Chemical inhibitors are often implemented for the functional characterization of genes to overcome the limitations associated with genetic approaches. Although it is well established that the specificity of the compound is key to success of a pharmacological approach, off-target effects are often overlooked or simply neglected in a complex biological setting. Here we illustrate the cause and implications of such secondary effects by focusing on piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H) that is frequently used to investigate the involvement of lignin during plant growth and development. When supplied to plants, we found that PA is recognized as a substrate by GRETCHEN HAGEN 3.6 (GH3.6), an amido synthetase involved in the formation of the indole-3-acetic acid (IAA) conjugate IAA-Asp. By competing for the same enzyme, PA interferes with IAA conjugation, resulting in an increase in IAA concentrations in the plant. In line with the broad substrate specificity of the GH3 family of enzymes, treatment with PA increased not only IAA levels but also those of other GH3-conjugated phytohormones, namely jasmonic acid and salicylic acid. Finally, we found that interference with the endogenous function of GH3s potentially contributes to phenotypes previously observed upon PA treatment. We conclude that deregulation of phytohormone homeostasis by surrogate occupation of the conjugation machinery in the plant is likely a general phenomenon when using chemical inhibitors. Our results hereby provide a novel and important basis for future reference in studies using chemical inhibitors.
- MeSH
- benzoáty MeSH
- cinnamáty farmakologie MeSH
- kyseliny indoloctové * farmakologie MeSH
- oxygenasy se smíšenou funkcí genetika MeSH
- regulace genové exprese u rostlin MeSH
- regulátory růstu rostlin * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- benzoáty MeSH
- cinnamáty MeSH
- kyseliny indoloctové * MeSH
- oxygenasy se smíšenou funkcí MeSH
- piperonylic acid MeSH Prohlížeč
- regulátory růstu rostlin * MeSH
The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.
- Klíčová slova
- auxin, auxin metabolism, flow cytometry, nucleus, subcellular fractionation,
- MeSH
- Arabidopsis účinky léků metabolismus ultrastruktura MeSH
- buněčné jádro účinky léků metabolismus ultrastruktura MeSH
- buněčné kultury MeSH
- centrifugace metody MeSH
- frakcionace buněk přístrojové vybavení metody MeSH
- hmotnostní spektrometrie MeSH
- homeostáza fyziologie MeSH
- indoly metabolismus farmakologie MeSH
- kyseliny indoloctové metabolismus MeSH
- protoplasty chemie MeSH
- průtoková cytometrie MeSH
- regulátory růstu rostlin metabolismus MeSH
- rostlinné buňky účinky léků metabolismus ultrastruktura MeSH
- tabák účinky léků metabolismus ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- indole MeSH Prohlížeč
- indoly MeSH
- kyseliny indoloctové MeSH
- regulátory růstu rostlin MeSH
In over 40 years of research on the cellular uptake of auxin it is somewhat chastening that we have elaborated so little on the original kinetic descriptions of auxin uptake by plant cells made by Rubery and Sheldrake in 1974. Every aspect of that seminal work has been investigated in detail, and the uptake activity they measured is now known to be attributed to the AUX1/LAX family of permeases. Recent pharmacological studies have defined the substrate specificity of AUX1, biochemical studies have evaluated its permeability to auxin in plant cell membranes, and rigourous kinetic studies have confirmed the affinity of AUX1 for IAA and synthetic auxins. Advances in genome sequencing have provided a rich resource for informatic analysis of the ancestry of AUX1 and the LAX proteins and, along with models of topology, suggest mechanistic links to families of eukaryotic proton co-transporters for which crystal structures have been presented. The insights gained from all the accumulated research reflect the brilliance of Rubery and Sheldrake's early work, but recent biochemical analyses are starting to advance further our understanding of this vitally important family of auxin transport proteins.
- Klíčová slova
- auxin transport, development, hormone, kinetics, permeability, structure,
- MeSH
- aktivní transport fyziologie MeSH
- buněčná membrána * genetika metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- membránové transportní proteiny * genetika metabolismus MeSH
- rostliny * genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- kyseliny indoloctové MeSH
- membránové transportní proteiny * MeSH
Plant hormones are master regulators of plant growth and development. Better knowledge of their spatial signaling and homeostasis (transport and metabolism) on the lowest structural levels (cellular and subcellular) is therefore crucial to a better understanding of developmental processes in plants. Recent progress in phytohormone analysis at the cellular and subcellular levels has greatly improved the effectiveness of isolation protocols and the sensitivity of analytical methods. This review is mainly focused on homeostasis of two plant hormone groups, auxins and cytokinins. It will summarize and discuss their tissue- and cell-type specific distributions at the cellular and subcellular levels.
- Klíčová slova
- auxin, cellular level, cytokinin, phytohormone metabolism, phytohormone transport, subcellular level,
- MeSH
- biologický transport MeSH
- cytokininy metabolismus MeSH
- fyziologie rostlin * MeSH
- homeostáza * MeSH
- intracelulární prostor metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- metabolické sítě a dráhy MeSH
- organely metabolismus MeSH
- regulátory růstu rostlin metabolismus MeSH
- rostlinné buňky metabolismus MeSH
- vývoj rostlin * MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- cytokininy MeSH
- kyseliny indoloctové MeSH
- regulátory růstu rostlin MeSH
Many proteins and cargoes in eukaryotic cells are secreted through the conventional secretory pathway that brings proteins and membranes from the endoplasmic reticulum to the plasma membrane, passing through various cell compartments, and then the extracellular space. The recent identification of an increasing number of leaderless secreted proteins bypassing the Golgi apparatus unveiled the existence of alternative protein secretion pathways. Moreover, other unconventional routes for secretion of soluble or transmembrane proteins with initial endoplasmic reticulum localization were identified. Furthermore, other proteins normally functioning in conventional membrane traffic or in the biogenesis of unique plant/fungi organelles or in plasmodesmata transport seem to be involved in unconventional secretory pathways. These alternative pathways are functionally related to biotic stress and development, and are becoming more and more important in cell biology studies in yeast, mammalian cells and in plants. The city of Lecce hosted specialists working on mammals, plants and microorganisms for the inaugural meeting on "Unconventional Protein and Membrane Traffic" (UPMT) during 4-7 October 2016. The main aim of the meeting was to include the highest number of topics, summarized in this report, related to the unconventional transport routes of protein and membranes.
- Klíčová slova
- autophagy, exosomes, intercellular channels, leaderless proteins, protein secretion, trafficking mechanisms, unconventional secretion,
- MeSH
- biologie buňky * MeSH
- lidé MeSH
- membránové proteiny metabolismus MeSH
- transport proteinů MeSH
- vývojová biologie * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- kongresy MeSH
- Názvy látek
- membránové proteiny MeSH
