents ws oserved in UV-B nd UV-C treted plnts y 11 nd 2%, ut in UV-A treted plnts there ws no signifint derese (1%) in omprison with the ontrol (Figure 2). Chlorophyll ontent (mg/gfw) Tretment Chl-T Chl Chl Figure 1. Levels of hl, hl nd hl (+) in leves of C.nnuum treted with supplementry UV-A, UV- B nd UV-C. Vlues re the mens of four replites, nd rs indite SEM signifint differene t P <.5 ording to Dunn, s test, (hl = hlorophyll). 27

4 The Effets of Ultrviolet Rdition on the Contents of Chlorophyll, Flvonoid, Anthoynin nd Proline in Cpsium nnuum L. Crotenoid ontent (mg/gfw) Tretment Figure 2. Effet of UV-A, UV-B nd UV-C tretment on rotenoid ontent. Vlues re the mens of four replites, nd rs indites, nd rs indite SEM signifint differene t P <.5 ording to Dunn, s test. Anthoynin onentrtions ws signifintly inresed, in UV-B nd UV-C treted pepper plnts y 4 nd 7% respetively ut in UV-A treted plnts there ws only (1%) inrese in omprison with the ontrol (Figure 3). This study reveled inreses in flvonoid levels (27, 3 nd 33 nm) in pepper leves exposed to UV-A (respetively 6%, 4%, 5%), nd UV-B (respetively 1%, 8%, 7%) nd UV-C (respetively 12%, 8%, 8%) in omprison with ontrol (Figure 4). HPLC nlyses showed tht rutin ontent ws signifintly inresed, in UV-A, UV-B nd UV-C respetively y 8, 17 nd 27% in omprison with the ontrol (Figure 5, 7). Asorne Tretment Figure 4. Influene of UV-A, UV-B nd UV-C on the sorne of the methnoli extrt of leves t 27, 3 nd 33 nm. Vlues re the mens of four replites, nd rs indite SEM signifint differene t P <.5 ording to Dunn, s test. Rutin ontent (mg/gfw) Tretment Figure 5. Influene of UV-A, UV-B nd UV-C on the ontent of rutin. Vlues re the mens of four replites, nd rs indite SEM signifint differene t P <.5 ording to Dunn, s test. d Anthoynin onentrtion (mmol/gfw) Tretment Figure 3. Influene of UV-A, UV-B nd UV-C on onentrtion of nthoynins t 55. Vlues re the mens of four replites, nd rs indite SEM signifint differene t P <.5 ording to Dunn, s test. Queretin ontent (mg/gfw) Tretment Figure 6. Influene of UV-A, UV-B nd UV-C on the ontent of queretin in pepper leves. Vlues re the mens of four replites, nd rs indite SEM signifint differene t P <.5 ording to Dunn, s test. d 28

5 K. MAHDAVIAN, M. GHORBANLI, Kh. M. KALANTARI Rutin Queretin min Stndrd Rutin Queretin min Control Rutin Queretin min UV-B Queretin 125 Rutin min UV-A 16 Queretin Rutin min UV-C Figure 7. An HPLC-hromtogrm of pepper (Cpsium nnuum L.) lef extrt t 355 nm grown under ontrol, UV-A, UV-B nd UV-C tretments, respetively. Rutin eluted t 7.3 min nd the pek re ws ompred to the stndred. Queretin eluted t 12.5 min nd the pek re ws ompred to the stndrd. Our results y HPLC nlyses reveled tht queretin ontent is synthesized in lrge mounts in response to UV-A, UV-B nd UV-C respetively y 7, 12 nd 17% in omprison with the ontrol (Figure 6,7). Mlonedildehyde onentrtion ws signifintly inresed, in UV-B nd UV-C treted pepper leves y 13 nd 23% respetively ut in UV-A treted plnts there ws no signifint inrese (1%) in omprison with the ontrol. Mlonedildehyde onentrtion ws signifintly inresed, in UV-A, UV-B nd UV-C treted pepper roots y 2, 16 nd 38% respetively in omprison with the ontrol. Other ldehyde onentrtion ws signifintly inresed, in UV-B nd UV-C treted pepper leves y 27 nd 31% respetively nd in UV-A treted plnts there ws no signifint inrese (2%) in omprison with the ontrol. Other ldehyde onentrtion ws signifintly inresed, in UV-B nd UV-C treted pepper roots y 13 nd 22% respetively ut in UV-A treted plnts there ws no signifint inrese (5%) in omprison with the ontrol (Figure 8). Lekge of eletrolytes ws signifintly inresed, in UV-A, UV-B nd UV-C treted pepper leves y 1, 2 nd 38% respetively in omprison with the ontrol (Figure 9). 29

