بررسی الگوی پروتئینی، سنجش آنزیم‌های آنتی‌اکسیدان و میزان انباشتگی کادمیوم در گیاه آفتابگردان (Helianthus annuus L.) تحت تیمار کادمیوم و اسید سالیسیلیک
الموضوعات :
1 - گروه زیست شناسی، دانشکده علوم پایه، دانشگاه پیام نور(مرکز خوی) ، ایران
الکلمات المفتاحية: پروتئین, آسکوربات پراکسیداز, کاتالاز, الکتروفورز, اسید سالیسیلیک, سوپر اکسید دیسموتاز, انباشتگی کادمیوم, گلوتاتیون ردوکتاز, آفتابگردان (Helianthus annuus L.),
ملخص المقالة :
به‌منظور بررسی اثر برهم کنش کادمیوم به‌عنوان یک فلز سنگین و اسید سالیسیلیک به‌عنوان یک ماده آنتی‌اکسیدان و ضد تنش در گیاه آفتابگردان، بذرها پس از ضدعفونی با محلول هیپوکلریت سدیم یک درصد و شستشو با آب مقطر در گلدان‌های حاوی خاک ماسه در اتاق کشت، با دمای 34 درجه سانتی‌گراد در روز و 25 درجه سانتی‌گراد در شب و فتوپریود 16 ساعت روشنایی و 8 ساعت تاریکی قرار گرفتند. این پژوهش به‌صورت آزمایش فاکتوریل در قالب طرح کاملاً تصادفی با چهار تکرار در شرایط گلخانه ای انجام شد. گیاهان در مرحله دو برگی هر هفته در معرض تیمار کادمیوم (غلظت‌های 0، 50 ، 100، 150 و 200 میکرومولار) قرارگرفتند. یک هفته پس از پایان تیمار کادمیوم برگ گیاهان با اسید سالیسیلیک (غلظت‌های 0، 250، 500 میکرومولار) اسپری شدند. بعد از برداشت، غلظت کادمیوم در ریشه و اندام هوایی اندازه گیری شد. سپس سنجش آنزیم‌های آنتی اکسیدان انجام گرفت. پروتئین کل نمونه‌های برگی استخراج و بررسی الگوی تغییرات پروتئینی با استفاده از سیستم الکتروفورز به روش SDS-PAGE انجام شد. غلظت کادمیوم در ریشه و اندام هوایی با افزایش کادمیوم و اسید سالیسیلیک، افزایش یافت. فعالیت کاتالاز با افزایش غلظت کادمیوم، کاهش و با افزایش اسید سالیسیلیک کاهش بسیار جزئی یافت. فعالیت آنزیم‌های سوپر اکسید دیسموتاز، آسکوربات پراکسیداز و گلوتاتیون ردوکتاز با افزایش کادمیوم، افزایش یافت و و با افزایش اسید سالیسیلیک کاهش یافت. الکتروفورز پروتئین‌های برگ تحت تیمارهای کادمیوم و اسید سالیسیلیک نشان دهنده اختلافاتی در شدت نقاط پروتئینی برگ نسبت به گیاهان شاهد بود. به طوری که بیان برخی از پروتئین‌ها افزایش یا کاهش یافت. بنابراین تیمار کادمیوم و اسید سالیسیلیک میزان غلظت کادمیوم، فعالیت آنزیم‌های آنتی اکسیدان و الگوی پروتئین‌های برگ آفتابگردان را تغییر داد.
Arora, A., Sairam, R.K. and Srivastava, G.C. (2002). Oxidative stress and antioxidant system
in plants. Plant Physiology. 82: 1227-1237.
Baker, A.J.M., Reeves, R.D. and Hajar, A.S.M. (1994). Heavy metal accumulation and tolerance in British populations of the metallophyte Thlaspi caerulescens J. & C. Presl. (Brassicaceae). New Phytologist. 127: 61-68.
Barandeh, F., Kavousi, H.R., Pourseyedi, S.H. (2014). Activity of antioxidant enzymes, PAL and prolin content of lentil seedlings under cadmium stress. 1th Conference on New Finding in Environment and Agriculture Ecosystems. Tehran, Iran
Burkhanova E.A., Fedina A.B. and Kulaeva O.N. (1999). Effect of salicylic acid and 2, 5-oligodenylates on protein synthesis in tobacco leaves under heat shock conditions: a comparative study. Russ Journal Plant Physiol. 46:16-22.
