Plant vasculature transports phloem sap from source organs to meristems via the interconnected sieve elements of the phloem. In Arabidopsis thaliana root, stem cells form two files of protophloem sieve elements (PPSEs), whose differentiation relies on regulators like BREVIS RADIX (BRX). brx loss-of-function mutants disrupt protophloem differentiation, impair phloem transport, and cause short roots, partly due to hyperactive autocrine CLAVATA3/EMBRYO SURROUNDING REGION 45 (CLE45)-BARELY ANY MERISTEM 3 (BAM3) signaling.
Notably, protophloem differentiation in wild-type seedlings can be suppressed by external application of nanomolar concentrations of CLE45. Since developing PPSEs express CLE45-BAM3 pathway components from initial stages until terminal differentiation, how they evade autocrine CLE45 inhibition was unclear. Our investigation reveals that wild-type plants become insensitive to CLE45 treatment under neutral to alkaline pH medium or with a specific proton pump inhibitor at standard pH (5.7). This insensitivity is not due to pH-dependent CLE45 uptake or charge, but rather a pH-dependent response of developing PPSEs that requires the CLE45 R4 residue.
Ratio-metric pH measurements in the root apical meristem showed a pH gradient along the protophloem, absent in neighboring cell files, suggesting a pH- dependent response to CLE45 in developing PPSEs. Further studies revealed that extracellular pH alkalinization correlates with PPSE elongation and differentiation, as well as lateral root cap development, indicating a connection with controlled autophagy and rapid differentiation. In contrast, extracellular acidification in the epidermis correlates with cell elongation, while no consistent relationship is observed in other tissues. These findings highlight tissue-specific roles of extracellular pH dynamics in root tip development.
In summary, we conclude that developing PPSEs self-organize their transition to differentiation by desensitizing themselves against autocrine CLE45 signaling through apoplastic pH alkalinization, and that there is no general firm correlation between apoplastic acidification and cell elongation across tissues.