The pentose phosphate pathway coordinates cytokinin homeostasis and CpG island methylation to control radicle development in maize

The pentose phosphate pathway (PPP) is a crucial metabolic route that influences various physiological processes in plants. In this study, we examined the effects of PPP inhibition on maize radicle growth using the PPP inhibitors and mutant. Our results showed that PPP inhibition significantly reduces radicle length, accompanied by a substantial decrease of energy level. Furthermore, molecular analyses revealed that PPP inhibition downregulates the cytokinin biosynthesis gene IPT while upregulating cytokinin oxidase/dehydrogenase (CKX) protein abundance. Bioinformatic and biochemical investigations identified a flavin adenine dinucleotide (FAD)-binding domain in CKX and showed that PPP blockade reduces root FAD content, impairing CKX enzymatic activity. Transcriptomic and metabolomics analysis revealed that PPP inhibition downregulates ZmRR3 , a cytokinin signaling related response regulator, through DNA methylation in its promoter region. Notably, DNA methylation inhibition via 5-Azacytidine treatment restores radicle growth and ZmRR3 expression. These findings highlight the essential role of PPP in regulating energy metabolism, cytokinin content, and gene expression during maize radicle development. This study advances understanding of the metabolic–cytokinin interplay critical for radicle growth and offers potential molecular targets for maize improvement.