Plant roots navigate in the soil environment following the gravity vector. Cell divisions in the meristem and rapid cell growth in the elongation zone propel the root tips through the soil. Actively elongating cells acidify their apoplast to enable cell wall extension by the activity of plasma membrane AHA H+-ATPases. The phytohormone auxin, central regulator of gravitropic response and root development, inhibits root cell growth, likely by rising the pH of the apoplast. However, the role of auxin in the regulation of the apoplastic pH gradient along the root tip is unclear. Here, we show, by using an improved method for visualization and quantification of root surface pH, that the Arabidopsis thaliana root surface pH shows distinct acidic and alkaline zones, which are not primarily determined by the activity of AHA H+-ATPases. Instead, the distinct domain of alkaline pH in the root transition zone is controlled by a rapid auxin response module, consisting of the AUX1 auxin influx carrier, the AFB1 auxin co-receptor, and the CNCG14 calcium channel. We demonstrate that the rapid auxin response pathway is required for an efficient navigation of the root tip.

• Klíčová slova
• A. thaliana, auxin, pH, plant biology, root,
• MeSH
• adenosintrifosfatasy metabolismus   MeSH
• Arabidopsis * metabolismus   MeSH
• kationtové kanály řízené cyklickými nukleotidy metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• kořeny rostlin MeSH
• kyseliny indoloctové metabolismus   MeSH
• proteiny huseníčku * metabolismus   MeSH
• půda MeSH
• regulace genové exprese u rostlin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• AUX1 protein, Arabidopsis MeSH Prohlížeč
• CNGC14 protein, Arabidopsis MeSH Prohlížeč
• kationtové kanály řízené cyklickými nukleotidy MeSH
• kyseliny indoloctové MeSH
• proteiny huseníčku * MeSH
• půda MeSH

Inducible systems for transgene expression activated by a chemical inducer or an inducer of non-plant origin are desirable tools for both basic plant research and biotechnology. Although, the technology has been widely exploited in dicotyledonous model plants such as Arabidopsis, it has not been optimised for use with the monocotyledonous model species, namely rice. We have adapted the dexamethasone-inducible pOp6/LhGR system for rice and the results indicated that it is fast, sensitive and tightly regulated, with high levels of induction that remain stable over several generations. Most importantly, we have shown that the system does not cause negative growth defects in vitro or in soil grown plants. Interestingly in the process of testing, we found that another steroid, triamcinolone acetonide, is a more potent inducer in rice than dexamethasone. We present serious considerations for the construct design to avoid undesirable effects caused by the system in plants, leakiness and possible silencing, as well as simple steps to maximize translation efficiency of a gene of interest. Finally, we compare the performance of the pOp6/LhGR system with other chemically inducible systems tested in rice in terms of the properties of an ideal inducible system.

• Klíčová slova
• Chemically inducible gene expression, Dexamethasone, GVG, Glucocorticoid, Monocots, Promotor, XVE,
• MeSH
• dexamethason metabolismus   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• rostlinné geny MeSH
• rýže (rod) genetika   růst a vývoj   metabolismus   MeSH
• transgeny MeSH
• vývoj rostlin účinky léků   genetika   MeSH
• zemědělské plodiny genetika   růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dexamethason 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.

• MeSH
• aminokyseliny metabolismus   MeSH
• dioxygenasy metabolismus   MeSH
• oxidace-redukce MeSH
• rostlinné proteiny metabolismus   MeSH
• tabák enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny MeSH
• dioxygenasy MeSH
• rostlinné proteiny MeSH

The plant-specific receptor-like cytoplasmic kinases (RLCKs) form a large, poorly characterized family. Members of the RLCK VI_A class of dicots have a unique characteristic: their activity is regulated by Rho-of-plants (ROP) GTPases. The biological function of one of these kinases was investigated using a T-DNA insertion mutant and RNA interference. Loss of RLCK VI_A2 function resulted in restricted cell expansion and seedling growth. Although these phenotypes could be rescued by exogenous gibberellin, the mutant did not exhibit lower levels of active gibberellins nor decreased gibberellin sensitivity. Transcriptome analysis confirmed that gibberellin is not the direct target of the kinase; its absence rather affected the metabolism and signalling of other hormones such as auxin. It is hypothesized that gibberellins and the RLCK VI_A2 kinase act in parallel to regulate cell expansion and plant growth. Gene expression studies also indicated that the kinase might have an overlapping role with the transcription factor circuit (PIF4-BZR1-ARF6) controlling skotomorphogenesis-related hypocotyl/cotyledon elongation. Furthermore, the transcriptomic changes revealed that the loss of RLCK VI_A2 function alters cellular processes that are associated with cell membranes, take place at the cell periphery or in the apoplast, and are related to cellular transport and/or cell wall reorganisation.

