PIN proteins establish the auxin concentration gradient, which coordinates plant growth. PIN1-4 and 7 localized at the plasma membrane (PM) and facilitate polar auxin transport while the endoplasmic reticulum (ER) localized PIN5 and PIN8 maintain the intracellular auxin homeostasis. Although an antagonistic activity of PIN5 and PIN8 proteins in regulating the intracellular auxin homeostasis and other developmental events have been reported, the membrane topology of these proteins, which might be a basis for their antagonistic function, is poorly understood. In this study we optimized digitonin based PM-permeabilizing protocols coupled with immunocytochemistry labeling to map the membrane topology of PIN5 and PIN8 in Arabidopsis thaliana root cells. Our results indicate that, except for the similarities in the orientation of the N-terminus, PIN5 and PIN8 have an opposite orientation of the central hydrophilic loop and the C-terminus, as well as an unequal number of transmembrane domains (TMDs). PIN8 has ten TMDs with groups of five alpha-helices separated by the central hydrophilic loop (HL) residing in the ER lumen, and its N- and C-terminals are positioned in the cytoplasm. However, the topology of PIN5 comprises nine TMDs. Its N-terminal end and the central HL face the cytoplasm while its C-terminus resides in the ER lumen. Overall, this study shows that PIN5 and PIN8 proteins have a divergent membrane topology while introducing a toolkit of methods for studying membrane topology of integral proteins including those localized at the ER membrane.

• Klíčová slova
• Arabidopsis thaliana, Auxin homeostasis and transport, Digitonin PM-permeabilizing protocols, Endoplasmic reticulum, Immunocytochemistry, Membrane topology determination methods, PIN auxin efflux carriers,
• Publikační typ
• časopisecké články MeSH

The identification of genes involved in salinity tolerance has primarily focused on model plants and crops. However, plants naturally adapted to highly saline environments offer valuable insights into tolerance to extreme salinity. Salicornia plants grow in coastal salt marshes, stimulated by NaCl. To understand this tolerance, we generated genome sequences of two Salicornia species and analyzed the transcriptomic and proteomic responses of Salicornia bigelovii to NaCl. Subcellular membrane proteomes reveal that SbiSOS1, a homolog of the well-known SALT-OVERLY-SENSITIVE 1 (SOS1) protein, appears to localize to the tonoplast, consistent with subcellular localization assays in tobacco. This neo-localized protein can pump Na+ into the vacuole, preventing toxicity in the cytosol. We further identify 11 proteins of interest, of which SbiSALTY, substantially improves yeast growth on saline media. Structural characterization using NMR identified it as an intrinsically disordered protein, localizing to the endoplasmic reticulum in planta, where it can interact with ribosomes and RNA, stabilizing or protecting them during salt stress.

• MeSH
• Chenopodiaceae * metabolismus   genetika   účinky léků   MeSH
• chlorid sodný farmakologie   metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• proteomika MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• rostlinné proteiny * metabolismus   genetika   MeSH
• salinita MeSH
• solný stres MeSH
• tabák metabolismus   genetika   účinky léků   MeSH
• tolerance k soli * genetika   MeSH
• transkriptom MeSH
• vakuoly metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

The endoplasmic reticulum (ER) is a dynamic organelle that is amenable to major restructuring. Introduction of recombinant ER-membrane-resident proteins that form homo oligomers is a known method of inducing ER proliferation: interaction of the proteins with each other alters the local structure of the ER network, leading to the formation large aggregations of expanded ER, sometimes leading to the formation of organized smooth endoplasmic reticulum (OSER). However, these membrane structures formed by ER proliferation are poorly characterized and this hampers their potential development for plant synthetic biology. Here, we characterize a range of ER-derived membranous compartments in tobacco and show how the nature of the polyproteins introduced into the ER membrane affect the morphology of the final compartment. We show that a cytosol-facing oligomerization domain is an essential component for compartment formation. Using fluorescence recovery after photobleaching, we demonstrate that although the compartment retains a connection to the ER, a diffusional barrier exists to both the ER and the cytosol associated with the compartment. Using quantitative image analysis, we also show that the presence of the compartment does not disrupt the rest of the ER network. Moreover, we demonstrate that it is possible to recruit a heterologous, bacterial enzyme to the compartment, and for the enzyme to accumulate to high levels. Finally, transgenic Arabidopsis constitutively expressing the compartment-forming polyproteins grew and developed normally under standard conditions.

