Hexokinases (HXKs), which sense and catalyze cellular sugar, play a critical role in the growth and development of various plants, including wheat, a primary source of human calories frequently attacked by fungal pathogens. However, the evolutionary dynamics and functional diversification of HXKs in wheat, particularly their roles in plant defense, remain unclear. Here, we discovered that the wheat hexokinase gene family originated through multiple ancient gene duplications across different plant lineages and has undergone comprehensive, multidimensional functional specialization in gene expression, subcellular localization, enzyme activity, and regulation of plant defense responses. Gene expression analysis suggests that two-thirds of the TaHXK genes are responsive to fungal infection. Subcellular analysis reveals that while six TaHXKs are localized in mitochondria, three TaHXKs from different phylogenetic branches are sorted into other cellular compartments. Notably, biochemical analysis shows that TaHXKs in mitochondria differ in their glucose-catalyzing activity, with TaHXK5 and TaHXK3 exhibiting the highest and lowest enzyme activity, respectively. Consistently, transient expression analysis suggests that TaHXK5 induces various plant defense responses, while TaHXK3 is defective in activating some plant defense responses. Furthermore, inactivation of the glucokinase activity of TaHXK5 compromised its function in defense activation, suggesting that mitochondrial TaHXKs display functional divergence in both enzyme activity and defense-inducing activity that are intrinsically connected. Overall, our findings reveal that the multidimensional specialization events following the ancient duplication events may have shaped the functional diversity of HXKs in wheat, shedding light on their evolutionary dynamics and potentially contributing to the improvement of wheat defense.
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