بررسی تاثیر محلول‌پاشی 5-آمینولوولونیک اسید (5-ALA) بر خصوصیات مورفولوژیک و بیوشیمیایی ریحان (Ocimum basilicum L.) در شرایط تنش آرسنیک

نوع مقاله : مقاله کامل علمی پژوهشی

نویسندگان

1 دانش‌آموخته دکتری گروه علوم باغبانی، دانشکده کشاورزی و منابع طبیعی، دانشگاه محقق اردبیلی، اردبیل، ایران.

2 نویسنده مسئول، استاد گروه علوم باغبانی، دانشکده کشاورزی و منابع طبیعی، دانشگاه محقق اردبیلی، اردبیل، ایران.

3 دانشیار گروه علوم خاک، دانشکده کشاورزی، دانشگاه ارومیه، ارومیه، ایران.

4 دانشیار گروه علوم خاک، دانشکده کشاورزی و منابع طبیعی، دانشگاه محقق اردبیلی، اردبیل، ایران.

5 دانشیار گروه علوم باغبانی، دانشکده کشاورزی و منابع طبیعی، دانشگاه محقق اردبیلی، اردبیل، ایران.

چکیده

سابقه و هدف: ریحان (Ocimum basilicum L.) یک گیاه یکساله از خانواده نعناعیان (Lamiacea) است که به عنوان سبزی و گیاه دارویی و معطر استفاده از آن در سراسر جهان روزبروز رو به افزایش است. فلزات و یا شبه‌فلزاتی که وزن مخصوص آنها بالاتر از 5 گرم بر سانتی‌متر مکعب باشند به عنوان فلزات سنگین در نظر گرفته می‌شوند و آرسنیک به عنوان یکی از مهمترین فلزات سنگین شناخته می‌شود. این عناصر به عنوان یکی از مهمترین آلوده کننده‌های محیط زیست مطرح بوده که برای همه موجودات زنده خطرناک هستند. این فلزات موجب جلوگیری از فرایندهای فیزیولوژیکی مانند فتوسنتز، تنفس، طویل شدن سلول، روابط آبی گیاه، متابولیسم نیتروژن و تغذیه معدنی گیاهان می‌شود. استفاده از تنظیم کننده رشدی به نام 5-ALA به عنوان یک راهکار نوین برای مقابله با برخی تنش‌های محیطی و تعدیل اثرات مخرب محیطی بر رشد و نمو گیاهان قبلاً مطالعه شده است. 5-ALA به عنوان پیش‌ماده برای سنتز ترکیبات تتراپیرولی مانند کلروفیل، پورفیرین، ویتامین B12، بیلین‌ها و هم است که در جلبک‌ها، باکتری‌ها، گیاهان و حیوانات یافت می‌شود. پژوهش حاضر به منظور بررسی تاثیر 5-ALA بر کاهش و یا تعدیل اثرات مخرب تنش آرسنیک بر صفات مورفولوژیکی و فیزیولوژیکی ریحان انجام شد.

مواد روش‌ها: مطالعه اثر محلول‌پاشی 5-ALA در سه غلظت (صفر، 10 و 20 میلی‌گرم در لیتر) بر خصوصیات مورفولوژیک و فیزیولوژیک گیاه ریحان در دو نوع آلوده به آرسنیک و خاک فاقد آلودگی به آرسنیک (شاهد) یک آزمایش فاکتوریل در قالب کاملاً تصادفی در سه تکرار در گلخانه انجام شد.

