The phytohormone auxin controls many processes in plants, at least in part through its regulation of cell expansion¹. The acid growth hypothesis has been proposed to explain auxin-stimulated cell expansion for five decades, but the mechanism that underlies auxin-induced cell-wall acidification is poorly characterized. Auxin induces the phosphorylation and activation of the plasma membrane H⁺-ATPase that pumps protons into the apoplast², yet how auxin activates its phosphorylation remains unclear. Here we show that the transmembrane kinase (TMK) auxin-signalling proteins interact with plasma membrane H⁺-ATPases, inducing their phosphorylation, and thereby promoting cell-wall acidification and hypocotyl cell elongation in Arabidopsis. Auxin induced interactions between TMKs and H⁺-ATPases in the plasma membrane within seconds, as well as TMK-dependent phosphorylation of the penultimate threonine residue on the H+-ATPases. Our genetic, biochemical and molecular evidence demonstrates that TMKs directly phosphorylate plasma membrane H⁺-ATPase and are required for auxin-induced H⁺-ATPase activation, apoplastic acidification and cell expansion. Thus, our findings reveal a crucial connection between auxin and plasma membrane H⁺-ATPase activation in regulating apoplastic pH changes and cell expansion through TMK-based cell surface auxin signalling.

Accurate sensing and responding to physical microenvironment are crucial for cell function and survival, but the underlying molecular mechanisms remain elusive. Pollen tube (PT) provides a perfect single-cell model for studying mechanobiology since it’s naturally subjected to complex mechanical instructions from the pistil during invasive growth. Recent reports have revealed discrepant PT behaviors between in vivo and flat, two-dimensional in vitro cultures. Here, we established the Stigma-style-transmitting tract (TT) Physical microenvironment Assay (SPA) to recapitulate pressure changes in the pistil. This biomimetic assay has enabled us to swiftly identify highly redundant genes, GEF8/9/11/12/13, as new regulators for maintaining PTs integrity during style-to-TT emergence. In contrast to normal growth on solid medium, SPA successfully phenocopied gef8/9/11/12/13 PT in vivo growth-arrest deficiency. Our results suggest the existence of distinct signaling pathways regulating in vivo and in vitro PT integrity maintenance, underscoring the necessity of faithfully mimicking the physical microenvironment for studying plant cell biology.

The development of multicellular tissues requires both local and global coordination of cell polarization, however, the mechanisms underlying their interplay are poorly understood. In Arabidopsis, leaf epidermal pavement cells (PC) develop a puzzle-piece shape locally coordinated through apoplastic auxin signaling. Here we show auxin also globally coordinates interdigitation by activating the TIR1/AFB-dependent nuclear signaling pathway. This pathway promotes a transient maximum of auxin at the cotyledon tip, which then moves across the leaf activating local PC polarization, as demonstrated by locally uncaged auxin globally rescuing defects in tir1;afb1;afb2;afb4;afb5 mutant but not in tmk1;tmk2;tmk3;tmk4 mutants. Our findings show that hierarchically integrated global and local auxin signaling systems, which respectively depend on TIR1/AFB-dependent gene transcription in the nucleus and TMK-mediated rapid activation of ROP GTPases at the cell surface, control PC interdigitation patterns in Arabidopsis cotyledons, revealing a mechanism for coordinating a local cellular process with the development of whole tissues.

CRISPR-associated protein 9 (Cas9)-mediated editing generates numerous mutations. Existing detection methods, such as ACT-PCR, T7EI endonuclease cleavage, HRM analysis, and high-throughput sequencing, often require stringent conditions, expensive reagents, or specialized instruments. Here, we introduce Mismatch-Enhanced Specific PCR (MES-PCR), a method that offers sensitivity for detecting mutations under non-stringent experimental conditions. Combined with quantitative PCR (MES-qPCR), it facilitates the calculation of sgRNA efficiency and enables screening for heterozygous mutations. We validated this method in soybean (Glycine max (L.) Merr.) and Arabidopsis thaliana, confirming its practicality. This approach significantly enhances the efficiency and reduces the cost of mutation screening, presenting a powerful tool to accelerate precision breeding and functional genomics research in crops.