Cytokinins control secondary cell wall formation in the inflorescence stem of Arabidopsis

Spatiotemporal control over developmental programs is vital to all organisms. Here we show that deficiency in cytokinin signaling or biosynthesis leads to early secondary cell wall (SCW) formation in Arabidopsis inflorescence stem that associates with precocious upregulation of a SCW transcriptional cascade controlled by NAC TFs (NSTs/VNDs). We demonstrate that cytokinin signaling through the AHK2/3 and the ARR1/10/12 suppresses the expression of several NSTs/VNDs and SCW formation in the apical portions of stems. Exogenous cytokinin application led to fast downregulation of NST1 and NST3 (NST1/3) and VND6/7 in the WT and reconstituted both proper development and apical-basal gradient of NST1/3 expression in a cytokinin biosynthesis-deficient mutant. We show that AHK2 and AHK3 required functional NST1 or NST3 to control SCW initiation in the interfascicular fibers, further evidencing that cytokinins act upstream of NSTs transcription factors. The premature onset of a rigid SCW biosynthesis and altered expression of NST1/3 and VND6/7 due to cytokinin deficiency led to the formation of smaller tracheary elements (TEs) and impaired hydraulic conductivity. We conclude that cytokinins downregulate NSTs to inhibit premature SCW formation in the apical part of the inflorescence stem, facilitating thus the development of fully functional TEs and interfascicular fibers. Overall design: RNA-seq was performed on dissected stem internodes (apical and basal) from wild-type plants and various cytokinin-related mutants or overexpressors, including mutants in cytokinin receptors (ahk2,3), response regulators (arr1,10; arr1,12; arr10,12; arr1,10,12), cytokinin biosynthesis genes (ipt3,5,7; ipt1,3,5,7), phosphotransfer proteins (ahp1,2,3,4,5), and cytokinin-degrading enzymes (p35S:CKX2; p35S:CKX3). Plants were grown under long-day conditions until stage 1 of reproductive development, corresponding to the first elongated silique. Stem segments were harvested, total RNA was isolated, and sequencing libraries were prepared using the Illumina TruSeq protocol. Each genotype and tissue type was sampled in biological replicates to enable differential expression analysis. The aim was to identify gene expression changes associated with altered cytokinin pathways that may influence secondary cell wall formation and vascular development.