Multistep phosphorelay (MSP) signaling integrates hormonal and environmental signals to control both plant development and adaptive responses. Type-A RESPONSE REGULATOR (RRA) genes, the downstream members of the MSP cascade and cytokinin primary response genes, are thought to mediate primarily the negative feedback regulation of (cytokinin-induced) MSP signaling. However, transcriptional data also suggest the involvement of RRA genes in stress-related responses. By employing evolutionary conservation with the well-characterized Arabidopsis thaliana RRA genes, we identified five and 38 novel putative RRA genes in Brassica oleracea and Brassica napus, respectively. Our phylogenetic analysis suggests the existence of gene-specific selective pressure, maintaining the homologs of ARR3, ARR6, and ARR16 as singletons during the evolution of Brassicaceae. We categorized RRA genes based on the kinetics of their cytokinin-mediated up-regulation and observed both similarities and specificities in this type of response across Brassicaceae species. Using bioinformatic analysis and experimental data demonstrating the cytokinin and abiotic stress responsiveness of the A. thaliana-derived TCSv2 reporter, we unveil the mechanistic conservation of cytokinin- and stress-mediated up-regulation of RRA genes in B. rapa and B. napus. Notably, we identify partial cytokinin dependency of cold stress-induced RRA transcription, thus further demonstrating the role of cytokinin signaling in crop adaptive responses.

• Klíčová slova
• Arabidopsis thaliana, Brassica napus, Brassica oleracea, Brassica rapa, cytokinins, multistep phosphorelay, osmotic stress, salinity, two-component signaling, type-A response regulator,
• MeSH
• Arabidopsis genetika   fyziologie   metabolismus   MeSH
• Brassica napus genetika   fyziologie   metabolismus   MeSH
• Brassica * genetika   fyziologie   metabolismus   MeSH
• cytokininy * metabolismus   MeSH
• fylogeneze MeSH
• fyziologický stres * MeSH
• regulace genové exprese u rostlin MeSH
• regulátory růstu rostlin metabolismus   MeSH
• rostlinné proteiny * genetika   metabolismus   MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokininy * MeSH
• regulátory růstu rostlin MeSH
• rostlinné proteiny * 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

To properly assess promoter activity, which is critical for understanding biosynthetic pathways in different plant species, we use agroinfiltration-based transient gene expression assay. We compare the activity of several known promoters in Nicotiana benthamiana with their activity in Cannabis sativa (both hemp and medicinal cannabis), which has attracted much attention in recent years for its industrial, medicinal, and recreational properties. Here we describe an optimized protocol for transient expression in Cannabis combined with a ratiometric GUS reporter system that allows more accurate evaluation of promoter activity and reduces the effects of variable infiltration efficiency.

• Klíčová slova
• Cannabis sativa, GUS, Transient expression, Vacuum infiltration,
• MeSH
• Cannabis * genetika   metabolismus   MeSH
• exprese genu genetika   MeSH
• geneticky modifikované rostliny * genetika   MeSH
• glukuronidasa genetika   metabolismus   MeSH
• promotorové oblasti (genetika) * MeSH
• regulace genové exprese u rostlin * MeSH
• reportérové geny MeSH
• tabák * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glukuronidasa MeSH

We extended the applicability of the BY-2 cell line as a model by introducing two new selection systems. Our protocol provides guidelines for optimising Basta selection in other recalcitrant models. Tobacco BY-2 cell line is the most commonly used cytological model in plant research. It is uniform, can be simply treated by chemicals, synchronised and easily transformed. However, only a few selection systems are available that complicate advanced studies using multiple stacked transgenes and extensive gene editing. In our work, we adopted for BY-2 cell line two other selection systems: sulfadiazine and phosphinothricin (PPT, an active ingredient of Basta herbicide). We show that sulfadiazine can be used in a wide range of concentrations. It is suitable for co-transformation and subsequent double selection with kanamycin or hygromycin, which are standardly used for BY-2 transformation. We also have domesticated the sulfadiazine resistance for the user-friendly GoldenBraid cloning system. Compared to sulfadiazine, establishing selection on phosphinothricin was considerably more challenging. It did not work in any concentration of PPT with standardly cultured cells. Since the selection is based on blocking glutamine synthetase and consequent ammonium toxicity and deficiency of assimilated nitrogen, we tried to manipulate nitrogen availability. We found that the PPT selection reliably works only with nitrogen-starved cells with reduced nitrate reserves that are selected on a medium without ammonium nitrate. Both these adjustments prevent the release of large amounts of ammonium, which can toxify the entire culture in the case of standardly cultured cells. Since high nitrogen reserves can be a common feature of in vitro cultures grown on MS media, nitrogen starvation could be a key step in establishing phosphinothricin resistance in other plant models.

