Understanding the role of glucose-sensor HEXOKINASE in seedling establishment in Arabidopsis thaliana

Abstract

This study investigates the complex role of HEXOKINASE (HXK) genes, with a focus on HXK1, in Arabidopsis thaliana seedling development and glucose sensing mechanisms. HEXOKINASEs are a family of glucose-phosphorylating enzymes that facilitate the breakdown of glucose to produce energy. Through a series of experiments employing mutant lines and biochemical assays, we explore the multifaceted functions of HXK1 in regulating seedling growth. Initial experiments establish HXK1 as a key glucose response factor, demonstrating its role as a glucose sensor/signal. Mutant seedlings exhibit reduced sensitivity to high glucose concentrations, implicating HXK1 in glucose signaling. Further analyses reveal growth deficiencies in HXK family genetic mutants, particularly in dark and low-light environments, emphasizing the crucial role of HXK1-mediated signaling in hypocotyl elongation regulation. Genetic and biochemical approaches elucidate the dependence of HXK1-mediated growth on its enzymatic function, highlighting the interactions between signaling components and glucose metabolism. Additionally, mutants of enzymes downstream of HXK1 in glycolysis show phenotypic similarities, underscoring the significance of glycolysis in seedling development. Investigation into the signaling mechanisms underlying HXK1 function, particularly in response to exogenous glucose and glucose-6-phosphate (G6P), reveals that HXK1 signaling primarily operates under high-glucose conditions, indicating complex regulatory networks in seedling growth. Moreover, analysis of catalytically inactive HXK1 alleles display unexpected findings, implicating glucose homeostasis in seedling development. Furthermore, this study explores the molecular basis of gene misregulation in the gin2-1 mutant, highlighting the role of HXK1-mediated signaling pathways in plant growth and stress responses. Transcriptomic analyses uncover significant patterns of misregulation in gin2-1, suggesting a starvation-type response and diminished sugar catabolism capacity consistent with findings outlined in our investigation of the HXK1-mediated growth phenotype. Investigation into the intersection between HXK1, PHTOCHROME INTERACTING FACTORS (PIFs), and SNF-RELATED KINASE 1 (SnRK1) by analysing the patterns laid out in the transcriptomes of key mutant lines revealed overlap in misregulated genes, providing insights into cross-regulatory processes. Lastly, this study investigates the regulatory mechanisms between PHYTOCHROME INTERACTING FACTOR 7 (PIF7) and HXK1 under End of Day Far Red (EoDFR) conditions, revealing unexpected induction of HXK family genes in wild-type (Wt) seedlings and implicating PIF7 in G6P-mediated hypocotyl elongation. Examination of genes relevant to SnRK1 signaling in EoDFR conditions corroborates trends outlined in our transcriptomic analysis and also suggest some form of novel interaction between PIF7 and SnRK1. The study provides insights into the regulatory networks governing plant growth and highlights potential interactions between sugar signaling pathways and light perception mechanisms mediated by PIF transcription factors, demonstrates PIF7 dependence on HXK1 in a previously undescribed manner, and begins to shape a model to lay a framework to further test the hypotheses laid out.