Loss of the plastidial hexokinase <i>CsHXK3</i> triggers metabolic starvation and <scp>ROS</scp> imbalance leading to pollen abortion in cucumber

SUMMARY Male sterility is a vital trait for hybrid seed production. However, the synergistic coordination between sugar metabolism and reactive oxygen species (ROS) during pollen development remains poorly understood. Here, we identified a plastid‐localized hexokinase, CsHXK3, that participates in both processes in cucumber ( Cucumis sativus L.). CsHXK3 is localized to the tapetum, microspores, and vascular tissues of the anther. The significant downregulation of CsHXK3 expression in cell wall invertase 3 ‐silenced ( CsCWIN3 ‐RNAi) lines supports its essential role in downstream hexose utilization. Biochemical analysis confirmed that CsHXK3 is a glucose‐preferring enzyme that is essential for hexose phosphorylation. The CRISPR/Cas9‐mediated knockout of CsHXK3 resulted in severe male sterility, characterized by defective tapetal programmed cell death, collapsed pollen grains, and severely impaired pollen germination. Loss of CsHXK3 led to reduced accumulation of sugars (glucose, fructose, and sucrose) and starch in the anthers, accompanied by substantially downregulated expression of sugar transporter genes, including Sugars Will Eventually be Exported Transporters ( CsSWEETs ) and Sugar Transport Protein 13 ( CsSTP13 ). CsHXK3 deficiency disrupted ROS homeostasis by reducing hydrogen peroxide (H 2 O 2 ) levels, which was accompanied by the downregulated expression of the ROS‐generating gene Respiratory Burst Oxidase Homolog B ( CsRBOHB ) and upregulated expression of genes encoding ROS‐scavenging peroxidases. Our findings indicate that CsHXK3 may coordinate carbohydrate metabolism with ROS homeostasis during pollen development, offering insights into the metabolic regulation of male reproductive success in plants.