یافته‌ها: نتایج تجزیه واریانس داده‌ها نشان داد که تنش آرسنیک موجب کاهش محتوای صفات مورفولوژیکی ریحان شد امًا تیمار 5-ALA تا حدودی موجب بهبود این صفات حتی در شرایط آلودگی آرسنیک می‌گردد. همچنین صفات فیزیولوژیکی مانند آنتوسیانین، فنول، فلاونوئید، پرولین، قندهای محلول، DPPH، MDA، گائیکول پراکسیداز، کاتالاز، آسکوربات پراکسیداز، RWC و نشت یونی به‌طور معنی‌داری تحت تاثیر آلودگی خاک به آرسنیک و محلول‌پاشی 5-ALA و اثر متقابل این دو قرار گرفتند. در مورد صفات رنگیزه‌های فتوسنتزی (کلروفیل a، b، کلروفیل کل و کارتنوئید) اثر آلودگی آرسنیک و محلول‌پاشی 5-ALA بر این صفات معنی‌دار بود امّا اثر متقابل این دو فاکتور بر صفات مذکور معنی‌دار نبود. تنش آرسنیک باعث کاهش رشد ریحان و میزان رنگیزه‌های فتوسنتزی آن و از طرف دیگر موجب افزایش نشت الکترولیت و MDA شد. در واقع محلول‌پاشی با 5-ALA با افزایش پرولین و آنزیم‌های آنتی اکسیدان باعث کاهش اثرات منفی تنش آرسنیک شد.

نتیجه‌گیری: با توجه به تاثیر کاهندگی و بازدارندگی آرسنیک بر رشد و نمو ریحان و اثرات 5-ALA در کاهش و تعدیل اثرات مذکور می-توان گفت که استفاده از این ترکیب می‌تواند به عنوان یک راهکار نو تا حدی موثر در کاهش اثرات مخرب محیطی در گیاهان در پژوهش‌آتی بیشتر مورد توجه قرار گیرد.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Investigation of the effects of 5-aminolevulinic acid (ALA) on morphological, physiological, and antioxidative enzymes of sweet basil (Ocimum basilicum L.) under arsenic stress

نویسندگان [English]

  • Ali Abdollahi 1
  • Behrooz Esmaielpour 2
  • Mohsen barin 3
  • Ali ashraf Soltani-toolarood 4
  • Mousa Torabi Gayklou 5
1 Ph.D. Graduate, Dept. of Horticulture, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran.
2 Corresponding Author, Professor, Dept. of Horticulture, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran.
3 Associate Prof., Dept. of Soil Science, Faculty of Agriculture, Urmia University, Urmia, Iran.
4 Associate Prof., Dept. of Soil Science, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran.
5 Associate Prof., Dept. of Horticulture, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran.
چکیده [English]

Introduction: Sweat basil (Ocimum basilicum L. ) is an annual herb belongs to Lamiaceae family and demands for its in increasing because of uses as vegetable, medicinal and cosmetic plant in the world wide. Metals and/or metalloids that have specific weights higher than about 5g/cm3 are known as heavy metals. Arsenic (As) is recognized as one of the most important heavy metal in the world. Heavy metals are significant environmental contaminants, and their toxicity is a problem for all living organisms. Heavy metals inhibit physiological processes such as respiration, photosynthesis, cell elongation, plant-water relationship, N-metabolism and mineral nutrition in plants. According to history of application of plant growth regulators as new procedure for confronting with environment stresses and adjustment of its dangerous effects on plant growth and development, this study carried out to investigation the reduction or balancing effects of 5-ALA on hazard effects of As on morphological and physiological properties of basil.

Material and methods: In this examination basil plant cultivated in two groups of As-contaminated and As-noncontaminated soils. For study of foliar application of 5-ALA in three concentrations (0, 10 and 20 mg/L) on morphological and physiological traits of basil conducted in the greenhouse in form of completely randomized design in four replication carried out.

Results: The analysis of results showed that arsenic stress decreased the content of basil morphological properties but the treatment of 5-ALA to some extent improved the content of these properties. Also the physiological properties such as anthocyanin, phenol, flavonoid, proline, soluble sugar, DPPH, MDA, guaicol peroxidase, catalase, ascorbate peroxidase, RWC and ion leakage significantly affected by arsenic contamination and 5-ALA spraying and reaction of these. In the case of photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll and carotenoids) were affected by arsenic contamination and spraying of 5-ALA but the reaction of these factors on these adjectives was not significant. Arsenic stress decreased the basil growth and photosynthetic pigments and on the other hand electrolyte leakage and MDA content were increased. Actually, the spraying of 5-ALA with the increase of proline and antioxidant enzymes reduced the negative effects of arsenic stress.

