Although telomerase (EC 2.7.7.49) is important for genome stability and totipotency of plant cells, the principles of its regulation are not well understood. Therefore, we studied subcellular localization and function of the full-length and truncated variants of the catalytic subunit of Arabidopsis thaliana telomerase, AtTERT, in planta. Our results show that multiple sites in AtTERT may serve as nuclear localization signals, as all the studied individual domains of the AtTERT were targeted to the nucleus and/or the nucleolus. Although the introduced genomic or cDNA AtTERT transgenes display expression at transcript and protein levels, they are not able to fully complement the lack of telomerase functions in tert -/- mutants. The failure to reconstitute telomerase function in planta suggests a more complex telomerase regulation in plant cells than would be expected based on results of similar experiments in mammalian model systems.

• MeSH
• Arabidopsis enzymologie   genetika   MeSH
• buněčné jadérko enzymologie   genetika   MeSH
• buněčné jádro enzymologie   genetika   MeSH
• geneticky modifikované rostliny MeSH
• jaderné lokalizační signály genetika   MeSH
• katalytická doména genetika   MeSH
• listy rostlin genetika   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteosyntéza MeSH
• regulace genové exprese u rostlin MeSH
• sestřih RNA MeSH
• tabák genetika   MeSH
• telomerasa chemie   genetika   metabolismus   MeSH
• terciární struktura proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

MAIN CONCLUSION: In tobacco, three sequence variants of the TERT gene have been described. We revealed unbalanced levels of TERT variant transcripts in vegetative tobacco tissues and enhanced TERT transcription and telomerase activity in reproductive tissues. Telomerase is a ribonucleoprotein complex responsible for the maintenance of telomeres, structures delimiting ends of linear eukaryotic chromosomes. In the Nicotiana tabacum (tobacco) allotetraploid plant, three sequence variants (paralogs) of the gene coding for the telomerase reverse transcriptase subunit (TERT) have been described, two of them derived from the maternal N. sylvestris genome (TERT_Cs, TERT_D) and one originated from the N. tomentosiformis paternal genome (TERT_Ct). In this work, we analyzed the transcription of TERT variants in correlation with telomerase activity in tobacco tissues. High and approximately comparable levels of TERT_Ct and TERT_Cs transcripts were detected in seedlings, roots, flower buds and leaves, while the transcript of the TERT_D variant was markedly underrepresented. Similarly, in N. sylvestris tissues, TERT_Cs transcript significantly predominated. A specific pattern of TERT transcripts was found in samples of tobacco pollen with the TERT_Cs variant clearly dominating particularly at the early stage of pollen development. Detailed analysis of TERT_C variants representation in functionally distinct fractions of pollen transcriptome revealed their prevalence in large ribonucleoprotein particles encompassing translationally silent mRNA; only a minority of TERT_Ct and TERT_Cs transcripts were localized in actively translated polysomes. Histones of the TERT_C chromatin were decorated predominantly with the euchromatin-specific epigenetic modification in both telomerase-positive and telomerase-negative tobacco tissues. We conclude that the existence and transcription pattern of tobacco TERT paralogs represents an interesting phenomenon and our results indicate its functional significance. Nicotiana species have again proved to be appropriate and useful model plants in telomere biology studies.

• MeSH
• buněčné jádro genetika   MeSH
• chromatinová imunoprecipitace MeSH
• euchromatin metabolismus   MeSH
• genetická transkripce MeSH
• genetická variace * MeSH
• histony metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• orgánová specificita genetika   MeSH
• polyribozomy metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• pylová láčka růst a vývoj   MeSH
• regulace genové exprese u rostlin * MeSH
• tabák genetika   MeSH
• telomerasa genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

Oxylipins of the jasmonate family are active as signals in plant responses to biotic and abiotic stresses as well as in development. Jasmonic acid (JA), its precursor cis-12-oxo-phytodienoic acid (OPDA) and the isoleucine conjugate of JA (JA-Ile) are the most prominent members. OPDA and JA-Ile have individual signalling properties in several processes and differ in their pattern of gene expression. JA-Ile, but not OPDA, is perceived by the SCFCOI1-JAZ co-receptor complex. There are, however, numerous processes and genes specifically induced by OPDA. The recently identified OPDA-Ile suggests that OPDA specific responses might be mediated upon formation of OPDA-Ile. Here, we tested OPDA-Ile-induced gene expression in wild type and JA-deficient, JA-insensitive and JA-Ile-deficient mutant background. Tests on putative conversion of OPDA-Ile during treatments revealed only negligible conversion. Expression of two OPDA-inducible genes, GRX480 and ZAT10, by OPDA-Ile could be detected in a JA-independent manner in Arabidopsis seedlings but less in flowering plants. The data suggest a bioactivity in planta of OPDA-Ile.