Cho, U.H. and Sohn, J.Y. (2004). Cadmium-induced changes in antioxidative systems, hydrogen peroxide content and lipid peroxidation in Arabidopsis thaliana L. Journal of Plant Biology. 47(3): 262-269.
Das, P., Samantary, S. and Rout, R. (1998). Studies on cadmium toxity in plants: A review. Environ Pollut. 98: 29-36.
Dazy, M., Jung, V., Ferard, J. and Masfaraud, J. (2008). Ecological recovery of vegetation on a coke-factory soil: Role of plant antioxidant enzymes and possible implication in site restoration. Chemosphere. 74: 57-63.
Deponte, M. (2013). Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes. Biochimica et Biophysica Acta, 1830: 3217–3266.
Dolatabadian, A., Modares sanavi, S.A.M. and Etemadi, F. (2008). Effects of salicylic acid pretreatment on wheat seeds germination under salt stress. Iranian Journal of Biology. 3 (21): 692-702.
Eftekhar, N., Fallah, S., Abbasi Sooraki, A., Khodaverdiloo, H. And Rahimi, A. (2019). Effect of salicylic acid and potassium nitrate pretreatment on enhancing the sunflower tolerance in contaminated soils with cadmium. Iranian Journal of Seed Science and Research. 6(2): 161-175.
Esterbauer, H. and Grill, D. (1978). Seasonal variation of glutathione and glutathione reductase in needles of Picea abies. Plant Physiology. 61: 119-121.
Eyidogan, F., Oz, M.T. (2005). Effect of salinity on antioxidant responses of chickpea seedlings.
Acta Physiology Plant. 29: 485–493.
Gallego, S.M., Benavides, M.P. and Tomaro, M.L. (1996). Effect of heavy metal ion excess on sunflower leaves: evidence for involvement of oxidative stress. Plant Science. 121: 151–159.
Giannopolitis, C.N. and Ries, S.K. (1977). Superoxide dismutase: occurrence in higher plants. Plant Physiology. 59: 309–314.
Gondor, O.K., Pal, M., Darko, E., Janda, T. and Szalai, G.(2016). Salicylic Acid and Sodium Salicylate Alleviate Cadmium Toxicity to Different Extents in Maize (Zea mays L.). Plos One. 11(8):1-18.
Jadia, C.H. and Fulekar, M.H. (2008). The application of vermicompost to remove zinc, cadmium, copper, nickel and lead by sunflower plant. Environmental Engineering and Management Journal. 7: 547-558.
Jing, C., Cheng, Z., Li-Ping, L., Zhong-Yang, S. and Xue-Bo, P. (2007). Effects of exogenous salicylic acid on growth and H2O2-metabolizing enzymes in rice seedlings under lead stress. Journal of Environmental Sciences. 19: 44-49.
Khosravi, F. (2010). The effect of different doses of potassium on phytoremediation of soil contaminated with cadmium. Journal of Agriculture. 90:57-64.
Krantev, A., Yordanova, R. and Popova, L. (2006). Salicylic acid decreases Cd toxicity in maize plants.Gen.Appl. Plant Physiology. Special Issue: 45-52.
Laemmli, U.K. (1970). Cleaving of the structural proteins during the assembly of the head of bacteriophage T4. Nature (London). 227: 680- 685.
Lasat, M.M. (2002). Phytoexteraction of toxic metals: A review of biological mechanisms. Journal of Environmental Quality. 31:109-120.
Leon, A.M., Palma, J.M., Corpas, F.J., Gomez, M., Romero-Puertas, M.C., Chatterjee, D., Mateos, R.M., Del Rio, L.A. and Sandalio, L.M. (2002). Antioxidative enzymes in cultivars of pepper plants with different sensitivity to cadmium. Plant Physiology and Biochemistry. 40: 813-820.
Lu, L.L., Tian, S.K., Yang, X.E., Wang, X.C., Brown, P., Li, T.Q. and He, Z.L.,(2008). Enhanced root-to-shoot translocation of cadmium in the hyperaccumulating ecotype of Sedum alfredii. Journal of Experimental Botany . 59(11): 3203–3213.
