Involvement of phenolics in allelopathy and submergence tolerance of rice (oryza sativa l )

Involvement of Phenolics in Allelopathy and Submergence Tolerance of Rice (Oryza sativa L.) D141228 DO TAN KHANG A Dissertation Submitted to the Graduate School for International Development and Cooperation of Hiroshima University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy September 2017 We hereby recommend that the dissertation by (Mr DO TAN KHANG entitled “Involvement of Phenolics in Allelopathy and Submergence Tolerance or Rice (Oryza sativa L.)” be accepted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Committee on Final Examination: XUAN Tran Dang, Associate Professor Chairperson NAKAGOSHI Nobukazu, Professor TSUDZUKI Masaoki, Professor MAEDA Teruo, Professor TAWATA Shinkichi, Professor PAK Research Center, University of the Ryukyus Date: Approved: Date: Baba Takuya, Professor Dean Graduate School for International Development and Cooperation Hiroshima University Contents LIST OF FIGURES vi LIST OF TABLES viii ABSTRACT CHAPTER INTRODUCTION 1.1 Background 1.1.1 Problem in rice production 1.1.2 Allelopathy in rice 11 1.1.3 Phenolics 13 1.2 Research objectives 17 1.3 Thesis structure 17 CHAPTER ALLELOPATHIC ACTIVITY OF DEHULLED RICE AND ITS EFFECTS ON WEED GERMINATION 19 2.1 Introduction 19 2.2 Materials and methods 21 2.2.1 Plant materials 21 2.2.2 Chemicals 21 i 2.2.3 Effects of dehulled rice and hulls on germination of test plants 21 2.2.3 Identification of phenolics released from root exudates 23 2.2.4 Data analysis 25 2.3 Results and discussion 26 2.3.1 Effects of rice, dehulled rice and hulls on germination of test plants 26 2.3.2 Identification of allelopathic phenolics 30 2.4 Conclusions 34 CHAPTER EFFECTS OF SELECTED PHENOLICS ON GERMINATION AND SEEDLING GROWTH OF PLANTS 35 3.1 Introduction 35 3.2 Materials and Methods 36 3.2.1 Plant materials 36 3.2.2 Effects of phenolic acids on germination of rice and test plants 36 3.2.3 Data analysis 37 3.3 Results and discussion 37 3.4 Conclusions 44 CHAPTER INVOLVEMENT OF PHENOLIC COMPOUNDS IN ANAEROBIC FLOODING GERMINATION OF RICE 45 ii 4.1 Introduction 45 4.2 Materials and methods 47 4.2.1 Plant materials 47 4.2.1 Chemicals and reagents 47 4.2.2 Screening of germination under flooding conditions 47 4.2.3 Screening of sub-1 gene using SSR markers 48 4.2.4 Phenolic extraction 50 4.2.5 Estimation of total phenolic content 51 4.2.6 Determination of total flavonoid content 52 4.2.7 Identification of phenolic acids of seedling extracts 53 4.2.8 Evaluation of antioxidant activity 53 4.2.9 Statistical analysis 54 4.3 Results and discussion 54 4.3.1 Screening anaerobic germination of different varieties under flooding conditions 54 4.3.2 Screening of sub-1 gene in rice varieties 60 4.3.3 The effect of flooding conditions on the total contents of phenolic compounds in anaerobic germination of rice varieties 65 iii 4.4 Conclusions 74 CHAPTER ENHANCEMENT OF CHLOROPHYLL, TOTAL PHENOLICS AND ANTIOXIDANT ENZYMES OF RICE DURING SUBMERGENCE USING EXOGENOUS PHENOLICS 75 5.1 Introduction 75 5.2 Materials and methods 77 5.2.1 Plant material and treatments 77 5.2.2 Measurement of lipid peroxidation 78 5.2.3 Determination of chlorophyll contents 79 5.2.4 Estimation of total phenolic and flavonoid contents 79 5.2.5 Evaluation of antioxidant activity 82 5.2.6 Enzyme extractions and assays 83 5.2.7 Gene expression of antioxidant enzymes using quantitative real-time PCR 85 5.2.8 Statistical analyses 86 5.3 Results and discussion 87 5.3.1 Effect of exogenous phenolics on stem elongation 87 iv 5.3.2 Effect of exogenous phenolics on photosynthetic pigments and lipid peroxidation 87 5.3.3 Effect of exogenous phenolics on total phenolic and flavonoid contents 89 5.3.5 5.4 Effect of exogenous phenolics on antioxidant enzymes 92 Conclusions 99 CHAPTER ISOLATION OF ALLELOCHEMICALS FROM RICE HUSKS 100 6.1 Introduction 100 6.2 Materials and methods 102 6.2.1 Materials 102 6.2.1 Methods 102 6.3 Results and discussion 104 6.3.1 Isolation of compounds 104 6.3.2 6.4 Allelopathic potential