The researchers first examined how auxin regulates MAKR6 transcription. Quantitative reverse-transcription PCR showed that a four-hour treatment with 10 micromolar 1-naphthaleneacetic acid increased MAKR6 messenger RNA levels by 4.6-fold. A similar response occurred after treatment with 1 micromolar indole-3-acetic acid, but it was strongly reduced by 10 micromolar alpha-phenylethyl-2-oxo-indole-3-acetic acid, an antagonist associated with TIR1/AFB-mediated auxin perception. Auxin induction was also diminished in plants expressing dominant-negative WRKY23. These observations placed MAKR6 downstream of TIR1/AFB signalling and within the Aux/IAA–ARF–WRKY23 transcriptional axis. The related gene MAKR4 showed a larger response to 10 micromolar NAA, increasing by 52.7-fold, and was also auxin inducible, but promoter-reporter analysis revealed a different distribution across tissues.

MAKR6 promoter activity was detected primarily in vascular tissues, beginning from the root tip and extending into cotyledon vasculature, leaf tips and young stems. Auxin treatment enhanced the reporter signal and expanded some expression domains. MAKR4 was expressed mainly in lateral root primordia and mature vascular tissues, with no detectable expression in leaves, whereas MAKR6 showed robust expression in leaf vasculature and tips. CRISPR–Cas9 loss-of-function mutants helped separate the proteins’ developmental roles. Primary root length was comparable among wild-type seedlings, MAKR4 mutants, MAKR6 mutants and the double mutant. MAKR4 loss reduced lateral-root number, but MAKR6 loss did not. Leaf venation showed the opposite pattern: normal four-loop cotyledon venation occurred in 76.2% of wild-type seedlings, 78.9% and 74.0% of two MAKR4 mutant lines, and 67.8% and 65.4% of two MAKR6 lines. Complementation with MAKR6 restored the venation defect, and the double mutant resembled the MAKR6 single mutant in this assay.

Two vascular assays tested whether MAKR6 participates directly in auxin canalization. Following transverse wounding of young inflorescence stems, 90% of wild-type plants formed continuous vascular strands that reconnected the separated tissues when examined six days later by Toluidine Blue O staining. In MAKR4 mutants, 60% of stems showed full regeneration and 30% showed partial regeneration. Continuous strands were rare in both MAKR6 mutants and MAKR4/MAKR6 double mutants, which instead displayed incomplete vasculature with few lignified cells or no regenerated vessels. In a second assay, lanolin containing 10 micromolar indole-3-acetic acid was placed below the incision. Most wild-type and MAKR4-mutant stems developed new vascular strands connecting the auxin source to existing vasculature, whereas MAKR6-deficient plants largely failed to establish those connections. The defects were rescued by a functional MAKR6 construct.

Reporter imaging indicated that the vascular phenotypes were accompanied by defects in the formation of auxin channels. In the local auxin assay, no MAKR6 mutant formed a complete PIN1–GFP channel, and 20% showed a complete DR5rev::GFP channel. In the regeneration assay, most wild-type plants formed complete or partial PIN1–GFP channels, while 100% showed continuous DR5rev::GFP channels around the wound. MAKR6 mutants rarely formed complete channels in either reporter background. These observations place the protein at an early stage of channel initiation and progression in the tested Arabidopsis tissues, rather than indicating a role limited to the maturation of vascular strands. The assays do not identify every cellular step affected by MAKR6, but they show a clear association between its loss and defective auxin-channel formation.

Cellular measurements linked MAKR6 to auxin-dependent PIN1 polarity and trafficking. In wild-type root endodermal cells, 10 micromolar NAA increased the ratio of lateral to rootward basal PIN1 signal from 0.79 to 1.13. The corresponding ratio in MAKR6 mutant roots changed more weakly, from 0.80 to 0.95, indicating impaired auxin-induced lateralization. Brefeldin A, which blocks PIN1 endomembrane trafficking and induces intracellular BFA bodies, revealed a related difference. Auxin strongly suppressed BFA-body size and density in wild-type roots, whereas this suppression was less pronounced in MAKR6 mutants. Immunolocalization in first leaves showed PIN1 polarity irregularities in 5 of 41 wild-type leaves, 14 of 49 leaves in a MAKR6 loss-of-function line and 23 of 53 leaves in an overexpression line. These comparable defects in loss- and gain-of-function lines mean that the experiments do not establish whether MAKR6 stimulates or inhibits PIN1 activity in every context. Instead, MAKR6 abundance appears to be important for maintaining coherent, auxin-sensitive PIN1 organization.