Conclusion: According to the reduction and inhibition effects of arsenic on the growth and development of basil, and the effects of 5-ALA decline and adjustment on these factors, it can be said that the use of this component could be as a new way to some extent in reduction of dangerous effects of these environment contaminations on plants in the future studies should be considered.

کلیدواژه‌ها [English]

  • Ocimum basilicum L
  • Arsenic stress
  • 5-aminolevulinic acid (5-ALA)
  • Antioxidant enzymes
1.Burdina, I., & Priss, O. (2016). Effect of the substrate composition on yield and quality of basil (Ocimum basilicum L.). Journal of Horticultural Research, 24 (2), 109-118.2.Zare Dehabadi, S., Asrar, Z., & Namaki, Shoushtari, A. (2014). Investigation of synergistic action between coronatine and nitric oxide in alleviating arsenic-induced toxicity in sweet basil seedlings. Journal of Plant Growth Regulation, 74 (2), 119-130.3.Shahid, M., Dumat, C., Khalid, S., Schreck, E., Xiong, T., & Niazi, N. K. (2016). Foliar heavy metal uptake, toxicity and detoxification in plants: A comparison of foliar and root metal uptake. Hazardous Materials, 325, 36-58.4.Akram, N. A., & Ashraf, M. (2013). Regulation in plant stress tolerance
by a potential plant growth regulator, 5-aminolevulinic acid. Journal of Plant Growth Regulation, 32 (3), 663-679.5.Wu, Y., Jin, X., Liao, W., Hu, L., Dawuda, M. M., Zhao, X., Tang, Z., Gong, T., & Yu, J. (2018). 5-Aminolevulinic acid (ALA) alleviated salinity stress in cucumber seedlings by enhancing chlorophyll synthesis pathway. Frontiers in Plant Science, 9, 635-645.6.Wang, L. J., Jiang, W. B., & Huang, B. J. (2004). Promotion of 5-aminolevulinic acid on photosynthesis of melon (Cucumis melo) seedlings under low light and chilling stress conditions. Plant Physiology ,121 (2), 258–264.7.Gupta, D., & Prasad, S. M. (2020). 5-Aminolevulinic acid: an emerging signaling molecule involved in improving abiotic stress tolerance in plants. Biochemical and Cellular Archives,20 (2), 6267-6287.8.Ali, B., Wang, B., Ali, S., Ghani, M. A., Hayat, M. T., Yang, C., Xu, L., & Zhou, W. J. (2013a). 5-Aminolevulinic acid ameliorates the growth, photosynthetic gas exchange capacity, and ultrastructural changes under cadmium stress in Brassica napus L. Journal of Plant Growth Regulation, 32, 604–614.9.Ali, B., Tao, Q., Zhou, Y., Gill, R. A., Ali, S., Rafiq, M. T., Xu, L., & Zhou, W. (2013b). 5-Aminolevolinic acid mitigates the cadmium-induced changes in Brassica napus as revealed by the biochemical and ultra-structural evaluation of roots. Ecotoxicology and Environmental Safety, 92, 271–280.10.Maghsoudi, K., Ashrafi, Dehkordi, E., & Mazloumi, S. M. (2021). The role of brassinosteroids and salicylic acid on spinach growth and cadmium accumulation under cadmium stress. Vegetable Science, 4 (2), 15-33.11.Gill, R. A., Ali, B., Islam, F., Farooq, M. A., Gill, M. B., Mwamba, T. M., & Zhou, W. (2015). Physiological and molecular analyses of black and yellow seeded Brassica napus regulated by 5-aminolivulinic acid under chromium stress. Plant Physiology and Biochemistry, 94, 130-143.12.Tian, T., Ali, B., Qin, Y., Malik, Z., Gill, R. G., Ali, S. H., & Zhou, W. (2014). Alleviation of lead toxicity by 5-aminolevulinic acid is related to elevated growth, photosynthesis, and suppressed ultrastructural damages in oilseed rape. BioMed Research International,1-11.13.Abbasi, B., Maleki, R., & Pirkharrati, H. (2017). Study effects of mining and gold extraction on amount of water contamination to As and Hg in Zarshoran area of Takab. Journal of Environmental Geology, 11 (40), 39-48.14.Watanabe, F. S., & Olsen, S. R. (1965). Test of an ascorbic acid method for determining phosphorus in water and NaHCO3 extracts from soil. Soil Science Society of America Journal,
