Investigating the role of tetrapyrrole biosynthesis under drought stress in cereal transgenics

Abstract

The tetrapyrrole biosynthesis pathway leads to chlorophyll and heme production and plays a key role in primary physiological processes such as photosynthesis and respiration. Recent studies have shed light on heme as a potential candidate molecule for triggering stress defence responses. However, detailed investigations are yet to be conducted to elucidate the potential role of heme in regulating responses to complex abiotic stress conditions such as drought. The terminal enzyme of heme biosynthesis is Ferrochelatase (FC), for which there are two isoforms encoded by separate genes (FC1 and FC2). Previous studies propose that the two FCs synthesize two physiologically distinct heme pools with different cellular functions. The overall scientific goal of this thesis was to investigate the roles of the two FCs in photosynthesis, drought and oxidative stress tolerance. In this study, barley (Hordeum vulgare) was used as both a major cereal crop and also as a model plant for other commercially relevant rain-fed cereal crops. Two FCs in barley (HvFC1 and HvFC2) were identified and their tissue-specific and stress-responsive expression patterns were investigated. These genes were cloned from the cultivar Golden Promise (GP) and transgenic lines ectopically overexpressing either HvFC1 or HvFC2 were generated. From 29 independent T₀ transgenic lines obtained for each FC construct, three single-copy transgenic lines ectopically overexpressing either HvFC1 or HvFC2 were evaluated for photosynthetic performance, oxidative and drought stress tolerance. The two HvFC isoforms share a common catalytic FC domain, while HvFC2 additionally contains C-terminal chlorophyll a/b binding (CAB) domain. The two genes are differentially expressed in photosynthetic and non-photosynthetic tissues and have distinct stress responsive expression profiles, implying that they may have distinct roles. Transgenic plants ectopically overexpressing either HvFC1 or HvFC2 exhibited significantly higher chlorophyll content, stomatal conductance (gs) [s subscript], carboxylation efficiency (CE) and photosynthetic rate relative to controls under both non-stressed and drought stress conditions. Furthermore, these transgenics, showed wilting avoidance and maintained higher leaf water content and water use efficiency relative to control plants when subjected to drought stress. Overexpression of HvFCs significantly up-regulated nuclear genes associated with ROS detoxification under drought stress. It also reduced photo-oxidative damage caused by perturbation of tetrapyrrole biosynthesis in tigrinaᵈ¹² mutants. Taken together, this study indicates that both HvFCs play roles in photosynthesis and improving oxidative and drought stress tolerance. The results reported in this thesis suggest that both HvFC derived heme pools are likely to be involved in chloroplast-to-nuclear retrograde signaling to trigger drought and oxidative stress tolerance. This study also highlights the tetrapyrrole pathway as an important target for engineering improved crop performance in both non-stressed and stressed environments.