Anna Pretova

High-resolution two-dimensional electrophoresis separation of proteins from metal-stressed rice (Oryza sativa L.) leaves: drastic reductions/fragmentation of ribulose-1,5-bisphosphate carboxylase/oxygenase and induction of stress-related proteins.

Employing high‐resolution two‐dimensional electrophoresis (2‐DE), we studied changes in the rice leaf protein patterns, in response to applied heavy and alkaline metals, important environmental pollutants in our surroundings. Drastic changes in 2‐DE protein patterns after treatment with copper, cadmium, and mercury, over control were found, including changes in the morphology of the leaf segments. Changes in the major leaf photosynthetic protein, ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO, both suppression and fragmentation), and induction of proteins are reported. A total of 33 proteins, which were highly reproducible in repeated experiments, were visually identified as changed over the control, and taken for N‐terminal or internal amino acid sequencing. Among these, nine proteins were N‐terminally blocked, and six proteins could not be sequenced. Most of the proteins showed homology to RuBisCO protein, and some to defense/stress‐related proteins, like the pathogenesis related class 5 protein (OsPR5), the probenazole‐inducible protein (referred to as the OsPR10), superoxide dismutase, and the oxygen evolving protein. Results presented here strongly indicate a highly specific action of some of these metals in disturbing the photosynthetic machinery, as evidenced by prominent reductions/fragmentation of the major photosynthetic protein, RuBisCO, and resulting in stress.

Proteomics analysis of flax grown in Chernobyl area suggests limited effect of contaminated environment on seed proteome.

The accident at the Chernobyl Nuclear Power Plant (CNPP) on April 26, 1986 is the most serious nuclear disaster in human history. Surprisingly, while the area proximal to the CNPP remains substantially contaminated with long-lived radioisotopes including (90)Sr and (137)Cs, the local ecosystem has been able to adapt. To evaluate plant adaptation, seeds of a local flax (Linum usitatissimum) variety Kyivskyi were sown in radio-contaminated and control fields of the Chernobyl region. A total protein fraction was isolated from mature seeds, and analyzed using 2-dimensional electrophoresis combined with tandem-mass spectrometry. Interestingly, growth of the plants in the radio-contaminated environment had little effect on proteome and only 35 protein spots differed in abundance (p-value of ≤0.05) out of 720 protein spots that were quantified for seeds harvested from both radio-contaminated and control fields. Of the 35 differentially abundant spots, 28 proteins were identified using state-of-the-art MS(E) method. Based on the observed changes, the proteome of seeds from plants grown in radio-contaminated soil display minor adjustments to multiple signaling pathways.

Flax anther culture: effect of genotype, cold treatment and media

We report on screening of wide range of flax cultivars for androgenic response and on testing of induction conditions for flax (Linum usitatissimum L.) anther culture and plant regeneration. Anthers were cultured on four different media: Mo, N6, MS and N&N supplemented with various combinations of growth regulators. The induction of callus formation from cultured anthers was the highest on N6 (with cultivar PR FGL 77 – 12 %) and N&N media (with cultivar Carolin – 2.8 %), preferentially after cold pretreatment (7days at 8 °C). Shoots were formed on calli derived from the microspores inside the cultured anthers on media N&N and N6 supplemented with 1 mg l−1 zeatin or 1 mg l−1BAP + 1 mg l−1NAA, respectively and elongated on MS medium supplemented with 2 mg l−1 zeatin. The highest number of shoots (120) was observed with cultivar Red Wing. Shoots were rooted on MS medium supplemented with 2 mg l−1IAA. Our experiments resulted in total in 62 % anther response and 155 plants regenerated and transferred into soil.