29 (6), 677-678.15.Fatemi, H., Esmaielpour, B., Sefidkon, F., Soltani, A. A., & Nematollahzadeh, A. (2020). Investigation the effects of nanoparticles and biostimulants on morphological and biochemical charecteristic and secondary metabolites of coriander under heavy metal contamitioned soils. PhD. Thesis University of Mohaghegh Ardabili, Ardabil, Iran.16.Davoodi, M., Esmaielpour, B., Fatemi, H., & Maleki Lajair, H. (2018). Effect of silicon nutrition on alleviation the detrimental effects of nickel stress in (Ocimum basilicum L.). Journal of Plant Process and Function, 7 (24), 25-32.17.Irigoyen, J. J., Einerich, D. W., & Sánchez‐Díaz, M. (1992). Water stress induced changes in concentrations of proline and total soluble sugars in nodulated alfalfa (Medicago sativa) plants. plants. Plant Physiology, 84 (1), 55-60.18.Bradford, M. M. (1976). A rapid
and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72 (1-2), 248-254.19.Singleton, V. L., Orthofer, R., & Lamuela-Raventós, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. I Methods Enzymol, 299, 152-178.20.Chang, C. C., Yang, M. H., Wen, H. M., & Chern, J. C. (2002). Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis, 10 (3), 178-182.21.Sudhakar, C., Lakshmi, A., & Giridarakumar, S. (2001). Changes in the antioxidant enzyme efficacy in two high yielding genotypes of mulberry (Morus alba L.) under NaCl salinity. Plant Science, 161 (3), 613-619.22.Siddiqui, F., Krishna, S. K., Tandon, P. K., & Srivastava, S. (2013). Arsenic accumulation in Ocimum spp. and its effect on growth and oil constituents. Acta Physiologiae Plantarum, 35, 1071-1079.23.Nabi, A., Naeem, M., Aftab, T., Masroor, M., & Khan, A. (2019). Arsenic toxicity induced changes in growth, photosynthetic pigments, antioxidant machinery, essential oil, menthol and other active constituents of menthol mint (Mentha arvensis L.). Journal of Essential Oil Bearing Plants, 22 (5), 1333-1348.24.Hekmati, J., Hamidoghli, Y., Esmaielpour, B., & Ghasemnezhad, M. (2021). Effect of arsenic and phosphate Biofertilizer on physiological and Biochemical properties of green mint (Mentha spicata L.). Journal of Plant Production Research, 28 (1), 127-139.25.Asghari, M., Masoumi Zavariyan, A., & Yousefi Rad, M. (2020). Investigating the effect of sodium nitroprusside in reducing cadmium toxicity in basil (Ocimum basilicum L.). Environmental Stresses in Crop Sciences, 13 (3), 1009-1018.26.Fattahi, B., Arzani, K., Souri, M. K., & Barzegar, M. (2020). Effect of cadmium and lead on morpho-physiological traits and Photosynthesis of sweet basil (Ocimum basilicum L.). Iranian Journal of Horticultural Science, 50(4), 839-849.27.Ahmad, B., Dar, T. A., Khan, M. M. A., Ahmad, A., Rinklebe, J., Chen, Y., & Ahmad, P. (2022). Oligochitosan fortifies antioxidative and photosynthetic metabolism and enhances secondary metabolite accumulation in arsenic-stressed peppermint. Plant Science, 13, 987746.28.Nahar, K., Rhaman, M. S., Parvin, K., Bardhan, K., Marques, D. N., García-Caparrós, P., & Hasanuzzaman, M. (2022). Arsenic-induced oxidative stress and antioxidant defense in plants. Stress, 2 (2), 179-209.29.Ahmad, R., Ali, S., Hannan, F., Rizwan, M., Iqbal, M., Hassan, Z., ... & Abbas, F. (2017). Promotive role of