• Klíčová slova
• Arabidopsis thaliana, cell expansion, gibberellins, hypocotyl growth, plant hormones, plant size, receptor-like cytoplasmic kinase, skotomorphogenesis, transcriptomic analysis,
• MeSH
• Arabidopsis účinky léků   enzymologie   genetika   růst a vývoj   MeSH
• DNA bakterií genetika   metabolismus   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• geneticky modifikované rostliny MeSH
• gibereliny metabolismus   farmakologie   MeSH
• hypokotyl účinky léků   enzymologie   genetika   růst a vývoj   MeSH
• inzerční mutageneze MeSH
• kotyledon účinky léků   enzymologie   genetika   růst a vývoj   MeSH
• kyseliny indoloctové metabolismus   farmakologie   MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin * MeSH
• regulátory růstu rostlin farmakologie   MeSH
• semenáček účinky léků   enzymologie   genetika   růst a vývoj   MeSH
• stanovení celkové genové exprese MeSH
• transkripční faktory bHLH genetika   metabolismus   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• transkriptom MeSH
• vývojová regulace genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• auxin response factor 6, Arabidopsis MeSH Prohlížeč
• BZR1 protein, Arabidopsis MeSH Prohlížeč
• DNA bakterií MeSH
• DNA vazebné proteiny MeSH
• gibereliny MeSH
• kyseliny indoloctové MeSH
• PIF4 protein, Arabidopsis MeSH Prohlížeč
• protein-serin-threoninkinasy MeSH
• proteiny huseníčku MeSH
• regulátory růstu rostlin MeSH
• T-DNA MeSH Prohlížeč
• transkripční faktory bHLH MeSH
• transkripční faktory MeSH

BACKGROUND: Methylation of cytosines is an evolutionarily conserved epigenetic mark that is essential for the control of chromatin activity in many taxa. It acts mainly repressively, causing transcriptional gene silencing. In plants, de novo DNA methylation is established mainly by RNA-directed DNA-methylation pathway. Even though the protein machinery involved is relatively well-described, the course of the initial phases remains covert. RESULTS: We show the first detailed description of de novo DNA-methylation dynamics. Since prevalent plant model systems do not provide the possibility to collect homogenously responding material in time series with short intervals, we developed a convenient system based on tobacco BY-2 cell lines with inducible production of siRNAs (from an RNA hairpin) guiding the methylation machinery to the CaMV 35S promoter controlling GFP reporter. These lines responded very synchronously, and a high level of promoter-specific siRNAs triggered rapid promoter methylation with the first increase observed already 12 h after the induction. The previous presence of CG methylation in the promoter did not affect the methylation dynamics. The individual cytosine contexts reacted differently. CHH methylation peaked at about 80% in 2 days and then declined, whereas CG and CHG methylation needed more time with CHG reaching practically 100% after 10 days. Spreading of methylation was only minimal outside the target region in accordance with the absence of transitive siRNAs. The low and stable proportion of 24-nt siRNAs suggested that Pol IV was not involved in the initial phases. CONCLUSIONS: Our results show that de novo DNA methylation is a rapid process initiated practically immediately with the appearance of promoter-specific siRNAs and independently of the prior presence of methylcytosines at the target locus. The methylation was precisely targeted, and its dynamics varied depending on the cytosine sequence context. The progressively increasing methylation resulted in a smooth, gradual inhibition of the promoter activity, which was entirely suppressed in 2 days.

• Klíčová slova
• Epigenetics, RNA interference, RdDM, Transcriptional gene silencing, sRNA sequencing,
• MeSH
• Caulimovirus genetika   MeSH
• estradiol farmakologie   MeSH
• malá interferující RNA genetika   metabolismus   MeSH
• metylace DNA * účinky léků   MeSH
• plazmidy genetika   metabolismus   MeSH
• promotorové oblasti (genetika) MeSH
• RNA interference MeSH
• rostlinné buňky metabolismus   MeSH
• tabák cytologie   MeSH
• zelené fluorescenční proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• estradiol MeSH
• malá interferující RNA MeSH
• zelené fluorescenční proteiny 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