• Klíčová slova
• Bialaphos, Glufosinate ammonium, Selectable markers, Suspension cell lines, Tobacco (Nicotiana tabacum),
• MeSH
• amoniové sloučeniny * MeSH
• dusík MeSH
• geneticky modifikované rostliny genetika   MeSH
• sulfadiazin MeSH
• tabák * genetika   MeSH
• transformace genetická MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• amoniové sloučeniny * MeSH
• dusík MeSH
• phosphinothricin MeSH Prohlížeč
• sulfadiazin MeSH

Methods for simple and fast assembly of exchangeable standard DNA parts using Type II S restriction enzymes are becoming more and more popular in plant synthetic and molecular biology. These methods enable routine construction of large and complex multigene DNA structures. Two available frameworks emphasize either high cloning capacity (Modular Cloning, MoClo) or simplicity (GoldenBraid, GB). Here we present a set of novel α-level plasmids compatible with the GB convention that extend the ability of GB to rapidly assemble more complex genetic constructs, while maintaining compatibility with all existing GB parts as well as most MoClo parts and GB modules. With the use of our new plasmids, standard GB parts can be assembled into complex assemblies containing 1, 5, 10 and up to theoretically 50 units in each successive level of infinite loop assembly. Assembled DNA constructs can be also combined with conventional binary GB-assemblies (1, 2, 4, 8… units). We demonstrate the usefulness of our framework on single tube assembly of replicating plant expression constructs based on the geminivirus Bean yellow dwarf virus (BeYDV).

• Klíčová slova
• Agrobacterium tumefaciens, Nicotiana benthamiana, cloning, plant virus vector, synthetic biology, transient expression,
• Publikační typ
• časopisecké články MeSH

Microtubules of all eukaryotic cells are formed by α- and β-tubulin heterodimers. In addition to the well known cytoplasmic tubulins, a subpopulation of tubulin can occur in the nucleus. So far, the potential function of nuclear tubulin has remained elusive. In this work, we show that α- and β-tubulins of various organisms contain multiple conserved nuclear export sequences, which are potential targets of the Exportin 1/CRM1 pathway. We demonstrate exemplarily that these NES motifs are sufficient to mediate export of GFP as model cargo and that this export can be inhibited by leptomycin B, an inhibitor of the Exportin 1/CRM1 pathway. Likewise, leptomycin B causes accumulation of GFP-tagged tubulin in interphase nuclei, in both plant and animal model cells. Our analysis of nuclear tubulin content supports the hypothesis that an important function of nuclear tubulin export is the exclusion of tubulin from interphase nuclei, after being trapped by nuclear envelope reassembly during telophase.

• MeSH
• aktivní transport - buněčné jádro fyziologie   MeSH
• buněčné jádro metabolismus   MeSH
• buněčné linie MeSH
• cytoplazma metabolismus   MeSH
• eukaryotické buňky metabolismus   MeSH
• karyoferiny metabolismus   MeSH
• lidé MeSH
• mikrotubuly metabolismus   MeSH
• protein exportin 1 MeSH
• receptory cytoplazmatické a nukleární metabolismus   MeSH
• tabák metabolismus   MeSH
• transport proteinů fyziologie   MeSH
• tubulin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• karyoferiny MeSH
• receptory cytoplazmatické a nukleární MeSH
• tubulin MeSH

BACKGROUND: Plant transformation via Agrobacterium tumefaciens is characterized by integration of commonly low number of T-DNAs at random positions in the genome. When integrated into an active gene region, promoterless reporter genes placed near the T-DNA border sequence are frequently transcribed and even translated to reporter proteins, which is the principle of promoter- and gene-trap lines. RESULTS: Here we show that even internal promotorless regions of T-DNAs are often transcribed. Such spontaneous transcription was observed in the majority of independently transformed tobacco BY-2 lines (over 65%) and it could effectively induce silencing if an inverted repeat was present within the T-DNA. We documented that the transcription often occurred in both directions. It was not directly connected with any regulatory elements present within the T-DNAs and at least some of the transcripts were initiated outside of the T-DNA. The likeliness of this read-through transcription seemed to increase in lines with higher T-DNA copy number. Splicing and presence of a polyA tail in the transcripts indicated involvement of Pol II, but surprisingly, the transcription was able to run across two transcription terminators present within the T-DNA. Such pervasive transcription was observed with three different T-DNAs in BY-2 cells and with lower frequency was also detected in Arabidopsis thaliana. CONCLUSIONS: Our results demonstrate unexpected pervasive read-through transcription of T-DNAs. We hypothesize that it was connected with a specific chromatin state of newly integrated DNA, possibly affected by the adjacent genomic region. Although this phenomenon can be easily overlooked, it can have significant consequences when working with highly sensitive systems like RNAi induction using an inverted repeat construct, so it should be generally considered when interpreting results obtained with the transgenic technology.