5-aminolevulinic acid on chromium-induced morphological, photosynthetic, and oxidative changes in cauliflower (Brassica oleracea var. botrytis L.). Environmental Science and Pollution Research, 24, 8814-8824.30.Naeem, M. S., Jin, Z. L., Wan, G. L., Liu, D., Liu, H. B., Yoneyama, K., & Zhou, W. J. (2010). 5-Aminolevulinic acid improves photosynthetic gas exchange capacity and ion uptake under salinity stress in oilseed rape (Brassica napus L.). Plant Soil, 332, 405-415.31.Ali, B., Xu, X., Gill, R. A., Yang, S., Ali, S., Tahir, M., & Zhou, W. (2014). Promotive role of 5-aminolevulinic acid on mineral nutrients and antioxidative defense system under lead toxicity in Brassica napus. Industrial Crops and Products, 52, 617-626.32.Akram, N. A., & Ashraf, M. (2013). Regulation in plant stress tolerance by a potential plant growth regulator, 5-aminolevulinic acid. Journal of Plant Growth Regulation, 32, 663-679.33.Farooq, M. A., Islam, F., Ali, B., Najeeb, U., Mao, B., Gill, R. A., & Zhou, W. (2016). Arsenic toxicity in plants: cellular and molecular mechanisms of its transport and metabolism. Environmental and Experimental Botany, 132, 42-52.34.Singh, R., Kesavan, A. K., Landi, M., Kaur, S., Thakur, S., Zheng, B., ... & Sharma, A. (2020). 5-aminolevulinic acid regulates Krebs cycle, antioxidative system and gene expression in Brassica juncea L. to confer tolerance against lead toxicity. Journal of Biotechnology, 323, 283-292.35.Sharma, R. K., & Agrawal, M. (2006). Single and combined effects of cadmium and zinc on carrots: uptake and bioaccumulation. Journal of Plant Nutrition, 29 (10), 1791-1804.36.Zhang, J., Li, D. M., Gao, Y., Yu, B., Xia, C. X., & Bai, J. G. (2012). Pretreatment with 5-aminolevulinic acid mitigates heat stress of cucumber leaves. Plant Biology, 56, 780-784.37.Sher, A., Nawaz, A., Ul-Allah, S., Sattar, A., Ijaz, M., Qayyum, A., & Manaf, A. (2024). Foliar application of 5-aminolevulinic acid improves the salt tolerance in sunflower (Helianthus annuus L.) by enhancing the morphological attributes and antioxidant defense mechanism. Acta Physiologiae Plantarum, 46 (3), 1-7.38.Ali, S., Rizwan, M., Zaid, A., Arif, M. S., Yasmeen, T., Hussain, A., & Abbasi, G. H. (2018). 5-Aminolevulinic acid-induced heavy metal stress tolerance and underlying mechanisms in plants. Journal of Plant Growth Regulation, 37, 1423-1436.39.Xu, F., Chang, J., Cheng, S. Y., Zhu, J., Li, L. L., & Cheng, Y. W. H. (2009). Promotive effect of 5-aminolevulinic acid on the antioxidant system in Ginkgo biloba leaves. African Journal of. Biotechnology, 8 (16), 3769.40.Sheteiwy, M., Shen, H., Xu, J., Guan, Y., Song, W., & Hu, J. (2017). Seed polyamines metabolism induced by seed priming with spermidine and 5-aminolevulinic acid for chilling tolerance improvement in rice (Oryza sativa L.) seedlings. Environmental and Experimental Botany, 137, 58-72.41.Zhang, H. H., Tang, M., Chen, H., Zheng, C. L., & Niu, Z. C. (2010). Effect of inoculation with AM fungi on lead uptake, translocation and stress alleviation of Zea mays L. seedlings planting in soil with increasing lead concentrations. European Journal of Soil Biology, 46 (5), 306-311.42.Fatemi, H. (2020). Investigation the effects of Nanoparticles and Biostimulants on morphological and biochemical charecteristics and secondary metabolites of Coriandr under heavy metal inated soils (Doctoral dissertation, University of Mohaghegh Ardabili).43.Verma, S., & Dubey, R. S. (2001). Effect of cadmium on soluble