McDonald, M.B. (2000). Seed priming. In: Seed Technology and Biological Basis, M. Black and J. D. Bewley (Eds.), Sheffield Academic Press. England: 287-325.
Metraux, J.P. (2001). Systemic acquired and salicylic acid: current state of knowledge. European Journal of Plant Pathology. 107:13-18.
Metwally, A., Finkermeier, I., Georgi, M. and Dietz, K.J. (2003). Salicylic acid alleviates the cadmium toxicity in barley seedlings. Plant Physiology.132: 272-281.
Mittler, R. (2002). Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science. 7: 405-410.
Mohtadi, A. and Hoshyari, S. (2016). Study of cadmium and zinc interaction in Matthiola flavida Boiss. Journal of Plant Research. 29(1):210-220.
Motesharezadeh, B., Savaghebi-Firoozabadi, G.R., Mirseyed Hosseini, H. and Alikhani, H.A. (2010). Study of the enhanced phytoextraction of cadmium in a calcareous soil. International Journal of Environmental Research.4(3): 525-532.
Padash, A., Ghanbari, A., Sirus Mehr, A. and Asgharipoor, M. (2016). Effect of salicylic acid on resistance of Ocimum plant to Pb toxicity. Iranian Plant Biology. 27 (8): 17-32.
Pal, M., Szalai, G., Horvath, E., Janda, T. and Paldi, E. (2002). Effect of salicylic acid during heavy metal stress. Proc. 7th Hungarian Congress of Plant Physiol. Szeged, Hungarian. 46: 119-120.
Peluffo, L., Lia, V., Troglia, C., Maringolo, C., Norma, P., Escande, A., Hopp, H.E., Lytovchenko, A., Fernie, A.R., Heinz, R. and Carrari, F. (2010). Metabolic profiles of sunflower genotypes with contrasting response to Sclerotinia sclerotiorum infection. Phytochemistry. 71: 70–80.
Popova, L., Maslenkova, L., Yordanova, R., Krantev, A., Szalai, G. and Janda, T.( 2008). Salicylic acid protects photosynthesis against cadmium toxicity in pea plants. Gen. Appl. Plant Physiology. Special Issue. 34 (3-4): 133-148.
Reed, R., Holmes, D., Weyers, J. and Jones, A. (1998). Practical skills in biomolecular sciences. pp. 103-108. Longman, Hongkong.
Reeves, R.D., Kruckeberg, A.R., Adiguzel, N. and Kramer, U. (2001). Studies on the flora of serpentine and other metalliferous areas of western Turkey. South African Journal of Science. 97: 513-517.
Romero-Puertas, M.C., Corpas, F.J., Rodriguez-Serrano, M., Gomez, M., Sandalio, L.M. and Del Rio, L.A. (2007). Differential expression and regulation of antioxidative enzymes by cadmium in pea plants. Journal of Plant Physiology. 164: 1346-1357.
Rosa, G., Peralta-Videa, J.R., Montes, M., Parsons, J.G., Cano-Aguilera, I. and Gardea-Torresdey, J.L. (2004). Cadmium uptake and translocation in tumbleweed (Salsola kali), a potential Cd-hyperaccumulator desert plant species: ICP/OES and XAS studies. Chemosphere. 55: 1159-1168.
Senaranta, T., Touchell, D., Bumm, E. and Dixon, K. (2002). Acetylsalicylic (aspirin) and salicylic acid induce multiple stress tolerance bean and tomato plants. Plant Growth Regulation. 30: 157-161.
Shi, G.R., Cai, Q.S., Liu, Q.Q. and Wu, L. (2009). Salicylic acid-mediated alleviation of cadmium toxicity in hemp plants in relation to cadmium uptake, photosynthesis and antioxidant enzymes. Acta Physiologiae Plantarum. 31: 969-977.
Shigeru, S., Takahiro, I., Masahiro, T., Yoshiko, M., Toru, T., Yukinori, Y. and Kazuya, Y. (2002). Regu1ation and function of ascorbate peroxidase isoenzymes. Journal of Experimental Botany. 5: 1305–1319.
Simon, L.M., Fatrai, Z., Jonas, D.E. and Matkovics, B. (1974). Study of peroxide metabolism enzymes during the development of Phaseolus vulgaris. Plant Biochemistry and Physiology. 166: 387–392.
