Polar auxin transport in the Arabidopsis (Arabidopsis thaliana) root tip maintains high auxin levels around the stem cell niche that gradually decrease in dividing cells but increase again once they transition toward differentiation. Protophloem differentiates earlier than other proximal tissues and employs a unique auxin "canalization" machinery that is thought to balance auxin efflux with retention. It consists of a proposed activator of PIN-FORMED (PIN) auxin efflux carriers, the cAMP-, cGMP- and Calcium-dependent (AGC) kinase PROTEIN KINASE ASSOCIATED WITH BRX (PAX); its inhibitor, BREVIS RADIX (BRX); and PHOSPHATIDYLINOSITOL-4-PHOSPHATE-5-KINASE (PIP5K) enzymes, which promote polar PAX and BRX localization. Because of a dynamic PAX-BRX-PIP5K interplay, the net cellular output of this machinery remains unclear. In this study, we deciphered the dosage-sensitive regulatory interactions among PAX, BRX, and PIP5K by their ectopic expression in developing xylem vessels. The data suggest that the dominant collective output of the PAX-BRX-PIP5K module is a localized reduction in PIN abundance. This requires PAX-stimulated clathrin-mediated PIN endocytosis upon site-specific phosphorylation, which distinguishes PAX from other AGC kinases. An ectopic assembly of the PAX-BRX-PIP5K module is sufficient to cause cellular auxin retention and affects root growth vigor by accelerating the trajectory of xylem vessel development. Our data thus provide direct evidence that local manipulation of auxin efflux alters the timing of cellular differentiation in the root.

• MeSH
• Arabidopsis * metabolismus   genetika   růst a vývoj   MeSH
• biologický transport MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem metabolismus   genetika   MeSH
• kořeny rostlin metabolismus   růst a vývoj   genetika   MeSH
• kyseliny indoloctové * metabolismus   MeSH
• membránové transportní proteiny metabolismus   genetika   MeSH
• protein-serin-threoninkinasy * MeSH
• proteiny huseníčku * metabolismus   genetika   MeSH
• regulace genové exprese u rostlin MeSH
• xylém metabolismus   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AT1G66150 protein, Arabidopsis MeSH Prohlížeč
• BREVIS RADIX protein, Arabidopsis MeSH Prohlížeč
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• kyseliny indoloctové * MeSH
• membránové transportní proteiny MeSH
• protein-serin-threoninkinasy * MeSH
• proteiny huseníčku * MeSH

Phosphatidylinositol 4-kinases (PI4Ks) are the first enzymes that commit phosphatidylinositol into the phosphoinositide pathway. Here, we show that Arabidopsis thaliana seedlings deficient in PI4Kβ1 and β2 have several developmental defects including shorter roots and unfinished cytokinesis. The pi4kβ1β2 double mutant was insensitive to exogenous auxin concerning inhibition of root length and cell elongation; it also responded more slowly to gravistimulation. The pi4kß1ß2 root transcriptome displayed some similarities to a wild type plant response to auxin. Yet, not all the genes displayed such a constitutive auxin-like response. Besides, most assessed genes did not respond to exogenous auxin. This is consistent with data with the transcriptional reporter DR5-GUS. The content of bioactive auxin in the pi4kß1ß2 roots was similar to that in wild-type ones. Yet, an enhanced auxin-conjugating activity was detected and the auxin level reporter DII-VENUS did not respond to exogenous auxin in pi4kß1ß2 mutant. The mutant exhibited altered subcellular trafficking behavior including the trapping of PIN-FORMED 2 protein in rapidly moving vesicles. Bigger and less fragmented vacuoles were observed in pi4kß1ß2 roots when compared to the wild type. Furthermore, the actin filament web of the pi4kß1ß2 double mutant was less dense than in wild-type seedling roots, and less prone to rebuilding after treatment with latrunculin B. A mechanistic model is proposed in which an altered PI4K activity leads to actin filament disorganization, changes in vesicle trafficking, and altered auxin homeostasis and response resulting in a pleiotropic root phenotypes.

• MeSH
• Arabidopsis * metabolismus   MeSH
• fosfatidylinositolfosfáty MeSH
• fosfatidylinositoly metabolismus   MeSH
• kořeny rostlin genetika   metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfatidylinositolfosfáty MeSH
• fosfatidylinositoly MeSH
• kyseliny indoloctové MeSH
• phosphatidylinositol 4-phosphate MeSH Prohlížeč
• proteiny huseníčku * MeSH

Pollen grains show an enormous variety of aperture systems. What genes are involved in the aperture formation pathway and how conserved this pathway is in angiosperms remains largely unknown. INAPERTURATE POLLEN1 (INP1) encodes a protein of unknown function, essential for aperture formation in Arabidopsis, rice and maize. Yet, because INP1 sequences are quite divergent, it is unclear if their function is conserved across angiosperms. Here, we conducted a functional study of the INP1 ortholog from the basal eudicot Eschscholzia californica (EcINP1) using expression analyses, virus-induced gene silencing, pollen germination assay, and transcriptomics. We found that EcINP1 expression peaks at the tetrad stage of pollen development, consistent with its role in aperture formation, which occurs at that stage, and showed, via gene silencing, that the role of INP1 as an important aperture factor extends to basal eudicots. Using germination assays, we demonstrated that, in Eschscholzia, apertures are dispensable for pollen germination. Our comparative transcriptome analysis of wild-type and silenced plants identified over 900 differentially expressed genes, many of them potential candidates for the aperture pathway. Our study substantiates the importance of INP1 homologs for aperture formation across angiosperms and opens up new avenues for functional studies of other aperture candidate genes.

• Klíčová slova
• Eschscholzia californica, INAPERTURATE POLLEN1, Papaveraceae, RNA-seq, VIGS, pollen, pollen aperture, transcriptome analysis,
• Publikační typ
• časopisecké články MeSH

Polar subcellular localization of the PIN exporters of the phytohormone auxin is a key determinant of directional, intercellular auxin transport and thus a central topic of both plant cell and developmental biology. Arabidopsis mutants lacking PID, a kinase that phosphorylates PINs, or the MAB4/MEL proteins of unknown molecular function display PIN polarity defects and phenocopy pin mutants, but mechanistic insights into how these factors convey PIN polarity are missing. Here, by combining protein biochemistry with quantitative live-cell imaging, we demonstrate that PINs, MAB4/MELs, and AGC kinases interact in the same complex at the plasma membrane. MAB4/MELs are recruited to the plasma membrane by the PINs and in concert with the AGC kinases maintain PIN polarity through limiting lateral diffusion-based escape of PINs from the polar domain. The PIN-MAB4/MEL-PID protein complex has self-reinforcing properties thanks to positive feedback between AGC kinase-mediated PIN phosphorylation and MAB4/MEL recruitment. We thus uncover the molecular mechanism by which AGC kinases and MAB4/MEL proteins regulate PIN localization and plant development.

• Klíčová slova
• Arabidopsis, cell polarity, lateral diffusion, plant development, polar auxin transport, positive feedback, protein phosphorylation,
• MeSH
• Arabidopsis * genetika   metabolismus   MeSH
• biologický transport MeSH
• kořeny rostlin metabolismus   MeSH
• kyseliny indoloctové MeSH
• membránové transportní proteiny genetika   MeSH
• polarita buněk MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné buňky metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• membránové transportní proteiny MeSH
• proteiny huseníčku * MeSH