Physical and genetic experiments connected MAKR6 with the receptor systems implicated in canalization. Co-immunoprecipitation in Nicotiana leaves and Förster resonance energy transfer combined with fluorescence-lifetime imaging microscopy in Arabidopsis root protoplasts detected associations between MAKR6 and TMK1 or TMK4, as well as between MAKR6 and CAMEL or CANAR. FLS2 served as a negative control in the fluorescence-lifetime tests, and auxin treatment did not noticeably alter the detected MAKR6 interactions. MAKR6 was observed at the plasma membrane in overexpression lines. Phos-tag analysis produced a pronounced mobility shift when MAKR6 was co-expressed with TMK1, TMK4 or CAMEL, consistent with phosphorylation by these kinase-active receptors. Higher-order mutants combining MAKR6 loss with TMK4, CAMEL or CANAR defects had root PIN1 repolarization and trafficking abnormalities comparable to those in the MAKR6 single mutant, and their leaf-venation defects were not more severe. The results support genetic interaction among the pathways but do not resolve their temporal order or identify the relevant MAKR6 phosphorylation sites.

The receptor systems themselves also associated with one another. Co-immunoprecipitation showed that CAMEL and CANAR were recovered with TMK1 or TMK4, and FRET/FLIM measurements in Arabidopsis protoplasts supported those associations. Auxin had no detectable effect on the TMK1–CAMEL interaction. Adding MAKR6 did not measurably change energy transfer between TMK1 and CAMEL/CANAR, or between TMK1 and PIN1. The assays therefore do not support a model in which MAKR6 measurably assembles or stabilizes a single, stable complex containing all these components. The authors instead propose that MAKR6 may act dynamically or downstream, coordinating signals from distinct receptor complexes that converge on auxin–PIN1 feedback. The study did not identify the relevant ligands, co-receptors or downstream cascades, leaving the precise architecture and regulatory direction of this network unresolved.

MAKR6 also associated directly with the PIN1 auxin transporter. FRET/FLIM detected interactions with both the PIN1 hydrophilic loop and full-length PIN1, and co-immunoprecipitation in Arabidopsis seedlings confirmed that tagged MAKR6 co-precipitated with endogenous PIN1. The FLS2 and MAKR2 controls did not show the same interactions in the reported assays. MAKR4 also associated with PIN1 but did not detectably interact with TMK1 or TMK4, indicating that the two MAKR proteins participate in distinct interaction modules. In Arabidopsis, co-immunoprecipitation additionally recovered endogenous TMK1 with MAKR6. The direct MAKR6–PIN1 association supplies a physical link between a receptor-associated regulator and the transporter whose polarity and trafficking were impaired in MAKR6 mutants. The experiments did not establish whether MAKR6 binding alters PIN1 directly, changes its trafficking, or affects the stability or signalling competence of transporter complexes.

Taken together, the experiments portray auxin canalization as a coordinated regulatory system involving transcription, cell-surface receptors and polar auxin transport. MAKR6 is induced through the Aux/IAA–ARF–WRKY23 axis, accumulates in vascular tissues, associates with PIN1 and the TMK1/4 and CAMEL–CANAR systems, and can be phosphorylated by TMK or CAMEL proteins. Its loss disrupts auxin channels, vascular regeneration, PIN1 lateralization and auxin-sensitive PIN1 trafficking in the tested Arabidopsis models. The work also distinguishes MAKR6 from the related MAKR4, which has a stronger role in lateral-root development and lacks detectable MAKR4–TMK1/4 interaction in these tests. Important gaps remain: the functional significance of individual phosphorylation sites, the molecular effect of MAKR6 on PIN1 and the possible involvement of other MAKR proteins remain unresolved. What the evidence establishes is that MAKR6 provides a multifunctional connection between auxin-responsive transcription, receptor-like kinase pathways and PIN1-dependent transport in vascular patterning and regeneration.

Subject of Research: MAKR6 regulation of PIN1-dependent auxin canalization and vascular development in Arabidopsis

Article Title: MAKR6 integrates TMK and CAMEL/CANAR signalling for auxin canalization in Arabidopsis

Article References: Ge, Z., Koczka, L., Mazur, E., Molnar, G., Vladimirtsev, D., Kassem, N., Ikene, S. A., Fiedler, L., & Friml, J. (2026). MAKR6 integrates TMK and CAMEL/CANAR signalling for auxin canalization in Arabidopsis. Nature Plants. https://doi.org/10.1038/s41477-026-02435-0

Image Credits: AI Generated

DOI: 10.1038/s41477-026-02435-0

Keywords: MAKR6, Arabidopsis, auxin canalization, PIN1 transporter, vascular regeneration, leaf venation, receptor-like kinases, auxin signalling, cell polarity, plant development, integrates, CAMEL