Morpho-histology and genotype dependence of in vitro morphogenesis in mature embryo cultures of wheat

Cellular totipotency is one of the basic principles of plant biotechnology. Currently, the success of the procedure used to produce transgenic plants is directly proportional to the successful insertion of foreign DNA into the genome of suitable target tissue/cells that are able to regenerate plants. The mature embryo (ME) is increasingly recognized as a valuable explant for developing regenerable cell lines in wheat biotechnology. We have previously developed a regeneration procedure based on fragmented ME in vitro culture. Before we can use this regeneration system as a model for molecular studies of the morphogenic pathway induced in vitro and investigate the functional links between regenerative capacity and transformation receptiveness, some questions need to be answered. Plant regeneration from cultured tissues is genetically controlled. Factors such as age/degree of differentiation and physiological conditions affect the response of explants to culture conditions. Plant regeneration in culture can be achieved through embryogenesis or organogenesis. In this paper, the suitability of ME tissues for tissue culture and the chronological series of morphological data observed at the macroscopic level are documented. Genetic variability at each step of the regeneration process was evaluated through a varietal comparison of several elite wheat cultivars. A detailed histological analysis of the chronological sequence of morphological events during ontogeny was conducted. Compared with cultures of immature zygotic embryos, we found that the embryogenic pathway occurs slightly earlier and is of a different origin in our model. Cytological, physiological, and some biochemical aspects of somatic embryo formation in wheat ME culture are discussed.

Shoots and embryo-like structures regenerated from cultured flax (Linum usitatissimum L.) hypocotyl segments

Summary The effect of growth regulators and culture media on regeneration of flax from hypocotyl explants were tested. Shoot differentiation was strongly influenced by genotype, concentration of TDZ, combination of BAP and NAA. The highest number of shoots was obtained with the fibre cultivar Alex using TDZ (0.25 mg · L -1 ) on MS medium. Embryo-like structures were formed only on 2,4-D (5 mg · L -1 ) pretreated hypocotyl segments of cultivar Szegedi-30 (oil flax). As a consequence of 2,4-D pretreatment, the embryo-like structures had poorly defined or fused cotyledons and no apparent shoot apices. Morphologically not well developed ELS did not convert to plants.

Comparative quantitative proteomic analysis of embryogenic and non-embryogenic calli in maize suggests the role of oxylipins in plant totipotency.

UNLABELLED Totipotency, the ability of somatic plant cell to generate whole plant through somatic embryogenesis, is still not well understood. In this study, maize immature zygotic embryos were used to generate embryogenic (EC) and non-embryogenic (NEC) calli. In order to compare proteomes of EC and NEC, two-dimensional electrophoresis (2-DE) in combination with mass spectrometry was used. This approach resulted into 361 quantified 2-DE spots out of which 44 were found statistically significantly differentially abundant between EC and NEC. Mass spectrometry provided the identity for 23 proteins that were classified into 8 metabolic categories. The most abundant were proteins associated with energy followed by proteins associated with disease and defense. Based on the abundances of identified proteins in this and other studies, working model for plant totipotency was proposed. One aspect of this working model suggests that increased abundances of proteins associated with pyruvate biosynthesis and suppression of embryogenic genes might be responsible for differences between EC and NEC cells. Furthermore we speculate that the increased abundance of lipoxygenase in the NEC cells results in changes in the equilibrium levels of one or more signaling molecules and is at least partly responsible for somatic cell reprogramming during totipotency. BIOLOGICAL SIGNIFICANCE Totipotency, the ability of somatic plant cell to generate whole plant through somatic embryogenesis, is still not well understood. In order to further advance understanding of this biological phenomenon, proteomes of embryogenic and non-embryogenic callus, derived from immature zygotic embryos of inbred maize line A19, were compared using 2-DE based proteomic technology. Based on the abundances of identified proteins in this and other studies, working model for plant totipotency was proposed. One aspect of this working model suggests that increased abundances of proteins associated with pyruvate biosynthesis and suppression of embryogenic genes might be responsible for differences between EC and NEC cells. Furthermore we speculate that the increased abundance of lipoxygenase in the NEC cells results in changes in the equilibrium levels of one or more signaling molecules and is at least partly responsible for somatic cell reprogramming during totipotency. This article is part of a Special Issue entitled: Environmental and structural proteomics.