Snyman, M. and Cronje, M.J. (2008). Modulation of heat shock factors accompanies salicylic acid-mediated potentiation of Hsp70 in tomato seedlings. Experimental Botany. 59(8): 2125-2132.
Torres, E., Cid, A., Herrero, C. and Abalde. J. (2000). Effect of cadmium on growth, ATP content, carbon fixation and ultra structure in the marine diatom Phaeodactylum tricornutum Bohlin. Water, Air, Soil Pollution. 117: 1-14.
Wagner, G. J. (1993). Accumulation of cadmium in crop plants and its consequences to human health. Advances in Agronomy. 51: 173-212.
Wang, J.w., Zheng, L.P. and Tan, R.X. (2006). Involvement of nitric oxide in oxidative burst phenylalanine ammonia-lyase activation and taxol production induced by low- energy ultrasound in Taxus yannanensiscell suspension cultures. Nitric Oxide Biology and Chemistry. 15: 351-358.
Westerman, R.E.L. (1990). Soil testing and plant analysis. SSSA, Madison, Wisconsin, USA.
Wojcik, M., Vangronsveld, J. and Tukiendorf, A. (2005). Cadmium tolerance in Thlaspi caerulescens: Growth parameters, metal accumulation and phytochelatin synthesis in response to cadmium. Environmental and Experimental Botany. 53: 151-161.
Yannarelli, G.G., Fernandez-Alvarez, A.J., Santa-Cruz, D.M. and Tomaro, M.L. (2007). Glutathione reductase activity and isoforms in leaves and roots of wheat plants subjected to cadmium stress. Phytochemistry. 68: 505–512.
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Arora, A., Sairam, R.K. and Srivastava, G.C. (2002). Oxidative stress and antioxidant system
in plants. Plant Physiology. 82: 1227-1237.
Baker, A.J.M., Reeves, R.D. and Hajar, A.S.M. (1994). Heavy metal accumulation and tolerance in British populations of the metallophyte Thlaspi caerulescens J. & C. Presl. (Brassicaceae). New Phytologist. 127: 61-68.
Barandeh, F., Kavousi, H.R., Pourseyedi, S.H. (2014). Activity of antioxidant enzymes, PAL and prolin content of lentil seedlings under cadmium stress. 1th Conference on New Finding in Environment and Agriculture Ecosystems. Tehran, Iran
Burkhanova E.A., Fedina A.B. and Kulaeva O.N. (1999). Effect of salicylic acid and 2, 5-oligodenylates on protein synthesis in tobacco leaves under heat shock conditions: a comparative study. Russ Journal Plant Physiol. 46:16-22.
Cho, U.H. and Sohn, J.Y. (2004). Cadmium-induced changes in antioxidative systems, hydrogen peroxide content and lipid peroxidation in Arabidopsis thaliana L. Journal of Plant Biology. 47(3): 262-269.
Das, P., Samantary, S. and Rout, R. (1998). Studies on cadmium toxity in plants: A review. Environ Pollut. 98: 29-36.
Dazy, M., Jung, V., Ferard, J. and Masfaraud, J. (2008). Ecological recovery of vegetation on a coke-factory soil: Role of plant antioxidant enzymes and possible implication in site restoration. Chemosphere. 74: 57-63.
Deponte, M. (2013). Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes. Biochimica et Biophysica Acta, 1830: 3217–3266.
Dolatabadian, A., Modares sanavi, S.A.M. and Etemadi, F. (2008). Effects of salicylic acid pretreatment on wheat seeds germination under salt stress. Iranian Journal of Biology. 3 (21): 692-702.
Eftekhar, N., Fallah, S., Abbasi Sooraki, A., Khodaverdiloo, H. And Rahimi, A. (2019). Effect of salicylic acid and potassium nitrate pretreatment on enhancing the sunflower tolerance in contaminated soils with cadmium. Iranian Journal of Seed Science and Research. 6(2): 161-175.
Esterbauer, H. and Grill, D. (1978). Seasonal variation of glutathione and glutathione reductase in needles of Picea abies. Plant Physiology. 61: 119-121.
Eyidogan, F., Oz, M.T. (2005). Effect of salinity on antioxidant responses of chickpea seedlings.
