Search

Abstract 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.

Abstract Background Tuberization in potato (Solanum tuberosum L.) represents a morphogenetic transition of stolon growth to tuber formation, which is under complex environmental and endogenous regulation. In the present work, we studied the regulatory mechanisms and the role of different morphogenetic factors in a newly isolated potato mutant, which exhibited spontaneous tuberization (ST). The ST mutant was characterized in detail at morphological, physiological and biochemical levels. Results Tuberization of the ST mutant grown in the soil was photoperiod-insensitive; predominantly sessile tubers formed directly from axillary buds even under continuous light. Single-node cuttings of the ST mutant cultured in vitro frequently formed tubers or basal tuber-like swellings instead of normal shoots under conditions routinely used for shoot propagation. The tuberization response of ST cuttings under light was dependent on sucrose, the concentration of which had to exceed certain threshold that inversely correlated with irradiance. Gibberellic acid prevented tuberization of ST cuttings, but failed to restore normal shoot phenotype and caused severe malformations. Carbohydrate analysis showed increased levels of both soluble sugars and starch in ST plants, with altered carbohydrate partitioning and metabolism. Comparative proteomic analysis revealed only a few differences between ST- and wild-type plants, primary amongst which seemed to be the absence of an isoform of manganese-stabilizing protein, a key subunit of photosystem II. Conclusion ST mutant exhibits complex developmental and phenotypic modifications, with features that are typical for plants strongly induced to tuberize. These changes are likely to be related to altered regulation of photosynthesis and carbohydrate metabolism rather than impaired transduction of inhibitory gibberellin or photoperiod-based signals. The effect of gibberellins on tuberization of ST mutant suggests that gibberellins inhibit tuberization downstream of the inductive effects of sucrose and other positive factors.

Cadmium is a potent inducer of programmed cell death (PCD) in plants but the morphological changes in cells exposed to cadmium are poorly characterized. Using light and transmission electron microscopy (TEM) we have investigated the changes in ultrastructure of tobacco BY-2 cells treated with 50 µM CdSO4. The cadmium-induced alterations in cell morphology occurred gradually over a period of 3-4 days and the first stages of the response resembled vacuolar type of cell death. The initial formation of numerous small cytoplasmic vacuoles and dilation of endoplasmic reticulum was followed first by fusion of smaller vacuoles with each other and with big vacuoles, and then by the appearance of autophagic vacuoles containing autophagic bodies. The final stages of cell death were accompanied by necrotic features including loss of plasmalemma integrity, shrinkage of the protoplast and unprocessed cellular components. In addition, we observed a gradual degradation of nuclear material. Our results demonstrate that cadmium-induced plant cell death is a slow process featuring elements of vacuolar cell death and terminating with necrosis., (© 2013 Scandinavian Plant Physiology Society.)

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.

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.

The microtubule-associated protein, MAP65, is a member of a family of divergent microtubule-associated proteins from different organisms generally involved in maintaining the integrity of the central spindle in mitosis. The dicotyledon Arabidopsis thaliana and the monocotyledon rice (Oryza sativa) genomes contain 9 and 11 MAP65 genes, respectively. In this work, we show that the majority of these proteins fall into five phylogenetic clades, with the greatest variation between clades being in the C-terminal random coil domain. At least one Arabidopsis and one rice isotype is within each clade, indicating a functional specification for the C terminus. In At MAP65-1, the C-terminal domain is a microtubule binding region (MTB2) harboring the phosphorylation sites that control its activity. The At MAP65 isotypes show differential localization to microtubule arrays and promote microtubule polymerization with variable efficiency in a MTB2-dependent manner. In vivo studies demonstrate that the dynamics of the association and dissociation of different MAP65 isotypes with microtubules can vary up to 10-fold and that this correlates with their ability to promote microtubule polymerization. Our data demonstrate that the C-terminal variable region, MTB2, determines the dynamic properties of individual isotypes and suggest that slower turnover is conditional for more efficient microtubule polymerization.

The character of programmed cell death (PCD) in plants differs in connection with the context, triggering factors and differentiation state of the target cells. To study the interconnections between cell cycle progression and cell death induction, we treated synchronized tobacco BY-2 cells with cadmium ions that represent a general abiotic stressor influencing both dividing and differentiated cells in planta. Cadmium induced massive cell death after application in all stages of the cell cycle; however, both the progression and the forms of the cell death differed pronouncedly. Apoptosis-like PCD induced by cadmium application in the S and G2 was characterized by pronounced internucleosomal DNA fragmentation. In contrast, application of cadmium in M and G1 phases was not accompanied by DNA cleavage, indicating suppression of autolysis and non-programmed character of the death. We interpret these results in the context of the situation in planta, where the induction of apoptosis-like PCD in the S and G2 phase might be connected with a need to preserve genetic integrity of dividing meristematic cells, whereas suppression of PCD response in differentiated cells (situated in G1/G0 phase) might help to avoid death of the whole plant, and thus enable initiation of the recovery and adaptation processes.