Molecular investigations and gene editing approaches for sustainable agriculture; understanding and optimising nitrogen transporters in cereals

Abstract

Nitrogen is a critical macronutrient for plant growth, yet its uptake and regulation in cereal crops remain complex and inefficient under variable environments. This thesis investigates the molecular, regulatory and functional dynamics of nitrogen transporters in maize, focusing on nitrate/chloride (NPF6) and ammonium (AMF) transporter families. An integrated bioinformatic, gene‑editing and reverse‑genetics approach was used to uncover mechanisms underpinning nitrogen use efficiency (NUE), stress resilience and symbiotic interactions.

Structural and transcriptomic analyses of ZmNPF6.4 in the fast‑flowering maize line Tx40J revealed cultivar‑specific INDELs potentially influencing NO₃⁻/Cl⁻ specificity. miRNA interactions and methylation patterns indicated both developmental and environmental regulation. Functional studies using CRISPR‑Cas9 editing of ZmNPF6.4 and ZmNPF6.6 (SDN1/SDN2) achieved construct development and embryo transformation in Tx40J, forming the basis for future mutant evaluation. Complementary rice knockouts provided cross‑species insights, including a novel link between Osnpf6.3 and altered root‑disease susceptibility, suggesting an emerging role for NPF6 transporters in plant defence.

Reverse‑genetics characterisation of AMF transporters (ZmAMF1.1, ZmAMF1.2) using insertional mutants in B73 and Gaspe confirmed plasma‑membrane and tonoplast localisation. Phenotypic analyses under contrasting nitrogen regimes revealed altered growth, biomass allocation and reproductive traits. δ¹⁵N pulse‑chase assays showed modified NH₄⁺ uptake and remobilisation, while reduced root hairs and arbuscular mycorrhizal structures suggested disrupted root development and symbiosis, likely linked to intracellular NH₄⁺ toxicity.

Together, this thesis advances understanding of nitrogen transporter biology in maize and demonstrates how targeted transporter manipulation can inform future gene‑editing strategies to improve NUE, stress tolerance and crop productivity.