Acta Physiology Plant. 29: 485–493.
Gallego, S.M., Benavides, M.P. and Tomaro, M.L. (1996). Effect of heavy metal ion excess on sunflower leaves: evidence for involvement of oxidative stress. Plant Science. 121: 151–159.
Giannopolitis, C.N. and Ries, S.K. (1977). Superoxide dismutase: occurrence in higher plants. Plant Physiology. 59: 309–314.
Gondor, O.K., Pal, M., Darko, E., Janda, T. and Szalai, G.(2016). Salicylic Acid and Sodium Salicylate Alleviate Cadmium Toxicity to Different Extents in Maize (Zea mays L.). Plos One. 11(8):1-18.
Jadia, C.H. and Fulekar, M.H. (2008). The application of vermicompost to remove zinc, cadmium, copper, nickel and lead by sunflower plant. Environmental Engineering and Management Journal. 7: 547-558.
Jing, C., Cheng, Z., Li-Ping, L., Zhong-Yang, S. and Xue-Bo, P. (2007). Effects of exogenous salicylic acid on growth and H2O2-metabolizing enzymes in rice seedlings under lead stress. Journal of Environmental Sciences. 19: 44-49.
Khosravi, F. (2010). The effect of different doses of potassium on phytoremediation of soil contaminated with cadmium. Journal of Agriculture. 90:57-64.
Krantev, A., Yordanova, R. and Popova, L. (2006). Salicylic acid decreases Cd toxicity in maize plants.Gen.Appl. Plant Physiology. Special Issue: 45-52.
Laemmli, U.K. (1970). Cleaving of the structural proteins during the assembly of the head of bacteriophage T4. Nature (London). 227: 680- 685.
Lasat, M.M. (2002). Phytoexteraction of toxic metals: A review of biological mechanisms. Journal of Environmental Quality. 31:109-120.
Leon, A.M., Palma, J.M., Corpas, F.J., Gomez, M., Romero-Puertas, M.C., Chatterjee, D., Mateos, R.M., Del Rio, L.A. and Sandalio, L.M. (2002). Antioxidative enzymes in cultivars of pepper plants with different sensitivity to cadmium. Plant Physiology and Biochemistry. 40: 813-820.
Lu, L.L., Tian, S.K., Yang, X.E., Wang, X.C., Brown, P., Li, T.Q. and He, Z.L.,(2008). Enhanced root-to-shoot translocation of cadmium in the hyperaccumulating ecotype of Sedum alfredii. Journal of Experimental Botany . 59(11): 3203–3213.
McDonald, M.B. (2000). Seed priming. In: Seed Technology and Biological Basis, M. Black and J. D. Bewley (Eds.), Sheffield Academic Press. England: 287-325.
Metraux, J.P. (2001). Systemic acquired and salicylic acid: current state of knowledge. European Journal of Plant Pathology. 107:13-18.
Metwally, A., Finkermeier, I., Georgi, M. and Dietz, K.J. (2003). Salicylic acid alleviates the cadmium toxicity in barley seedlings. Plant Physiology.132: 272-281.
Mittler, R. (2002). Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science. 7: 405-410.
Mohtadi, A. and Hoshyari, S. (2016). Study of cadmium and zinc interaction in Matthiola flavida Boiss. Journal of Plant Research. 29(1):210-220.
Motesharezadeh, B., Savaghebi-Firoozabadi, G.R., Mirseyed Hosseini, H. and Alikhani, H.A. (2010). Study of the enhanced phytoextraction of cadmium in a calcareous soil. International Journal of Environmental Research.4(3): 525-532.
Padash, A., Ghanbari, A., Sirus Mehr, A. and Asgharipoor, M. (2016). Effect of salicylic acid on resistance of Ocimum plant to Pb toxicity. Iranian Plant Biology. 27 (8): 17-32.
Pal, M., Szalai, G., Horvath, E., Janda, T. and Paldi, E. (2002). Effect of salicylic acid during heavy metal stress. Proc. 7th Hungarian Congress of Plant Physiol. Szeged, Hungarian. 46: 119-120.
