Heat stress (HS) is a major abiotic stress that negatively impacts crop yields across the globe. Plants respond to elevated temperatures by changing gene expression, mediated by transcription factors (TFs) functioning to enhance HS tolerance. The involvement of Group I bZIP TFs in the heat stress response (HSR) is not known. In this study, bZIP18 and bZIP52 were investigated for their possible role in the HSR. Localization experiments revealed their nuclear accumulation following heat stress, which was found to be triggered by dephosphorylation. Both TFs were found to possess two motifs containing serine residues that are candidates for phosphorylation. These motifs are recognized by 14-3-3 proteins, and bZIP18 and bZIP52 were found to bind 14-3-3 ε, the interaction of which sequesters them to the cytoplasm. Mutation of both residues abolished 14-3-3 ε interaction and led to a strict nuclear localization for both TFs. RNA-seq analysis revealed coordinated downregulation of several metabolic pathways including energy metabolism and translation, and upregulation of numerous lncRNAs in particular. These results support the idea that bZIP18 and bZIP52 are sequestered to the cytoplasm under control conditions, and that heat stress leads to their re-localization to nuclei, where they jointly regulate gene expression.
- MeSH
- Arabidopsis genetika růst a vývoj MeSH
- buněčné jádro genetika MeSH
- proteiny 14-3-3 genetika MeSH
- proteiny huseníčku genetika MeSH
- reakce na tepelný šok genetika MeSH
- regulace genové exprese u rostlin genetika MeSH
- RNA dlouhá nekódující genetika MeSH
- transkripční faktory genetika MeSH
- Publikační typ
- časopisecké články MeSH
Plants, like other multicellular organisms, survive through a delicate balance between growth and defense against pathogens. Salicylic acid (SA) is a major defense signal in plants, and the perception mechanism as well as downstream signaling activating the immune response are known. Here, we identify a parallel SA signaling that mediates growth attenuation. SA directly binds to A subunits of protein phosphatase 2A (PP2A), inhibiting activity of this complex. Among PP2A targets, the PIN2 auxin transporter is hyperphosphorylated in response to SA, leading to changed activity of this important growth regulator. Accordingly, auxin transport and auxin-mediated root development, including growth, gravitropic response, and lateral root organogenesis, are inhibited. This study reveals how SA, besides activating immunity, concomitantly attenuates growth through crosstalk with the auxin distribution network. Further analysis of this dual role of SA and characterization of additional SA-regulated PP2A targets will provide further insights into mechanisms maintaining a balance between growth and defense.
- MeSH
- Arabidopsis růst a vývoj fyziologie MeSH
- imunita rostlin MeSH
- kořeny rostlin růst a vývoj metabolismus MeSH
- kyselina salicylová metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- proteinfosfatasa 2 metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- signální transdukce * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Auxin concentration gradients are informative for the transduction of many developmental cues, triggering downstream gene expression and other responses. The generation of auxin gradients depends significantly on cell-to-cell auxin transport, which is supported by the activities of auxin efflux and influx carriers. However, at the level of individual plant cell, the co-ordination of auxin efflux and influx largely remains uncharacterized. We addressed this issue by analyzing the contribution of canonical PIN-FORMED (PIN) proteins to the carrier-mediated auxin efflux in Nicotiana tabacum L., cv. Bright Yellow (BY-2) tobacco cells. We show here that a majority of canonical NtPINs are transcribed in cultured cells and in planta. Cloning of NtPIN genes and their inducible overexpression in tobacco cells uncovered high auxin efflux activity of NtPIN11, accompanied by auxin starvation symptoms. Auxin transport parameters after NtPIN11 overexpression were further assessed using radiolabelled auxin accumulation and mathematical modelling. Unexpectedly, these experiments showed notable stimulation of auxin influx, which was accompanied by enhanced transcript levels of genes for a specific auxin influx carrier and by decreased transcript levels of other genes for auxin efflux carriers. A similar transcriptional response was observed upon removal of auxin from the culture medium, which resulted in decreased auxin efflux. Overall, our results revealed an auxin transport-based homeostatic mechanism for the maintenance of endogenous auxin levels. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at http://osf.io/ka97b/.
- MeSH
- biologický transport MeSH
- buněčné linie MeSH
- fylogeneze MeSH
- homeostáza MeSH
- kyseliny indoloctové metabolismus MeSH
- membránové transportní proteiny genetika metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulátory růstu rostlin metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- tabák genetika fyziologie MeSH
- teoretické modely MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
Coordination of plant development requires modulation of growth responses that are under control of the phytohormone auxin. PIN-FORMED plasma membrane proteins, involved in intercellular transport of the growth regulator, are key to the transmission of such auxin signals and subject to multilevel surveillance mechanisms, including reversible post-translational modifications. Apart from well-studied PIN protein modifications, namely phosphorylation and ubiquitylation, no further post-translational modifications have been described so far. Here, we focused on root-specific Arabidopsis PIN2 and explored functional implications of two evolutionary conserved cysteines, by a combination of in silico and molecular approaches. PIN2 sequence alignments and modeling predictions indicated that both cysteines are facing the cytoplasm and therefore would be accessible to redox status-controlled modifications. Notably, mutant pin2C-A alleles retained functionality, demonstrated by their ability to almost completely rescue defects of a pin2 null allele, whereas high resolution analysis of pin2C-A localization revealed increased intracellular accumulation, and altered protein distribution within plasma membrane micro-domains. The observed effects of cysteine replacements on root growth and PIN2 localization are consistent with a model in which redox status-dependent cysteine modifications participate in the regulation of PIN2 mobility, thereby fine-tuning polar auxin transport.
- MeSH
- Arabidopsis genetika metabolismus MeSH
- cystein genetika MeSH
- konzervovaná sekvence * MeSH
- kořeny rostlin růst a vývoj metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- membránové mikrodomény metabolismus MeSH
- proteiny huseníčku chemie genetika metabolismus MeSH
- transport proteinů MeSH
- Publikační typ
- časopisecké články MeSH
The plant-specific DREPP protein family comprises proteins that were shown to regulate the actin and microtubular cytoskeleton in a calcium-dependent manner. Our phylogenetic analysis showed that DREPPs first appeared in ferns and that DREPPs have a rapid and plastic evolutionary history in plants. Arabidopsis DREPP paralogues called AtMDP25/PCaP1 and AtMAP18/PCaP2 are N-myristoylated, which has been reported as a key factor in plasma membrane localization. Here we show that N-myristoylation is neither conserved nor ancestral for the DREPP family. Instead, by using confocal microscopy and a new method for quantitative evaluation of protein membrane localization, we show that DREPPs rely on two mechanisms ensuring their plasma membrane localization. These include N-myristoylation and electrostatic interaction of a polybasic amino acid cluster. We propose that various plasma membrane association mechanisms resulting from the evolutionary plasticity of DREPPs are important for refining plasma membrane interaction of these signalling proteins under various conditions and in various cells.
A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.
