The heterooctameric vesicle-tethering complex exocyst is important for plant development, growth, and immunity. Multiple paralogs exist for most subunits of this complex; especially the membrane-interacting subunit EXO70 underwent extensive amplification in land plants, suggesting functional specialization. Despite this specialization, most Arabidopsis exo70 mutants are viable and free of developmental defects, probably as a consequence of redundancy among isoforms. Our in silico data-mining and modeling analysis, corroborated by transcriptomic experiments, pinpointed several EXO70 paralogs to be involved in plant biotic interactions. We therefore tested corresponding single and selected double mutant combinations (for paralogs EXO70A1, B1, B2, H1, E1, and F1) in their two biologically distinct responses to Pseudomonas syringae, root hair growth stimulation and general plant susceptibility. A shift in defense responses toward either increased or decreased sensitivity was found in several double mutants compared to wild type plants or corresponding single mutants, strongly indicating both additive and compensatory effects of exo70 mutations. In addition, our experiments confirm the lipid-binding capacity of selected EXO70s, however, without the clear relatedness to predicted C-terminal lipid-binding motifs. Our analysis uncovers that there is less of functional redundancy among isoforms than we could suppose from whole sequence phylogeny and that even paralogs with overlapping expression pattern and similar membrane-binding capacity appear to have exclusive roles in plant development and biotic interactions.

• Klíčová slova
• Arabidopsis thaliana, EXO70, biotic stress, exocyst, gene expression, lipid binding, redundancy, root hairs,
• Publikační typ
• časopisecké články MeSH

Plant cell growth and morphogenesis depend on remodelling of both actin and microtubule cytoskeletons. AtFH1 (At5g25500), the main housekeeping Arabidopsis formin, is targeted to membranes and known to nucleate and bundle actin. The effect of mutations in AtFH1 on root development and cytoskeletal dynamics was examined. Consistent with primarily actin-related formin function, fh1 mutants showed increased sensitivity to the actin polymerization inhibitor latrunculin B (LatB). LatB-treated mutants had thicker, shorter roots than wild-type plants. Reduced cell elongation and morphological abnormalities were observed in both trichoblasts and atrichoblasts. Fluorescently tagged cytoskeletal markers were used to follow cytoskeletal dynamics in wild-type and mutant plants using confocal microscopy and VAEM (variable-angle epifluorescence microscopy). Mutants exhibited more abundant but less dynamic F-actin bundles and more dynamic microtubules than wild-type seedlings. Treatment of wild-type seedlings with a formin inhibitor, SMIFH2, mimicked the root growth and cell expansion phenotypes and cytoskeletal structure alterations observed in fh1 mutants. The results suggest that besides direct effects on actin organization, the in vivo role of AtFH1 also includes modulation of microtubule dynamics, possibly mediated by actin-microtubule cross-talk.

• MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• forminy MeSH
• kořeny rostlin genetika   růst a vývoj   metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• mikrofilamenta genetika   metabolismus   MeSH
• mikrotubuly genetika   metabolismus   MeSH
• mutace * MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AFH1 protein, Arabidopsis MeSH Prohlížeč
• forminy MeSH
• membránové proteiny MeSH
• proteiny huseníčku 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
• Názvy látek
• prolin MeSH
• rostlinné proteiny MeSH

Activated cortical domains (ACDs) are regions of the plant cell cortex performing localized membrane turnover, delimited by concerted action of the cortical cytoskeleton and endomembrane compartments. Arabidopsis thaliana rhizodermis consists of two cell types differing by a single ACD (trichoblasts, carrying tip-growing root hairs, and hairless atrichoblasts), providing a model for the study of ACD determination. We compiled a set of genes specifically upregulated in root hairs from published transcriptome data, and compared it with a "virtual Arabidopsis root hair proteome", i.e. a list of computationally identified homologs of proteins from the published soybean root hair proteome. Both data sets were enriched in genes and proteins associated with root hairs in functional studies, but there was little overlap between the transcriptome and the proteome: the former captured gene products specific to root hairs, while the latter selected those abundant in root hairs but not necessarily specific to them. Decisive steps in ACD specification may be performed by signaling proteins of high expression specifity and low abundance. Nevertheless, 73 genes specifically transcribed in Arabidopsis trichoblasts or root hairs encode homologs of abundant root hair proteins from soybean. Most of them encode "housekeeping" proteins required for rapid tip growth. However, among the "candidates" is also a generative actin isoform, ACT11. Preliminary characterization of an act11 mutant allele indeed suggests a hitherto unexpected role for this gene in root and root hair development.

• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• fenotyp MeSH
• kořeny rostlin genetika   metabolismus   MeSH
• mutace MeSH
• proteiny huseníčku metabolismus   MeSH
• pyl metabolismus   MeSH
• regulace genové exprese u rostlin fyziologie   MeSH
• stanovení celkové genové exprese * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny huseníčku MeSH

BACKGROUND AND AIMS: Transgenic plants represent an excellent tool for experimental plant biology and are an important component of modern agriculture. Fully understanding the stability of transgene expression is critical in this regard. Most changes in transgene expression occur soon after transformation and thus unwanted lines can be discarded easily; however, transgenes can be silenced long after their integration. METHODS: To study the long-term changes in transgene expression in potato (Solanum tuberosum), the activity of two reporter genes, encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII), was monitored in a set of 17 transgenic lines over 5 years of vegetative propagation in vitro. KEY RESULTS: A decrease in transgene expression was observed mainly in lines with higher initial GFP expression and a greater number of T-DNA insertions. Complete silencing of the reporter genes was observed in four lines (nearly 25 %), all of which successively silenced the two reporter genes, indicating an interconnection between their silencing. The loss of GFP fluorescence always preceded the loss of kanamycin resistance. Treatment with the demethylation drug 5-azacytidine indicated that silencing of the NPTII gene, but probably not of GFP, occurred directly at the transcriptional level. Successive silencing of the two reporter genes was also reproduced in lines with reactivated expression of previously silenced transgenes. CONCLUSIONS: We suggest a hypothetical mechanism involving the successive silencing of the two reporter genes that involves the switch of GFP silencing from the post-transcriptional to transcriptional level and subsequent spreading of methylation to the NPTII gene.

• MeSH
• geneticky modifikované rostliny genetika   růst a vývoj   MeSH
• kanamycinkinasa genetika   MeSH
• metylace DNA MeSH
• regulace genové exprese u rostlin genetika   MeSH
• Solanum tuberosum genetika   růst a vývoj   MeSH
• transgeny genetika   MeSH
• umlčování genů fyziologie   MeSH
• zelené fluorescenční proteiny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kanamycinkinasa MeSH
• zelené fluorescenční proteiny MeSH

BACKGROUND: Somatic embryogenesis in spruce is a process of high importance for biotechnology, yet it comprises of orchestrated series of events whose cellular and molecular details are not well understood. In this study, we examined the role of actin cytoskeleton during somatic embryogenesis in Norway spruce line AFO 541 by means of anti-actin drugs. RESULTS: Application of low doses (50-100 nM) of latrunculin B (Lat B) during the maturation of somatic embryos predominantly killed suspensor cells while leaving the cells in meristematic centres alive, indicating differential sensitivity of actin in the two cell types. The treatment resulted in faster development of more advanced embryos into mature somatic embryos and elimination of insufficiently developed ones. In searching for the cause of the differential actin sensitivity of the two cell types, we analysed the composition of actin isoforms in the culture and isolated four spruce actin genes. Analysis of their expression during embryo maturation revealed that one actin isoform was expressed constitutively in both cell types, whereas three actin isoforms were expressed predominantly in suspensor cells and their expression declined during the maturation. The expression decline was greatly enhanced by Lat B treatment. Sequence analysis revealed amino-acid substitutions in the Lat B-binding site in one of the suspensor-specific actin isoforms, which may result in a different binding affinity for Lat B. CONCLUSIONS: We show that manipulating actin in specific cell types in somatic embryos using Lat B treatment accelerated and even synchronized the development of somatic embryos and may be of practical use in biotechnology.

• MeSH
• aktiny antagonisté a inhibitory   metabolismus   MeSH
• bicyklické sloučeniny heterocyklické farmakologie   MeSH
• cytoskelet účinky léků   MeSH
• embryonální vývoj MeSH
• fylogeneze MeSH
• protein - isoformy metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• RNA rostlin genetika   MeSH
• rostlinné geny MeSH
• sekvenční seřazení MeSH
• smrk embryologie   růst a vývoj   MeSH
• substituce aminokyselin MeSH
• techniky tkáňových kultur MeSH
• thiazolidiny farmakologie   MeSH
• vazebná místa MeSH
• vývojová regulace genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• bicyklické sloučeniny heterocyklické MeSH
• latrunculin B MeSH Prohlížeč
• protein - isoformy MeSH
• RNA rostlin MeSH
• thiazolidiny MeSH