Peluffo, L., Lia, V., Troglia, C., Maringolo, C., Norma, P., Escande, A., Hopp, H.E., Lytovchenko, A., Fernie, A.R., Heinz, R. and Carrari, F. (2010). Metabolic profiles of sunflower genotypes with contrasting response to Sclerotinia sclerotiorum infection. Phytochemistry. 71: 70–80.
Popova, L., Maslenkova, L., Yordanova, R., Krantev, A., Szalai, G. and Janda, T.( 2008). Salicylic acid protects photosynthesis against cadmium toxicity in pea plants. Gen. Appl. Plant Physiology. Special Issue. 34 (3-4): 133-148.
Reed, R., Holmes, D., Weyers, J. and Jones, A. (1998). Practical skills in biomolecular sciences. pp. 103-108. Longman, Hongkong.
Reeves, R.D., Kruckeberg, A.R., Adiguzel, N. and Kramer, U. (2001). Studies on the flora of serpentine and other metalliferous areas of western Turkey. South African Journal of Science. 97: 513-517.
Romero-Puertas, M.C., Corpas, F.J., Rodriguez-Serrano, M., Gomez, M., Sandalio, L.M. and Del Rio, L.A. (2007). Differential expression and regulation of antioxidative enzymes by cadmium in pea plants. Journal of Plant Physiology. 164: 1346-1357.
Rosa, G., Peralta-Videa, J.R., Montes, M., Parsons, J.G., Cano-Aguilera, I. and Gardea-Torresdey, J.L. (2004). Cadmium uptake and translocation in tumbleweed (Salsola kali), a potential Cd-hyperaccumulator desert plant species: ICP/OES and XAS studies. Chemosphere. 55: 1159-1168.
Senaranta, T., Touchell, D., Bumm, E. and Dixon, K. (2002). Acetylsalicylic (aspirin) and salicylic acid induce multiple stress tolerance bean and tomato plants. Plant Growth Regulation. 30: 157-161.
Shi, G.R., Cai, Q.S., Liu, Q.Q. and Wu, L. (2009). Salicylic acid-mediated alleviation of cadmium toxicity in hemp plants in relation to cadmium uptake, photosynthesis and antioxidant enzymes. Acta Physiologiae Plantarum. 31: 969-977.
Shigeru, S., Takahiro, I., Masahiro, T., Yoshiko, M., Toru, T., Yukinori, Y. and Kazuya, Y. (2002). Regu1ation and function of ascorbate peroxidase isoenzymes. Journal of Experimental Botany. 5: 1305–1319.
Simon, L.M., Fatrai, Z., Jonas, D.E. and Matkovics, B. (1974). Study of peroxide metabolism enzymes during the development of Phaseolus vulgaris. Plant Biochemistry and Physiology. 166: 387–392.
Snyman, M. and Cronje, M.J. (2008). Modulation of heat shock factors accompanies salicylic acid-mediated potentiation of Hsp70 in tomato seedlings. Experimental Botany. 59(8): 2125-2132.
Torres, E., Cid, A., Herrero, C. and Abalde. J. (2000). Effect of cadmium on growth, ATP content, carbon fixation and ultra structure in the marine diatom Phaeodactylum tricornutum Bohlin. Water, Air, Soil Pollution. 117: 1-14.
Wagner, G. J. (1993). Accumulation of cadmium in crop plants and its consequences to human health. Advances in Agronomy. 51: 173-212.
Wang, J.w., Zheng, L.P. and Tan, R.X. (2006). Involvement of nitric oxide in oxidative burst phenylalanine ammonia-lyase activation and taxol production induced by low- energy ultrasound in Taxus yannanensiscell suspension cultures. Nitric Oxide Biology and Chemistry. 15: 351-358.
Westerman, R.E.L. (1990). Soil testing and plant analysis. SSSA, Madison, Wisconsin, USA.
Wojcik, M., Vangronsveld, J. and Tukiendorf, A. (2005). Cadmium tolerance in Thlaspi caerulescens: Growth parameters, metal accumulation and phytochelatin synthesis in response to cadmium. Environmental and Experimental Botany. 53: 151-161.
Yannarelli, G.G., Fernandez-Alvarez, A.J., Santa-Cruz, D.M. and Tomaro, M.L. (2007). Glutathione reductase activity and isoforms in leaves and roots of wheat plants subjected to cadmium stress. Phytochemistry. 68: 505–512.