• MeSH
• Arabidopsis účinky léků   genetika   MeSH
• cyklopentany metabolismus   MeSH
• ekotyp MeSH
• isoleucin analogy a deriváty   metabolismus   MeSH
• listy rostlin účinky léků   metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• nenasycené mastné kyseliny metabolismus   farmakologie   MeSH
• oxylipiny metabolismus   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH

Immunodetection is a powerful tool in functional studies of all organisms. In plants, the gene redundancy and presence of gene families composed of highly homologous members often impedes the unambiguous identification of individual gene products. A family of eight sensor histidine kinases (HKs) mediates the transduction of diverse signals into Arabidopsis thaliana cells, thereby ensuring the initiation of appropriate adaptive responses. Antibodies recognizing specific members of the HK family would be valuable for studying their functions in Arabidopsis and other plant species including important crops. We have focused on developing and applying antibodies against CYTOKININ-INDEPENDENT 1 (CKI1), which encodes a constitutively active membrane-bound sensor HK that regulates the development of female gametophytes and vascular tissue in Arabidopsis. A coding sequence delimiting the C-terminal receiver domain of CKI1 (CKI1(RD)) was expressed in Escherichia coli using the IPTG-inducible expression system and purified to give a highly pure target protein. The purified CKI1(RD) protein was then used as an antigen for anti-CKI1(RD) antibody production. The resulting polyclonal antibodies had a detection limit of 10 ng of target protein at 1:20,000 dilution and were able to specifically distinguish CKI1, both in vitro and in situ, even in a direct comparison with highly homologous members of the same HK family AHK4, CKI2 and ETR1. Finally, anti-CKI1(RD) antibodies were able to selectively bind CKI1-GFP fusion protein in a pull-down assay using crude lysate from an Arabidopsis cell suspension culture. Our results suggest that the receiver domain is a useful target for the functional characterization of sensor HKs in immunological and biochemical studies.

• MeSH
• Arabidopsis cytologie   enzymologie   MeSH
• imunoprecipitace MeSH
• molekulární sekvence - údaje MeSH
• proteinkinasy chemie   imunologie   izolace a purifikace   metabolismus   MeSH
• proteiny huseníčku chemie   imunologie   izolace a purifikace   metabolismus   MeSH
• protilátky imunologie   MeSH
• sekvence aminokyselin MeSH
• signální transdukce MeSH
• specificita protilátek MeSH
• terciární struktura proteinů MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND AND AIMS: Hybrid proline-rich proteins (HyPRPs) represent a large family of putative cell-wall proteins characterized by the presence of a variable N-terminal domain and a conserved C-terminal domain that is related to non-specific lipid transfer proteins. The function of HyPRPs remains unclear, but their widespread occurrence and abundant expression patterns indicate that they may be involved in a basic cellular process. METHODS: To elucidate the cellular function of HyPRPs, we modulated the expression of three HyPRP genes in tobacco (Nicotiana tabacum) BY-2 cell lines and in potato (Solanum tuberosum) plants. KEY RESULTS: In BY-2 lines, over-expression of the three HyPRP genes with different types of N-terminal domains resulted in similar phenotypic changes, namely increased cell elongation, both in suspension culture and on solid media where the over-expression resulted in enhanced calli size. The over-expressing cells showed increased plasmolysis in a hypertonic mannitol solution and accelerated rate of protoplast release, suggesting loosening of the cell walls. In contrast to BY-2 lines, no phenotypic changes were observed in potato plants over-expressing the same or analogous HyPRP genes, presumably due to more complex compensatory mechanisms in planta. CONCLUSIONS: Based on the results from BY-2 lines, we propose that HyPRPs, more specifically their C-terminal domains, represent a novel group of proteins involved in cell expansion.

• MeSH
• buněčná stěna metabolismus   MeSH
• geneticky modifikované rostliny MeSH
• proliferace buněk MeSH
• prolin metabolismus   MeSH
• proteinové domény bohaté na prolin MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné buňky metabolismus   MeSH
• rostlinné geny MeSH
• rostlinné proteiny metabolismus   MeSH
• Solanum tuberosum cytologie   genetika   metabolismus   MeSH
• tabák cytologie   genetika   metabolismus   MeSH
• zvětšování buněk MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In most oxygenic phototrophs, including cyanobacteria, two independent enzymes catalyze the reduction of protochlorophyllide to chlorophyllide, which is the penultimate step in chlorophyll (Chl) biosynthesis. One is light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR) and the second type is dark-operative protochlorophyllide oxidoreductase (DPOR). To clarify the roles of both enzymes, we assessed synthesis and accumulation of Chl-binding proteins in mutants of cyanobacterium Synechocystis PCC 6803 that either completely lack LPOR or possess low levels of the active enzyme due to its ectopic regulatable expression. The LPOR-less mutant grew photoautotrophically in moderate light and contained a maximum of 20 % of the wild-type (WT) Chl level. Both Photosystem II (PSII) and Photosystem I (PSI) were reduced to the same degree. Accumulation of PSII was mostly limited by the synthesis of antennae CP43 and especially CP47 as indicated by the accumulation of reaction center assembly complexes. The phenotype of the LPOR-less mutant was comparable to the strain lacking DPOR that also contained <25 % of the wild-type level of PSII and PSI when cultivated under light-activated heterotrophic growth conditions. However, in the latter case, we detected no reaction center assembly complexes, indicating that synthesis was almost completely inhibited for all Chl-proteins, including the D1 and D2 proteins.