Somatic Embryogenesis in Solanum tuberosum L. cv. Désirée from Unripe Zygotic Embryos

Summary Somatic embryo formation in Solanum tuberosum L. was studied. Direct and indirect somatic embryos arose from unripe zygotic embryos of cv. Desiree. On B 5 culture medium supplemented with BAP, yeast extract and glutamine, direct formation of mostly trumpet-shaped somatic embryos was observed, but at a low frequency. On media (according to Murashige and Skoog or Monnier) supplemented with dicamba or 2,4-D, callus formation was observed. After frequent subculture of this callus on the same medium a granular embryogenic callus arose. Somatic embryos were formed on hormone free medium. The frequency of indirect formation of somatic embryos was comparatively higher than in direct ones.

Doubled haploid production in Flax (Linum usitatissimum L.).

There is a requirement of haploid and double haploid material and homozygous lines for cell culture studies and breeding in flax. Anther culture is currently the most successful method producing doubled haploid lines in flax. Recently, ovary culture was also described as a good source of doubled haploids. In this review we focus on tissue and plants regeneration using anther culture, and cultivation of ovaries containing unfertilized ovules. The effect of genotype, physiological status of donor plants, donor material pre-treatment and cultivation conditions for flax anthers and ovaries is discussed here. The process of plant regeneration from anther and ovary derived calli is also in the focus of this review. Attention is paid to the ploidy level of regenerated tissue and to the use of molecular markers for determining of gametic origin of flax plants derived from anther and ovary cultures. Finally, some future prospects on the use of doubled haploids in flax biotechnology are outlined here.

Dihaploid Production in Flax by Anther and Ovary Cultures

Abstract The response of flax anther cultures is still very genotype dependent. We screened different flax genotypes (Szegedi 30, Flanders, Carolin, PR FGL 77, Viking and Red Wing) for androgenic response. The highest androgenic response for each genotype was achieved on N&N medium supplemented with 6% sucrose and combination of growth hormones (1 mg l−1 NAA and 1 mg l−1 BAP) after the anthers were cold pretreated at 8°C for 7 days. The highest number of calli and shoots were derived with genotype Red Wing. In case of Red Wing even embryo-like structures were regenerated from the calli. For gynogenesis this is the first report on ovary cultures in flax. The obtained response (number of ovaries producing calli) ranged from 4% in genotype PR FGL 62 to 64% in genotype AC Emerson on N6 medium supplemented with the same sucrose and hormone combination and concentration as for anther cultures. Yellow calli were formed within two to four months and in course of cultivation turned green. Regeneration was realized via shoot formation. Cells in the yellow calli were preferentially diploid (2n) and in green ones tetraploid (4n) and also higher ploidy level was observed (up to 16n) in course of cultivation.

Analysis of expression profiles of selected genes associated with the regenerative property and the receptivity to gene transfer during somatic embryogenesis in Triticum aestivum L.

The physiological, biochemical and molecular mechanisms regulating the initiation of a regenerative pathway remain partially unknown. Efforts to identify the biological features that confer transformation ability, or the tendency of some cells to induce transgene silencing, would help to improve plant genetic engineering. The objective of our study was to monitor the evolution of plant cell competencies in relation to both in vitro tissue culture regeneration and the genetic transformation properties. We used a simple wheat regeneration procedure as an experimental model for studying the regenerative capacity of plant cells and their receptivity to direct gene transfer over the successive steps of the regenerative pathway. Target gene profiling studies and biochemical assays were conducted to follow some of the mechanisms triggered during the somatic-to-embryogenic transition (i.e. dedifferentiation, cell division activation, redifferentiation) and affecting the accessibility of plant cells to receive and stably express the exogenous DNA introduced by bombardment. Our results seem to indicate that the control of cell-cycle (S-phase) and host defense strategies can be crucial determinants of genetic transformation efficiency. The results from studies conducted at macro-, micro- and molecular scales are then integrated into a holistic approach that addresses the question of tissue culture and transgenesis competencies more broadly. Through this multilevel analysis we try to establish functional links between both regenerative capacity and transformation receptiveness, and thereby to provide a more global and integrated vision of both processes, at the core of defense/adaptive mechanisms and survival, between undifferentiated cell proliferation and organization.