Leaf senescence is an essential physiological process in plants that supports the recycling of nitrogen and other nutrients to support the growth of developing organs, including young leaves, seeds, and fruits. Thus, the regulation of senescence is crucial for evolutionary success in wild populations and for increasing yield in crops. Here, we describe the influence of a NAC transcription factor, SlNAP2 (Solanum lycopersicum NAC-like, activated by Apetala3/Pistillata), that controls both leaf senescence and fruit yield in tomato (S. lycopersicum). SlNAP2 expression increases during age-dependent and dark-induced leaf senescence. We demonstrate that SlNAP2 activates SlSAG113 (S. lycopersicum SENESCENCE-ASSOCIATED GENE113), a homolog of Arabidopsis (Arabidopsis thaliana) SAG113, chlorophyll degradation genes such as SlSGR1 (S. lycopersicum senescence-inducible chloroplast stay-green protein 1) and SlPAO (S. lycopersicum pheide a oxygenase), and other downstream targets by directly binding to their promoters, thereby promoting leaf senescence. Furthermore, SlNAP2 directly controls the expression of genes important for abscisic acid (ABA) biosynthesis, S. lycopersicum 9-cis-epoxycarotenoid dioxygenase 1 (SlNCED1); transport, S. lycopersicum ABC transporter G family member 40 (SlABCG40); and degradation, S. lycopersicum ABA 8'-hydroxylase (SlCYP707A2), indicating that SlNAP2 has a complex role in establishing ABA homeostasis during leaf senescence. Inhibiting SlNAP2 expression in transgenic tomato plants impedes leaf senescence but enhances fruit yield and sugar content likely due to prolonged leaf photosynthesis in aging tomato plants. Our data indicate that SlNAP2 has a central role in controlling leaf senescence and fruit yield in tomato.

• MeSH
• dioxygenasy genetika   metabolismus   MeSH
• geneticky modifikované rostliny MeSH
• genový knockdown MeSH
• kyselina abscisová genetika   metabolismus   MeSH
• listy rostlin fyziologie   MeSH
• ovoce genetika   růst a vývoj   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• Solanum lycopersicum genetika   růst a vývoj   MeSH
• systém (enzymů) cytochromů P-450 genetika   metabolismus   MeSH
• tma MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 9-cis-epoxy-carotenoid dioxygenase MeSH Prohlížeč
• abscisic acid 8'-hydroxylase MeSH Prohlížeč
• dioxygenasy MeSH
• kyselina abscisová MeSH
• rostlinné proteiny MeSH
• systém (enzymů) cytochromů P-450 MeSH
• transkripční faktory MeSH

BACKGROUND: Processes of anterograde and retrograde membrane trafficking play an important role in cellular homeostasis and dynamic rearrangements of the plasma membrane (PM) in all eukaryotes. These processes depend on the activity of adenosine ribosylation factors (ARFs), a family of GTP-binding proteins and their guanine exchange factors (GEFs). However, knowledge on the function and specificity of individual ARF-GEFs for individual steps of membrane trafficking pathways is still limited in plants. RESULTS: In this work, treatments with various trafficking inhibitors showed that the endocytosis of FM 4-64 is largely dynamin-dependent and relies on proteins containing endocytic tyrosine-based internalization motif and intact cytoskeleton. Interestingly, brefeldin A (BFA), reported previously as an inhibitor of anterograde membrane trafficking in plants, appeared to be the most potent inhibitor of endocytosis in tobacco. In concert with this finding, we demonstrate that the point mutation in the Sec7 domain of the GNOM-LIKE protein1a (NtGNL1a) confers intracellular trafficking pathway-specific BFA resistance. The internalization of FM 4-64 and trafficking of PIN-FORMED1 (PIN1) auxin efflux carrier in BY-2 tobacco cells were studied to reveal the function of the ARF-GEF NtGNL1a in these. CONCLUSIONS: Altogether, our observations uncovered the role of NtGNL1a in endocytosis, including endocytosis of PM proteins (as PIN1 auxin efflux carrier). Moreover these data emphasize the need of careful evaluation of mode of action of non-native inhibitors in various species. In addition, they demonstrate the potential of tobacco BY-2 cells for selective mapping of ARF-GEF-regulated endomembrane trafficking pathways.

• MeSH
• endocytóza MeSH
• kvartérní amoniové sloučeniny metabolismus   MeSH
• pyridinové sloučeniny metabolismus   MeSH
• rostlinné buňky fyziologie   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• tabák genetika   fyziologie   MeSH
• transport proteinů MeSH
• výměnné faktory guaninnukleotidů genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• FM 4-64 MeSH Prohlížeč
• kvartérní amoniové sloučeniny MeSH
• pyridinové sloučeniny MeSH
• rostlinné proteiny MeSH
• výměnné faktory guaninnukleotidů MeSH