of isolated compounds 108 Conclusions 112 CHAPTER GENERAL DISCUSSION 113 REFERENCES 120 v List of Figures Fig 1.1 Rate of changing flood risk (“R”) in main deltas in the world The color and size of the circles represent the level of risk (Tessier et al., 2015) Fig 1.2 The 12 most troublesome weeds of rice in Asia 10 Fig 1.3 The biosynthetic pathways of the common allelochemicals (Li et al., 2010) 12 Fig 1.4 Induction, production, release and transport of allelochemicals and their effects (Amb and Ahluwalia, 2016) 13 Fig 1.5 Phenolic classification 16 Fig 2.1 Intact rice (A), dehulled rice (B) and hull (C) 22 Fig 2.2 Diagram of seed distribution in a Petri dish 22 Fig 2.3 HPLC chromatogram of phenolic standards 24 Fig 2.4 Germination of tested plants after days 26 Fig 4.1 Submergence treatments of rice 48 Fig 4.2 Standard curve of gallic acid 51 Fig 4.3 Standard curve of rutin 52 Fig 4.4 DNA electrophoresis image of rice varities 60 Fig 4.5 PCR profile of the marker RM219 on rice varieties 61 Fig 4.6 HPLC chromatogram of standard phenolic acids (A) and rice leaves of the strong tolerant variety before (B) and after submergence treatment (B) 69 Fig 4.7 HPLC chromatogram of standard phenolic acids (A) and rice leaves of the weak tolerant variety before (B) and after submergence treatment (B) 70 Fig 4.8 Antioxidant activity (DPPH scavenging) of samples 72 vi Fig 5.1 HPLC chromatogram of protocatechuic acid (1) and vanillic acid (2) 81 Fig 5.2 HPLC standard curves of protocatechuic acid 81 Fig 5.3 HPLC standard curves of vanillic acid 82 Fig 5.4 Changes of chlorophyll a (A), chlorophyll b (B), and MDA (C) contents of rice after pre-treatment with phenolic acids 88 Fig 5.5 Variation of total phenolic (A), total flavonoid (B), protocatechuic acid (C), and vanillic acid (D) contents in rice after pre-treatment with phenolic acids 90 Fig 5.6 Antioxidant activity of rice extracts after phenolic acid treatment 91 Fig 5.7 Changes of antioxidant enzyme activities in rice after pre-treatment 93 Fig 5.8 Comparison of relative expression of antioxidant enzymes after phenolic acid pre-treatment (Error bars are based on standard errors) 94 Fig 6.1 Identified rice allelochemicals (Macías et al., 2007) 101 Fig 6.2 Bioassay test using Petri dish method 104 Fig 6.3 TLC photo of the compounds 105 Fig 6.4 TLC photo of the compounds after separation 105 Fig 6.5 GC chromatograms of compounds (1) and (2) 106 Fig 6.6 Mass spectra of compound (1) and (2) 107 Fig 6.7 Allelopathic potential of compound (1) and (2) on radish 111 Fig 6.8 Effect of compound (1) on early growth of radish 112 vii List of Tables Table 1.1 Rice area and yield in Asian countries by different areas (Swain et al., 2005) Table 1.2 Classes of phenolic compounds in plants 14 Table 2.1 Retention times and standard curve of individual phenolic acids 25 Table 2.2 Effects of intact rice, dehulled rice and hulls on germination and seedling growth of barnyardgrass, expressed as different percentages over controls 27 Table 2.3 Effects of intact rice, dehulled rice and hulls on germination and seedling growth of lettuce, expressed as different percentages over controls 28 Table 2.4 Effects of intact rice, dehulled rice and hulls on germination and seedling growth of radish, expressed as different percentages over controls 29 Table 2.5 Phenolic acids (µg ml-1 extract) in agar media of weeds and rice after oneweek germination 32 Table 3.1 Effects of allelochemicals on germination and seedling growth of barnyardgrass, expressed as different percentages over controls 38 Table 3.2 Effects of allelochemicals on germination and seedling growth of lettuce, expressed as different percentages over controls 39 Table 3.3 Effects of allelochemicals on germination and seedling growth of radish, expressed as different percentages over controls 40 Table 3.4 Effects of allelochemicals on germination and seedling growth of rice, expressed as different percentages over controls 41 Table 4.1 List of SSR markers linked to sub-1 gene 49 Table 4.2 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