Department of Environment and Agriculture Hormonal Regulation of Mango Fruit Ripening Siti Zaharah Sakimin This thesis is presented for the Degree of Doctor of Philosophy of Curtin University August 2011 Declaration Declaration To the best of my knowledge and belief this thesis contains no material previously published by any other person except where due the acknowledgement has been made. This thesis contains no material which has been accepted for the award of any other degree or diploma in any university. Signature: Date: 13 th December 2011 i Dedication Dedication To: My father , Hj. Sakimin Sakidin; My mother , Hajjah Poriah Hj. Mokti; My father-in-law , Mr. Iberahim Yusuf; My mother-in-law , Mrs. Tuan Minah Tuan Kadir; My husband , Dr. Ismail Iberahim; My son , Muhammad Zahin Iqbal Ismail; & All my family members For “A constant source of inspiration and doa’a during the entire period of my PhD study and my life...” ii Acknowledgements Acknowledgements In the name of Allah, the most Beneficent and the Merciful. I bow my head, with all the humility and modesty, before Allah Almighty, the Creator of everything, Who imbibed in man a feel for knowledge and desire to explore the unknown for the benefit of mankind, for granting me courage, patience, and perseverance to undertake and accomplish this prime task of research work which could not be possible without His Blessings and Grace. May peace and Blessings of Allah Almighty be upon His all Prophets including Muhammad (PBUH), His last messenger, who is the fountain of knowledge and guidance for the salvation of mankind in this world and the hereafter. I feel paucity of words and phrases to express my deepest and eternal gratitude to my supervisor, Dr. Zora Singh, Foundation Professor of Horticulture, Department of Agriculture and Environment, Curtin University, Western Australia (WA). He was very kind and generous in his auspicious help, scholarly guidance, great encouragement and affectionate support which were a colossal source of inspiration and solace for me in the quest of my PhD program. I extend my sincere thanks to my associate supervisor, Dr. Gregory M. Symons, Senior Scientist (Biotechnologist), Agricultural Research and Development, Tasmanian Alkaloids, Westbury, Tasmania, for his advice and help in estimation of the endogenous level of plant hormones and his suggestions on Chapter 4 and 5. I grateful to Dr. Razi Ismail and Dr Fauzi Ramlan, Professor of Plant Physiology, Universiti Putra Malaysia, Serdang for their invaluable guidance and shaping my career based on my interest of study. I gratefully acknowledge the Ministry of Higher Education of Malaysia for providing financial support under academic training scheme of Malaysia, Public Higher Education Institution and the Universiti Putra Malaysia for study leave during my PhD studies. I also gratefully acknowledge Curtin University, WA, for providing me with Completion Scholarship during my final year of PhD study. iii Acknowledgements I take this opportunity of expression my profound gratitude to Dr. Ahmad Sattar Khan, visiting Post Doctoral fellow for his suggestions on Chapter 6 of my thesis. My special appreciation and thanks to Ms. Susan Petersen, Curtin Horticulture Research Laboratory for technical support and assistance during my laboratory work. I also wish to thank my lab-mates, Dr. Ismail Iberahim, Dr. Wan Zaliha Wan Sembok, Dr. Tham Pham, Dr. Sukhvinder Pal Singh, Dr. Anirudh Thakur, Mrs. Maria Fransisca Kaligis-Sumual, Mr. Zahoor Hussain, Prof. Shamin Ahmed Kam Khan, Mrs. Diviya Rathinam Alagappan, and my friend Mrs. Noraini Jaafar for their help and support during the course of my studies. I owe a heartfelt debt of gratitude to my parents and parents-in-law, who endured all the strains and stress during the course of my study while their hearts were beating with prayers for my success. I dedicate this thesis to my most affectionate and loving parents, husband (Dr. Ismail Iberahim) for being my soul mate and strength and also to my little prince (Muhammad Zahin Iqbal) to cheer my life during the highs and lows in this course. Their patience and encouragement has made this experience more fulfilling. I am indebted and grateful to my brothers, sisters, brothers- and sisters-in-law who endured my long separation and always remembered me in their prayers. I am also thankful to my relatives, well-wishers and friends for their good wishes. Last but not least are to my lovely nephews and niches, whose were always raised in prayers for my success. I am thankful to Allah Almighty, without His help it was impossible to complete this research work. iv Abstract Abstract Mango fruit ripen quickly. It is highly perishable. Short shelf life of mango fruit limits its transportation to distant domestic and international markets. The objective of my