• Klíčová slova
• GFP, Inverted repeat, Promoterless, RNAi, Read-through transcription, T-DNA, Tobacco BY-2 cell line,
• MeSH
• Agrobacterium tumefaciens genetika   MeSH
• Arabidopsis genetika   MeSH
• buněčné linie MeSH
• DNA bakterií genetika   MeSH
• genetická transkripce * MeSH
• geneticky modifikované rostliny MeSH
• messenger RNA genetika   MeSH
• obrácené repetice genetika   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• reportérové geny MeSH
• tabák genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA bakterií MeSH
• messenger RNA MeSH
• T-DNA MeSH Prohlížeč

The dynamic microtubule cytoskeleton plays fundamental roles in the growth and development of plants including regulation of their responses to environmental stress. Plants exposed to hyper-osmotic stress commonly acclimate, acquiring tolerance to variable stress levels. The underlying cellular mechanisms are largely unknown. Here, we show, for the first time, by in vivo imaging approach that linear patterns of phospholipase Dδ match the localization of microtubules in various biological systems, validating previously predicted connection between phospholipase Dδ and microtubules. Both the microtubule and linear phospholipase Dδ structures were disintegrated in a few minutes after treatment with oryzalin or salt. Moreover, by using immunofluorescence confocal microscopy of the cells in the root elongation zone of Arabidopsis, we have shown that the cortical microtubules rapidly depolymerized within 30 min of treatment with 150 or 200 mM NaCl. Within 5 h of treatment, the density of microtubule arrays was partially restored. A T-DNA insertional mutant lacking phospholipase Dδ showed poor recovery of microtubule arrays following salt exposition. The restoration of microtubules was significantly retarded as well as the rate of root growth, but roots of overexpressor GFP-PLDδ prepared in our lab, have grown slightly better compared to wild-type plants. Our results indicate that phospholipase Dδ is involved in salt stress tolerance, possibly by direct anchoring and stabilization of de novo emerging microtubules to the plasma membrane, providing novel insight into common molecular mechanism during various stress events.

• Klíčová slova
• Arabidopsis roots, BY-2, Microtubule dynamics, Phospholipase Dδ, Salt stress,
• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• fosfolipasa D metabolismus   MeSH
• mikrotubuly metabolismus   MeSH
• tolerance k soli MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfolipasa D MeSH
• phospholipase D delta MeSH Prohlížeč

The proper timing of flowering is essential for the adaptation of plant species to their ever-changing environments. The central position in a complex regulatory network is occupied by the protein FT, which acts as a florigen. We found that light, following a permissive period of darkness, was essential to induce the floral promoter CrFTL1 and to initiate flowering in seedlings of the short-day plant Chenopodium rubrum L. We also identified two novel CONSTANS-like genes in C. rubrum and observed their rhythmic diurnal and circadian expressions. Strong rhythmicity of expression suggested that the two genes might have been involved in the regulation of photoperiod-dependent processes, despite their inability to complement co mutation in A. thaliana. The CrCOL1 and CrCOL2 genes were downregulated by dark-light transition, regardless of the length of a preceding dark period. The same treatment activated the floral promoter CrFTL1. Light therefore affected CrCOL and CrFTL1 in an opposite manner. Both CrCOL genes and CrFTL1 displayed expression patterns unique among short-day plants. Chenopodium rubrum, the subject of classical physiological studies in the past, is emerging as a useful model for the investigation of flowering at the molecular level.

• Klíčová slova
• CONSTANS-like, Chenopodium rubrum, FLOWERING LOCUS T-like, flowering, gene expression, light sensitivity, short-day plant.,
• MeSH
• Arabidopsis MeSH
• Chenopodium genetika   růst a vývoj   fyziologie   MeSH
• florigen metabolismus   MeSH
• fotoperioda MeSH
• květy růst a vývoj   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u rostlin * MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• semenáček růst a vývoj   MeSH
• testy genetické komplementace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• florigen MeSH
• rostlinné proteiny MeSH