Nejvíce citovaný článek - PubMed ID 31349380
Transcription of specific auxin efflux and influx carriers drives auxin homeostasis in tobacco cells
Auxin, indole-3-acetic acid (IAA), is a key phytohormone with diverse morphogenic roles in land plants, but its function and transport mechanisms in algae remain poorly understood. We therefore aimed to explore the role of IAA in a complex, streptophyte algae Chara braunii. Here, we described novel responses of C. braunii to IAA and characterized two homologs of PIN auxin efflux carriers: CbPINa and CbPINc. We determined their localization in C. braunii using epitope-specific antibodies and tested their function in heterologous land plant models. Further, using phosphoproteomic analysis, we identified IAA-induced phosphorylation events. The thallus regeneration assay showed that IAA promotes thallus elongation and side branch development. Immunolocalization of CbPINa and CbPINc confirmed their presence on the plasma membrane of vegetative and generative cells of C. braunii. However, functional assays in tobacco BY-2 cells demonstrated that CbPINa affects auxin transport, whereas CbPINc does not. The IAA is effective in the acceleration of cytoplasmic streaming and the phosphorylation of evolutionary conserved targets such as homolog of RAF-like kinase. These findings suggest that, although canonical PIN-mediated auxin transport mechanisms might not be fully conserved in Chara, IAA is involved in morphogenesis and fast signaling processes.
- Klíčová slova
- Chara, auxin transport, indole‐3‐acetic acid, plant evolution, streptophytes,
- MeSH
- biologický transport účinky léků MeSH
- buněčná membrána metabolismus účinky léků MeSH
- Chara * metabolismus účinky léků MeSH
- fosforylace účinky léků MeSH
- kyseliny indoloctové * metabolismus farmakologie MeSH
- membránové transportní proteiny * metabolismus MeSH
- rostlinné proteiny * metabolismus MeSH
- tabák metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- indoleacetic acid MeSH Prohlížeč
- kyseliny indoloctové * MeSH
- membránové transportní proteiny * MeSH
- rostlinné proteiny * MeSH
Fluorescence light microscopy provided convincing evidence for the domain organization of plant plasma membrane (PM) proteins. Both peripheral and integral PM proteins show an inhomogeneous distribution within the PM. However, the size of PM nanodomains and protein clusters is too small to accurately determine their dimensions and nano-organization using routine confocal fluorescence microscopy and super-resolution methods. To overcome this limitation, we have developed a novel correlative light electron microscopy method (CLEM) using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). Using this technique, we determined the number of auxin efflux carriers from the PINFORMED (PIN) family (NtPIN3b-GFP) within PM nanodomains of tobacco cell PM ghosts. Protoplasts were attached to coverslips and immunostained with anti-GFP primary antibody and secondary antibody conjugated to fluorochrome and gold nanoparticles. After imaging the nanodomains within the PM with TIRFM, the samples were imaged with A-ESEM without further processing, and quantification of the average number of molecules within the nanodomain was performed. Without requiring any post-fixation and coating procedures, this method allows to study details of the organization of auxin carriers and other plant PM proteins.
- Klíčová slova
- auxin carriers, correlative microscopy, nanodomains, plasma membrane,
- MeSH
- Arabidopsis genetika růst a vývoj MeSH
- buněčná membrána genetika metabolismus ultrastruktura MeSH
- konfokální mikroskopie MeSH
- kovové nanočástice chemie MeSH
- kyseliny indoloctové metabolismus MeSH
- mikroskopie elektronová rastrovací * MeSH
- počítačové zpracování obrazu MeSH
- protoplasty metabolismus ultrastruktura MeSH
- regulátory růstu rostlin genetika metabolismus MeSH
- tabák genetika metabolismus ultrastruktura MeSH
- zlato chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kyseliny indoloctové MeSH
- regulátory růstu rostlin MeSH
- zlato MeSH
Together with auxin transport, auxin metabolism is a key determinant of auxin signaling output by plant cells. Enzymatic machinery involved in auxin metabolism is subject to regulation based on numerous inputs, including the concentration of auxin itself. Therefore, experiments characterizing altered auxin availability and subsequent changes in auxin metabolism could elucidate the function and regulatory role of individual elements in the auxin metabolic machinery. Here, we studied auxin metabolism in auxin-dependent tobacco BY-2 cells. We revealed that the concentration of N-(2-oxindole-3-acetyl)-l-aspartic acid (oxIAA-Asp), the most abundant auxin metabolite produced in the control culture, dramatically decreased in auxin-starved BY-2 cells. Analysis of the transcriptome and proteome in auxin-starved cells uncovered significant downregulation of all tobacco (Nicotiana tabacum) homologs of Arabidopsis (Arabidopsis thaliana) DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1), at both transcript and protein levels. Auxin metabolism profiling in BY-2 mutants carrying either siRNA-silenced or CRISPR-Cas9-mutated NtDAO1, as well as in dao1-1 Arabidopsis plants, showed not only the expected lower levels of oxIAA, but also significantly lower abundance of oxIAA-Asp. Finally, ability of DAO1 to oxidize IAA-Asp was confirmed by an enzyme assay in AtDAO1-producing bacterial culture. Our results thus represent direct evidence of DAO1 activity on IAA amino acid conjugates.