research was to elucidate the role of changes in endogenous levels of brassinosteroids (BRs), ethylene, abscisic acid (ABA) and/or indole-3-acetic acid (IAA) in modulating the ripening processes of 'Kensington Pride' mango fruit. The endogenous levels of these regulators were regulated using inhibitors of their biosynthesis and/or action to unfold their mechanism in delaying/hastening mango fruit ripening, extending storage life and improving fruit quality as well as to underpin the mode of action of ABA and NO in modulating ethylene biosynthesis and activities of fruit softening enzymes in the pulp during ripening and/or alleviating chilling injury (CI) during cool storage. Higher endogenous level of ABA at the climacteric-rise stage triggered the climacteric peak of ethylene production coupled with a significant quadratic relationship between both of them; suggest that ABA play a key role in modulating mango fruit ripening. The exogenous application of ABA (1.0 - 2.0 mM) promoted skin colour development and fruit softening during ripening, and the trend was reversed with its inhibitor of biosynthesis - nordihydroguaiaretic acid (0.1 - 0.2 mM NDGA). The endogenous level of IAA was higher at the initial stage of ripening and decline over ripening period. The exogenous application of 45 - 60 ng g -1 FW Epi- BL increased the climacteric peak of ethylene and respiration, promoted skin colour, but the changes in the endogenous level of BRs (castasterone and brassinolide) are unlikely to modulate mango fruit ripening as it is present in a trace amounts in mango pulp tissues throughout the ripening period. Exogenous postharvest application of ABA (1.0 mM) increased the climacteric peak of ethylene production through promoting the activities of 1- aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), ACC oxidase (ACO) enzymes, and ACC content, decreased the fruit firmness with increased exo - polygalacturonase ( exo -PG), endo -PG and e ndo -1,4-β-D-glucanase (EGase) activities, decreased pectinesterase (PE) activity in the pulp, higher total sugars and v Abstract sucrose, advanced degradation of total organic acids, citric and fumaric acid. The application of 0.2 mM NDGA showed reverse trends for these ripening indicator parameters. NO fumigation (20 µL L -1 or 40 µL L -1) was more effective in delaying fruit ripening when applied at the pre-climacteric (PC) stage, than at the climacteric-rise (CR) stage. NO (20 µL L -1) fumigation delayed and suppressed the endogenous ethylene production, activities of ACS and ACO enzymes, and ACC content, rate of respiration, higher pulp rheological properties (firmness, springiness, cohesiveness, chewiness, adhesiveness, and stiffness) with lower activities of exo -, endo -PG, EGase, but maintained higher PE activity in pulp tissues during ripening at 21°C and cool storage (13°C). NO treatments (20 and 40 µL L -1) significantly alleviated CI index during ripening at ambient temperature following 2- or 4-week of cold-stored (5°C) period. All NO fumigation treatments significantly suppressed ethylene production and respiration rates irrespective of cold storage period. NO–fumigated with above 5 µL L -1 significantly delayed fruit softening up to 2 days and retarded colour development, reduced total sugar and fructose concentrations, increased tartaric and shikimic acid at fully ripe stage during ripening period irrespective of cold-stored fruit. In conclusion, the higher levels of endogenous IAA in fruit pulp during the PC stage and the accumulation of ABA prior to the climacteric stage might switch on ethylene production that triggers fruit ripening. There is a significant quadratic relationship between endogenous level of ABA in the pulp and ethylene production during fruit ripening period. Exogenous Epi-BL promoted fruit ripening, whilst, the changes in endogenous levels of BRs are unlikely to modulate mango fruit ripening. Moreover, the exogenous application of ABA (1.0 mM) promoted the activities of ethylene biosynthesis enzymes (ACS and ACO) and ACC content and ethylene biosynthesis as well as endo -PG activity in the pulp, whilst, the NDGA-treated fruit showed the reverse trends. The application of NO fumigation (20 µL L -1) at PC stage can be effectively used to delay the fruit ripening up to 2 days at ambient temperature (21°C) and cool-storage (13°C) through suppression the activity of ethylene biosynthesis and softening enzymes and alleviate CI following 2- and 4-week cold storage (5°C) without any adverse effects on fruit quality. vi Table of contents Table of contents Declaration ..................................................................................................................... i Acknowledgements......................................................................................................iii