• MeSH
• 2D gelová elektroforéza MeSH
• aktivace enzymů MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• buněčná membrána enzymologie   metabolismus   MeSH
• chlorofyl biosyntéza   genetika   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fenotyp MeSH
• fotosystém I - proteinový komplex genetika   metabolismus   MeSH
• fotosystém II - proteinový komplex genetika   metabolismus   MeSH
• fototrofní procesy MeSH
• oxidoreduktasy působící na CH-CH vazby genetika   metabolismus   MeSH
• protochlorofylid metabolismus   MeSH
• regulace genové exprese enzymů * MeSH
• regulace genové exprese u rostlin MeSH
• světlo MeSH
• Synechocystis enzymologie   genetika   metabolismus   účinky záření   MeSH
• transformace genetická MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

MAIN CONCLUSION: Resistant Lactuca spp. genotypes can efficiently modulate levels of S-nitrosothiols as reactive nitrogen species derived from nitric oxide in their defence mechanism against invading biotrophic pathogens including lettuce downy mildew. S-Nitrosylation belongs to principal signalling pathways of nitric oxide in plant development and stress responses. Protein S-nitrosylation is regulated by S-nitrosoglutathione reductase (GSNOR) as a key catabolic enzyme of S-nitrosoglutathione (GSNO), the major intracellular S-nitrosothiol. GSNOR expression, level and activity were studied in leaves of selected genotypes of lettuce (Lactuca sativa) and wild Lactuca spp. during interactions with biotrophic mildews, Bremia lactucae (lettuce downy mildew), Golovinomyces cichoracearum (lettuce powdery mildew) and non-pathogen Pseudoidium neolycopersici (tomato powdery mildew) during 168 h post inoculation (hpi). GSNOR expression was increased in all genotypes both in the early phase at 6 hpi and later phase at 72 hpi, with a high increase observed in L. sativa UCDM2 responses to all three pathogens. GSNOR protein also showed two-phase increase, with highest changes in L. virosa-B. lactucae and L. sativa cv. UCDM2-G. cichoracearum pathosystems, whereas P. neolycopersici induced GSNOR protein at 72 hpi in all genotypes. Similarly, a general pattern of modulated GSNOR activities in response to biotrophic mildews involves a two-phase increase at 6 and 72 hpi. Lettuce downy mildew infection caused GSNOR activity slightly increased only in resistant L. saligna and L. virosa genotypes; however, all genotypes showed increased GSNOR activity both at 6 and 72 hpi by lettuce powdery mildew. We observed GSNOR-mediated decrease of S-nitrosothiols as a general feature of Lactuca spp. response to mildew infection, which was also confirmed by immunohistochemical detection of GSNOR and GSNO in infected plant tissues. Our results demonstrate that GSNOR is differentially modulated in interactions of susceptible and resistant Lactuca spp. genotypes with fungal mildews and uncover the role of S-nitrosylation in molecular mechanisms of plant responses to biotrophic pathogens.

• MeSH
• aldehydoxidoreduktasy metabolismus   MeSH
• konfokální mikroskopie MeSH
• nemoci rostlin mikrobiologie   MeSH
• odolnost vůči nemocem fyziologie   MeSH
• oomycety patogenita   MeSH
• polymerázová řetězová reakce MeSH
• regulace genové exprese u rostlin MeSH
• S-nitrosothioly metabolismus   MeSH
• salát (hlávkový) enzymologie   fyziologie   MeSH
• western blotting MeSH
• Publikační typ
• časopisecké články MeSH