• Klíčová slova
• Compartment, OSER, endoplasmic reticulum, membrane, proliferation, synthetic biology,
• MeSH
• Arabidopsis * metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• intracelulární membrány metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• polyproteiny * analýza   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH
• polyproteiny * MeSH

Photosynthesis is among the first processes negatively affected by environmental cues and its performance directly determines plant cell fitness and ultimately crop yield. Primarily sites of photosynthesis, chloroplasts are unique sites also for the biosynthesis of precursors of the growth regulator auxin and for sensing environmental stress, but their role in intracellular auxin homeostasis, vital for plant growth and survival in changing environments, remains poorly understood. Here, we identified two ATP-binding cassette (ABC) subfamily B transporters, ABCB28 and ABCB29, which export auxin across the chloroplast envelope to the cytosol in a concerted action in vivo. Moreover, we provide evidence for an auxin biosynthesis pathway in Arabidopsis thaliana chloroplasts. The overexpression of ABCB28 and ABCB29 influenced stomatal regulation and resulted in significantly improved water use efficiency and survival rates during salt and drought stresses. Our results suggest that chloroplast auxin production and transport contribute to stomata regulation for conserving water upon salt stress. ABCB28 and ABCB29 integrate photosynthesis and auxin signals and as such hold great potential to improve the adaptation potential of crops to environmental cues.

• Klíčová slova
• ABC transporter, auxin, drought, hormone transport, photosynthesis, salinity, stress,
• Publikační typ
• časopisecké články MeSH

Actin-related protein (ARP2/3) complex is a heteroheptameric protein complex, evolutionary conserved in all eukaryotic organisms. Its conserved role is based on the induction of actin polymerization at the interface between membranes and the cytoplasm. Plant ARP2/3 has been reported to participate in actin reorganization at the plasma membrane during polarized growth of trichomes and at the plasma membrane-endoplasmic reticulum contact sites. Here we demonstrate that individual plant subunits of ARP2/3 fused to fluorescent proteins form motile spot-like structures in the cytoplasm that are associated with peroxisomes in Arabidopsis and tobacco. ARP2/3 is found at the peroxisome periphery and contains the assembled ARP2/3 complex and the WAVE/SCAR complex subunit NAP1. This ARP2/3-positive peroxisomal domain colocalizes with the autophagosome and, under conditions that affect the autophagy, colocalization between ARP2/3 and the autophagosome increases. ARP2/3 subunits co-immunoprecipitate with ATG8f and peroxisome-associated ARP2/3 interact in vivo with the ATG8f marker. Since mutants lacking functional ARP2/3 complex have more peroxisomes than wild type, we suggest that ARP2/3 has a novel role in the process of peroxisome degradation by autophagy, called pexophagy.

• MeSH
• aktiny MeSH
• Arabidopsis * metabolismus   MeSH
• komplex proteinů 2-3 souvisejících s aktinem metabolismus   MeSH
• makroautofagie MeSH
• peroxizomy metabolismus   MeSH
• proteiny huseníčku * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• komplex proteinů 2-3 souvisejících s aktinem MeSH
• proteiny huseníčku * MeSH

The central metabolic regulator SnRK1 controls plant growth and survival upon activation by energy depletion, but detailed molecular insight into its regulation and downstream targets is limited. Here we used phosphoproteomics to infer the sucrose-dependent processes targeted upon starvation by kinases as SnRK1, corroborating the relation of SnRK1 with metabolic enzymes and transcriptional regulators, while also pointing to SnRK1 control of intracellular trafficking. Next, we integrated affinity purification, proximity labelling and crosslinking mass spectrometry to map the protein interaction landscape, composition and structure of the SnRK1 heterotrimer, providing insight in its plant-specific regulation. At the intersection of this multi-dimensional interactome, we discovered a strong association of SnRK1 with class II T6P synthase (TPS)-like proteins. Biochemical and cellular assays show that TPS-like proteins function as negative regulators of SnRK1. Next to stable interactions with the TPS-like proteins, similar intricate connections were found with known regulators, suggesting that plants utilize an extended kinase complex to fine-tune SnRK1 activity for optimal responses to metabolic stress.

• MeSH
• cukerné fosfáty * metabolismus   MeSH
• protein-serin-threoninkinasy genetika   MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• rostliny metabolismus   MeSH
• signální transdukce MeSH
• trehalosa metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cukerné fosfáty * MeSH
• protein-serin-threoninkinasy MeSH
• proteiny huseníčku * MeSH
• trehalosa MeSH