sugars and enzymes of their metabolism in rice. Plant Biology, 44, 117-123.44.Rucińska-Sobkowiak, R. (2016). Water relations in plants subjected to heavy metal stresses. Acta Physiologiae Plantarum, 38, 1-13.45.Chandrakar, V., Naithani, S. C., & Keshavkant, S. (2016). Arsenic-induced metabolic disturbances and their mitigation mechanisms in crop plants: A review. Biologia, 71 (4), 367-377.46.Chakrabarty, D., Trivedi, P. K., Misra, P., Tiwari, M., Shri, M., Shukla, D., ... & Tuli, R. (2009). Comparative transcriptome analysis of arsenate and arsenite stresses in rice seedlings. Chemosphere, 74 (5), 688-702.47.Corpas, F. J., Aguayo-Trinidad, S., Ogawa, T., Yoshimura, K., & Shigeoka, S. (2016). Activation of NADPH-recycling systems in leaves and roots of Arabidopsis thaliana under arsenic-induced stress conditions is accelerated by knock-out of Nudix hydrolase 19 (AtNUDX19) gene. Journal of Plant Physiology, 192, 81-89.48.Aksakal, O., Algur, O. F., Icoglu Aksakal, F., & Aysin, F. (2017). Exogenous 5-aminolevulinic acid alleviates the detrimental effects of UV-B stress on lettuce (Lactuca sativa L) seedlings. Acta Physiologiae Plantarum, 39, 1-10.49.Ye JiaBao, Y. J., Chen QiangWen, C. Q., Tao TingTing, T. T., Wang Gui Yuan, W. G., & Xu Feng, X. F. (2016). Promotive effects of 5-aminolevulinic acid on growth, photosynthetic gas exchange, chlorophyll, and antioxidative enzymes under salinity stress in Prunnus persica L. Batseh seedling. Journal of Food and Agriculture, 28 (11), 1.50.Yang, H., Zhang, J., Zhang, H., Xu, Y., An, Y., & Wang, L. (2021). Effect of 5-aminolevulinic acid (5-ALA) on leaf chlorophyll fast fluorescence characteristics and mineral element content of Buxus megistophylla grown along urban roadsides. Horticulture, 7 (5), 95.51.Farid, M., Ali, S., Rizwan, M., Ali, Q., Saeed, R., Nasir, T., & Ahmad, T. (2018). Phyto-management of chromium contaminated soils through sunflower under exogenously applied 5-aminolevulinic acid. Ecotoxicology and Environmental Safety, 151, 255-265.52.El-Amier, Y., Elhindi, K., El-Hendawy, S., Al-Rashed, S., & Abd-ElGawad, A. (2019). Antioxidant system and biomolecules alteration in Pisum sativum under heavy metal stress and possible alleviation by 5-aminolevulinic acid. Molecules, 24 (22), 4194.53.Naeem, M. S., Warusawitharana, H., Liu, H., Liu, D., Ahmad, R., Waraich,
E. A., ... & Zhou, W. (2012). 5-Aminolevulinic acid alleviates the salinity-induced changes in Brassica napus as revealed by the ultrastructural study of chloroplast. Plant Physiology and Biochemistry, 57, 84-92.54.An, Y., Qi, L., & Wang, L. (2016). ALA pretreatment improves waterlogging tolerance of fig plants. PloS one, 11 (1), e0147202.55.Niu, K., Ma, X., Liang, G., Ma, H., Jia, Z., Liu, W., & Yu, Q. (2017). 5-Aminolevulinic acid modulates antioxidant defense systems and mitigates drought-induced damage in Kentucky bluegrass seedlings. Protoplasma, 254, 2083-2094.56.Sharma, P., & Dubey, R. S. (2005). Lead toxicity in plants. Brazilian Journal of Plant Physiology, 17, 35-52.57.Seregin, I. V., & Ivanov, V. B. (2001). Physiological aspects of cadmium and lead toxic effects on higher plants. Russian Journal of Plant Physiology, 48, 523-544.58.Khan, M. S., Zaidi, A., & Wani, P. A. (2009). Role of phosphate solubilizing microorganisms in sustainable agriculture-a review. Agronomy for Sustainable Development, 551-570.59.Yordanov, I. (1995). Responses of photosynthesis to stress and plant growth regulators. Journal of Plant Physiology, 21 (2-3), 51-70.