The nodulin/glutamine synthetase-like protein (NodGS) that we identified proteomically in Arabidopsis thaliana is a fusion protein composed of an N-terminal amidohydrolase domain that shares homology with nodulins and a C-terminal domain of prokaryotic glutamine synthetase type I. The protein is homologous to the FluG protein, a morphogenetic factor in fungi. Although genes encoding NodGS homologues are present in many plant genomes, their products have not yet been characterized. The Arabidopsis NodGS was present in an oligomeric form of ~700-kDa, mainly in the cytosol, and to a lesser extent in the microsomal membrane fraction. The oligomeric NodGS was incorporated into large heterogeneous protein complexes >700 kDa and partially co-immunoprecipitated with γ-tubulin. In situ and in vivo microscopic analyses revealed a NodGS signal in the cytoplasm, with endomembranes, particularly in the perinuclear area. NodGS had no detectable glutamine synthetase activity. Downregulation of NodGS by RNAi resulted in plants with a short main root, reduced meristematic activity and disrupted development of the root cap. Y2H analysis and publicly available microarray data indicated a role for NodGS in biotic stress signalling. We found that flagellin enhanced the expression of the NodGS protein, which was then preferentially localized in the nuclear periphery. Our results point to a role for NodGS in root morphogenesis and microbial elicitation. These data might help in understanding the family of NodGS/FluG-like fusion genes that are widespread in prokaryotes, fungi and plants.

• MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• flagelin genetika   metabolismus   MeSH
• glutaminsynthetasa genetika   metabolismus   fyziologie   MeSH
• kořeny rostlin genetika   růst a vývoj   metabolismus   MeSH
• membránové proteiny genetika   metabolismus   fyziologie   MeSH
• morfogeneze fyziologie   MeSH
• proteiny huseníčku genetika   metabolismus   fyziologie   MeSH
• regulace genové exprese u rostlin MeSH
• regulátory růstu rostlin metabolismus   MeSH
• rostlinné geny MeSH
• rostlinné proteiny genetika   metabolismus   fyziologie   MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

MAIN CONCLUSION: Nepenthes regulates enzyme activities by sensing stimuli from the insect prey. Protein is the best inductor mimicking the presence of an insect prey. Carnivorous plants of the genus Nepenthes have evolved passive pitcher traps for prey capture. In this study, we investigated the ability of chemical signals from a prey (chitin, protein, and ammonium) to induce transcription and synthesis of digestive enzymes in Nepenthes × Mixta. We used real-time PCR and specific antibodies generated against the aspartic proteases nepenthesins, and type III and type IV chitinases to investigate the induction of digestive enzyme synthesis in response to different chemical stimuli from the prey. Transcription of nepenthesins was strongly induced by ammonium, protein and live prey; chitin induced transcription only very slightly. This is in accordance with the amount of released enzyme and proteolytic activity in the digestive fluid. Although transcription of type III chitinase was induced by all investigated stimuli, a significant accumulation of the enzyme in the digestive fluid was found mainly after protein and live prey addition. Protein and live prey were also the best inducers for accumulation of type IV chitinase in the digestive fluid. Although ammonium strongly induced transcription of all investigated genes probably through membrane depolarization, strong acidification of the digestive fluid affected stability and abundance of both chitinases in the digestive fluid. The study showed that the proteins are universal inductors of enzyme activities in carnivorous pitcher plants best mimicking the presence of insect prey. This is not surprising, because proteins are a much valuable source of nitrogen, superior to chitin. Extensive vesicular activity was observed in prey-activated glands.

• MeSH
• Caryophyllales enzymologie   fyziologie   ultrastruktura   MeSH
• chitin metabolismus   MeSH
• chlorid amonný farmakologie   MeSH
• enzymy genetika   metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• masožravci MeSH
• membránové potenciály MeSH
• regulace genové exprese u rostlin * MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• sérový albumin hovězí metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

MAIN CONCLUSION: Chenopodium ficifoliumflowered under long days despite much lower expression ofFLOWERING LOCUS Thomolog than under short days. Frequent duplications of the FLOWERING LOCUS T (FT) gene across various taxonomic lineages resulted in FT paralogs with floral repressor function, whereas others duplicates maintained their floral-promoting role. The FT gene has been confirmed as the inducer of photoperiodic flowering in most angiosperms analyzed to date. We identified all FT homologs in the transcriptome of Chenopodium ficifolium and in the genome of Chenopodium suecicum, which are closely related to diploid progenitors of the tetraploid crop Chenopodium quinoa, and estimated their expression during photoperiodic floral induction. We found that expression of FLOWERING LOCUS T like 1 (FTL1), the ortholog of the sugar beet floral activator BvFT2, correlated with floral induction in C. suecicum and short-day C. ficifolium, but not with floral induction in C. ficifolium with accelerated flowering under long days. This C. ficifolium accession was induced to flowering without the concomitant upregulation of any FT homolog.

• MeSH
• aktivace transkripce MeSH
• Chenopodium genetika   růst a vývoj   MeSH
• fotoperioda MeSH
• květy genetika   růst a vývoj   MeSH
• Magnoliopsida genetika   růst a vývoj   MeSH
• regulace genové exprese u rostlin * MeSH
• upregulace * MeSH
• Publikační typ
• časopisecké články MeSH