Telomerase, an essential enzyme that maintains chromosome ends, is important for genome integrity and organism development. Various hypotheses have been proposed in human, ciliate and yeast systems to explain the coordination of telomerase holoenzyme assembly and the timing of telomerase performance at telomeres during DNA replication or repair. However, a general model is still unclear, especially pathways connecting telomerase with proposed non-telomeric functions. To strengthen our understanding of telomerase function during its intracellular life, we report on interactions of several groups of proteins with the Arabidopsis telomerase protein subunit (AtTERT) and/or a component of telomerase holoenzyme, POT1a protein. Among these are the nucleosome assembly proteins (NAP) and the minichromosome maintenance (MCM) system, which reveal new insights into the telomerase interaction network with links to telomere chromatin assembly and replication. A targeted investigation of 176 candidate proteins demonstrated numerous interactions with nucleolar, transport and ribosomal proteins, as well as molecular chaperones, shedding light on interactions during telomerase biogenesis. We further identified protein domains responsible for binding and analyzed the subcellular localization of these interactions. Moreover, additional interaction networks of NAP proteins and the DOMINO1 protein were identified. Our data support an image of functional telomerase contacts with multiprotein complexes including chromatin remodeling and cell differentiation pathways.

• Klíčová slova
• Arabidopsis, chromatin, folding, mitochondria, protein–protein interaction, replication, telomerase, transport,
• MeSH
• Arabidopsis metabolismus   MeSH
• genetická transkripce MeSH
• Golgiho aparát metabolismus   MeSH
• homeostáza telomer MeSH
• mapy interakcí proteinů MeSH
• mitochondrie metabolismus   MeSH
• multiproteinové komplexy metabolismus   MeSH
• nukleozomy metabolismus   MeSH
• peptidy metabolismus   MeSH
• posttranskripční úpravy RNA genetika   MeSH
• proteiny huseníčku chemie   metabolismus   MeSH
• proteiny vázající telomery metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• replikace DNA MeSH
• restrukturace chromatinu MeSH
• ribozomy metabolismus   MeSH
• telomerasa metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• multiproteinové komplexy MeSH
• nukleozomy MeSH
• peptidy MeSH
• proteiny huseníčku MeSH
• proteiny vázající telomery MeSH
• telomerasa MeSH

Growing evidence has highlighted the essential role of plant hormones, notably, cytokinins (CKs), in nitrogen-fixing symbiosis, both at early and late nodulation stages1,2. Despite numerous studies showing the central role of CK in nodulation, the importance of CK transport in the symbiosis is unknown. Here, we show the role of ABCG56, a full-size ATP-binding cassette (ABC) transporter in the early stages of the nodulation. MtABCG56 is expressed in roots and nodules and its messenger RNA levels increase upon treatment with symbiotic bacteria, isolated Nod factor and CKs, accumulating within the epidermis and root cortex. MtABCG56 exports bioactive CKs in an ATP-dependent manner over the plasma membrane and its disruption results in an impairment of nodulation. Our data indicate that ABCG-mediated cytokinin transport is important for proper establishment of N-fixing nodules.

• MeSH
• ABC transportér, podrodina G genetika   metabolismus   MeSH
• biologický transport MeSH
• cytokininy metabolismus   MeSH
• fixace dusíku MeSH
• Medicago truncatula genetika   mikrobiologie   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• Rhizobium fyziologie   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• symbióza genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ABC transportér, podrodina G MeSH
• cytokininy MeSH
• regulátory růstu rostlin MeSH
• rostlinné proteiny MeSH

Crop breeding for resistance to pathogens largely relies on genes encoding receptors that confer race-specific immunity. Here, we report the identification of the wheat Pm4 race-specific resistance gene to powdery mildew. Pm4 encodes a putative chimeric protein of a serine/threonine kinase and multiple C2 domains and transmembrane regions, a unique domain architecture among known resistance proteins. Pm4 undergoes constitutive alternative splicing, generating two isoforms with different protein domain topologies that are both essential for resistance function. Both isoforms interact and localize to the endoplasmatic reticulum when co-expressed. Pm4 reveals additional diversity of immune receptor architecture to be explored for breeding and suggests an endoplasmatic reticulum-based molecular mechanism of Pm4-mediated race-specific resistance.

• MeSH
• alternativní sestřih * MeSH
• Ascomycota imunologie   MeSH
• klonování DNA MeSH
• molekulární evoluce MeSH
• nemoci rostlin genetika   MeSH
• odolnost vůči nemocem genetika   MeSH
• proteinkinasy genetika   fyziologie   MeSH
• pšenice enzymologie   genetika   mikrobiologie   MeSH
• rekombinace genetická MeSH
• rostlinné geny MeSH
• rostlinné proteiny genetika   fyziologie   MeSH
• umlčování genů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteinkinasy MeSH
• rostlinné proteiny MeSH

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.

• Klíčová slova
• 14–3–3, Arabidopsis, bZIP, heat stress, localization, transcriptomics,
• 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
• Názvy látek
• proteiny 14-3-3 MeSH
• proteiny huseníčku MeSH
• RNA dlouhá nekódující MeSH
• transkripční faktory MeSH