Abstract
The pale-green barley mutant xan-h.chli-1 has a HvCHLI subunit of Mg-chelatase with an Arg-to-Lys substitution at position 298 and exhibits a unique cold-sensitive phenotype. Under winter field conditions, xan-h.chli-1 plants fail to survive, whereas they thrive under spring or greenhouse conditions. Controlled experiments show a specific cold-induced chlorosis gradient along leaf blades of the mutant that was not observed in other pale-green mutants with either altered chlorophyll biosynthesis, such as chlorina-f2.101 and chlorina.111, or defects in photosystem antenna protein assembly, such as hus1. Photosynthetic function in young leaf tissues was restored when exposed to optimal temperatures, emphasizing the reversibility of this cold stress. Molecular dynamics simulations revealed a temperature-dependent disruption of the interaction of ATP with Lys298 in the HvCHLI subunit, which correlates with the observed cold sensitivity. Transcriptomic analyses revealed distinct gene expression patterns under cold stress in xan-h.chli-1 leaves, which were characterized by marked inactivation of genes related to pigment biosynthesis, photosynthesis, cell-wall formation, and remodeling. These findings highlight the role of the xan-h.chli-1 mutation in conferring cold sensitivity and offer new insights into molecular strategies for introducing the pale-green trait into modern crop varieties.