6 The Effets of Ultrviolet Rdition on the Contents of Chlorophyll, Flvonoid, Anthoynin nd Proline in Cpsium nnuum L. other ldehyde onentrtion (mmol/gfw) Lef Root Tretment M DA onentrtion (mmol/gfw) Lef d Root Tretment Figure 8. Effet of UV-A, UV-B nd UV-C tretment on lipid peroxidtion. Vlues re the mens of four replites, nd rs indite SEM signifint differene t P <.5 ording to Dunn, s test. The omprison etween the men for lef nd root ontent of these ldehydes were not shown. Lef Eletrolyte Lekge (%) Tretment d Lef proline ontent (mm/gfw) Tretment Figure 9. Effet of UV-A, UV-B nd UV-C tretment on lekge of eletrolytes. Vlues re the mens of four replites, nd rs indite SEM signifint differene t P <.5 ording to Dunn, s test. Figure 1. Proline ontent of leves of Cpsium nnuum L. treted with supplementry UV-A, UV-B nd UV-C. Vlues re the mens of four replites, nd rs indite SEM signifint differene t P <.5 ording to Dunn, s test. In this study, proline ontent ws signifintly elevted, in UV-B nd UV-C treted pepper leves y 1 nd 1% respetively ut in UV-A treted plnts there ws no signifint inrese (1%) in omprison with the ontrol (Figure 1). Disussion The experimentl results showed tht inrese UV-B nd UV-C irrdine lso used the redution of the ontents of hlorophyll, nd ( + ) of pepper leves. The redution of the hlorophyll ontent hs negtive effet on plnt photosyntheti effiieny. Sine it hs een reported tht photosynthesis is dependent on the light hrvesting properties of the hlorophylls (Go et l., 24). UV-B indued redution in hlorophyll my e expeted to result in lower levels of iomss umultion, nd hene e useful inditor of UV-B sensitivity (Smith et l., 2). Reent studies hve shown tht rotenoids serve protetive funtion ginst UV-B (Ru et l., 1991) nd UV-C (Cmpos et l., 1991) rdition. The effiy of rotenoids in proteting the photosystems is likely due to their funtion s effiient quenhers of high energy short wve rdition. The 3

7 K. MAHDAVIAN, M. GHORBANLI, Kh. M. KALANTARI mehnism y whih this is omplished ws first proposed to involve photohemil stte hnge of singlet oxygen to triplet form y intertion with rotenoids, removing the potentilly dngerous oxygen rdils produed in photo oxidtive proesses (Krinsky, 1979). In the present study pepper leves responded to the UV-B tretment y inresing signifintly their flvonoid nd nthoynin ontents, whih presumly offers protetion from the high UV-B level thus suggesting tht pepper leves re not well proteted from exposure to high UV-B nd need to synthesize dditionl pigments to protet them from the UV-B. Mesuring rutin ws n ttempt to estimte the signifine of phenoli ompounds, umulted in response to UV rdition (Figure 5). The flvonoids ply mny defensive roles in plnts, nd intereption of UV-B y epiderml flvonoids often proposed s n dptive mehnism preventing UV- B from rehing the mesophyll nd ffeting photosynthesis (Liu et l., 1995). Thus, the pepper plnts my tivted defene mehnism ginst UV dmges y inresing non-photosyntheti pigments. The involvement of flvonols in the UV-B response hs een reported for severl plnt speies, inluding legume suh s soyens (Glyine mx) (Middleton & Termur, 1993). However, the ntioxidnt funtion of flvonoids is omplex nd depends on vriety of ftors, inluding omprtmentliztion, redox potentil, presene of doule nds, glyosyltion nd hydroxyltion (Bors et l., 1995; Rie et l., 1996; Cooper-Driver & Bhtthry, 1998). This omplexity therefore lso needs to e tken into ount in the onsidertion of possile ntioxidnt funtions for inresed flvonoid levels under UV tretment. In this reserh the results of spetrophotometri nd HPLC nlysis showed n inrese in flvonoids under UV tretment, espeilly UV-B nd UV-C tretments tht proly mens either inrement in flvonoid is the role of these ompounds in the protetion of oxidtive stress in plnts, or preventing the penetrtion of destrutive nds of UV light to the most sensitive tissue. Oxidtive dmge n e deteted y lipid peroxidtion. Hydroxyl rdils nd singlet oxygen n ret with lipids nd form lipid peroxy rdils nd hydroperoxide (Blokhin et l., 23; Hollosy 22). The peroxy rdils n strt hydrogen from other unsturted ftty ids, leding to hin retion of peroxidtion. The peroxidtion of memrne lipids leds to the rekdown of their struture nd funtion (Hollosy 22; Yun et l., 2). The inrese in TBARS ontent is more preisely n inditor of generl UV-indued oxidtive dmge, due to the impirment of ell defense system (Cost et l., 22; Brk et l., 2). Chnge in `TBARS (espeilly MDA) ws the first evidene tht under our experiment, UV (UV-B nd UV-C) indued oxidtive stress (Figure 8). In pepper, n inrese of lef memrne-permeility ws onsiderle in omprison with the ontrol. Indiret evidene from mny experiments suggests tht UV-B nd UV-C exposure genertes free rdils, whih inrese the lipid peroxidtion nd disruption of memrne integrity (Krmer et l., 1991). In the present study, mrked inrese in proline in UV-B nd UV-C tretment (Figure 1) represents dptive responses ginst oxidtive dmge indued y UV rdition. Proline is known to e involved in lleviting ytosoli idi ssoited with severl stresses (Kurkdjin & Guern, 1989). The removl of exess H + ourring s result of proline synthesis my hve positive effet on redution of the UV-B nd UV-C indued dmge. This led us to elieve tht UV rdition indued proline umultion protets plnts ginst UV rdition promoted peroxidtion proesses. Seondry metolite nlysis in our experiment showed inreses in queretin in pepper leves under UV- A, UV-B nd UV-C rdition (Figure 6, 7). Correltions etween queretin onentrtions nd lipid peroxidtion levels indited n ntioxidnt role of seondry metolites in pepper leves exposed to UV-A, UV-B nd UV-C rdition. These findings indite n importnt role of UV-B nd UV-C rdition in queretin synthesis. It seems tht pplied doses of UV rdition exert stte of stress, where limits of tolerne re exeeded nd dptive pity is overtxed, tht possily results in disturne of queretin synthesis. Conlusion By onsidering to otined results in this study we onluded tht UV-A often hrmful in plnt, ut UV-B nd UV-C hve serious effets on plnt. 31

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