臺灣博碩士論文加值系統

Micronutrient malnourishment is a serious problem in the developing nations especially among young children and pregnant women. One major concern is iron (Fe) deficiency which is a prevalent health disorder and almost 2 billion people worldwide are affected by this. Prolonged Fe deficiency results in Iron Deficiency Anemia. Micronutrient supplements, medicines and food diversification can abate severity of the condition but due to geographical or financial competence such treatments may not be available to everyone. Moreover, Fe-fortification of food is difficult because soluble Fe is unstable which alters the food taste and insoluble Fe is non-bioavailable and poorly absorbed. This reinforces the necessity for better interpretation of how plants acquire and process Fe as plants are the principal source of dietary Fe. Hence, the implementation of genetic biofortification of crops appear to be a self-emerged, rapid and simple solution. For successful Fe biofortification the molecular components and signaling networks involved in Fe acquisition from soil and transportation to destitute parts of plants are required to be studied comprehensively. Fe is abundant in environment but the availability in soil is limited, hence Fe acquisition is crucial. IRT1 (IRON REGULATED TRANSPORTER 1), transport Fe from soil, is most downstream gene in Fe signaling pathway. IRT1 was targeted to identify novel regulators involved in Fe homeostasis as IRT1 has broad substrate specificity under Fe deficient conditions and it can also uptake divalent heavy metals like Cd, Zn, Cu, Ni which affects plant growth and development. In order to alleviate IRT1 non-specificity and enhance Fe specificity to balance Fe homeostasis IRT1 promoter driven luciferase (PIRT1:LUC) construct was used for genetic screening. Ethyl methanesulfonate (EMS) mutant iron deficiency tolerant1 (idt1) was identified which can accumulate 4-7 times higher amounts of Fe than wild-type and exhibits the metal tolerance and iron accumulation (Metina) phenotype. The C to T transition mutation resulting in an alanine to valine substitution at amino acid position 320 of bHLH34 (IDT1A320V) is responsible for the dominant phenotype. The idt1 possesses constitutive activation of Fe regulatory pathway. Overexpression of IDT1A320V in Arabidopsis leads to the Metina phenotype and constitutive activation of IDT1A320V protein. Consequently, this allele controls upstream Fe homeostasis signaling pathway. The region of mutation is conserved among different agronomic species and expressing IDT1A320V in Nicotiana tabacum have escalated specificity towards Fe accumulation and enhanced tolerance to Cd. Similar results have found in rice transgenic lines showing higher Fe accumulation. Enhanced accumulation of Fe can serve in Fe-biofortification of edible crops. Moreover, its tolerance and accumulation of heavy metals in tobacco can aid the development of tools for phytoremediation of contaminants.

Acknowledgement i
Abstract iii
Table of Contents v
List of Tables vii
List of Figures vii
1 Iron and its irony in plants 1
1.1 Role of Fe in plants 1
1.2 Fe abundance to availability 2
1.3 Survival of the fittest: evolution of Fe acquisition strategies in plants 2
1.4 Strategy-I reduction-based strategy 3
1.4.1 Rhizosphere acidification 3
1.4.2 Fe3+ reduction 3
1.4.3 Transport of Fe2+ 4
1.4.4 Regulation of Fe acquisition in Strategy-I plants 4
1.5 Strategy-II chelation-based strategy 6
1.6 Factors affecting Fe homeostasis 8
1.7 Utilization of existing knowledge for Fe-biofortification and heavy metal phytoremediation 9
1.7.1 Fe-biofortification: lessons and challenges 9
1.7.2 Phytoremediation: role of Fe to abate heavy metal toxicity 11
1.8 Scope of the thesis 12
2 The dual benefit of a dominant mutation in Arabidopsis IRON DEFICIENCY TOLERANT1 for iron biofortification and heavy metal phytoremediation 13
2.1 Abstract 13
2.2 Introduction 14
2.3 Results 16
2.3.1 Screening and identification of Fe-specific EMS mutant idt1 16
2.3.2 The idt1-1 exhibits the Metina phenotype 16
2.3.3 Constitutive Fe deficiency response is observed in idt1-1 and IDT1A320V overexpression lines 17
2.3.4 Overexpression of IDT1A320V results in the Metina phenotype 18
2.3.5 High IDT1 accumulation and stability is positively correlated to the Metina phenotype 18
2.3.6 Overexpression of IDT1A320V results in the Metina phenotype in tobacco 19
2.3.7 Overexpression of IDT1A320V results in high iron accumulation in rice 19
2.4 Discussion 20
2.5 Future Prospects 23
2.5.1 Sufficient meal but insufficient calories 23
2.5.2 Significance of the current study in Fe-biofortification 23
2.5.3 Significance of the current study in heavy metal phytoremediation 23
2.5.4 Significance of this study as green manuring 24
2.6 Conclusion 25
2.7 Materials and methods 25
2.7.1 Plant materials and growth conditions 25
2.7.2 Generation of transgenic constructs and plant transformation 25
2.7.3 Genetic mapping of idt1 26
2.7.4 Determination of biomass and chlorophyll content 27
2.7.5 Gene expression and analysis 27
2.7.6 Antibody generation and immunoblot analysis 27
2.7.7 Ferric chelate reductase activity 28
2.7.8 Measurement of mineral concentration 28
2.7.9 Histochemical staining of Fe 28
2.7.10 Fluorescence imaging in protoplast and stable transgenic lines 28
2.7.11 Gene cloning and transformation of rice 29
References 52
Resume 57

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