Improvement of adventitious root formation in flax using hydrogen peroxide.

Flax (Linum usitatissimum L.) is an important crop for the production of oil and fiber. In vitro manipulations of flax are used for genetic improvement and breeding while improvements in adventitious root formation are important for biotechnological programs focused on regeneration and vegetative propagation of genetically valuable plant material. Additionally, flax hypocotyl segments possess outstanding morphogenetic capacity, thus providing a useful model for the investigation of flax developmental processes. Here, we investigated the crosstalk between hydrogen peroxide and auxin with respect to reprogramming flax hypocotyl cells for root morphogenetic development. Exogenous auxin induced the robust formation of adventitious roots from flax hypocotyl segments while the addition of hydrogen peroxide further enhanced this process. The levels of endogenous auxin (indole-3-acetic acid; IAA) were positively correlated with increased root formation in response to exogenous auxin (1-Naphthaleneacetic acid; NAA). Histochemical staining of the hypocotyl segments revealed that hydrogen peroxide and peroxidase, but not superoxide, were positively correlated with root formation. Measurements of antioxidant enzyme activities showed that endogenous levels of hydrogen peroxide were controlled by peroxidases during root formation from hypocotyl segments. In conclusion, hydrogen peroxide positively affected flax adventitious root formation by regulating the endogenous auxin levels. Consequently, this agent can be applied to increase flax regeneration capacity for biotechnological purposes such as improved plant rooting.

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

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.

Proteomic and biochemical analysis of maize anthers after cold pretreatment and induction of androgenesis reveals an important role of anti-oxidative enzymes.

In stress conditions, microspores and young pollen grains can be switched from their normal pollen development toward an embryogenic pathway via a process called androgenesis. Androgenic embryos can produce completely homozygous, haploid or double-haploid plants. This study aimed to investigate changes in the abundance of protein species during cold pretreatment and subsequent cultivation of maize anthers on induction media using gel-based proteomics. Proteins upregulated on the third day of anther induction were identified and discussed here. Simultaneous microscopic observations revealed that the first division occurred in microspores within this period. Using 2-D electrophoresis combined with MALDI TOF/TOF MS/MS analysis 19 unique proteins were identified and classified into 8 functional groups. Proteins closely associated with metabolism, protein synthesis and cell structure were the most abundant ones. Importantly, ascorbate peroxidase, an enzyme decomposing hydrogen peroxide, was also upregulated. Isozyme analysis of peroxidases validated the proteomic data and showed increased peroxidase activities during androgenic induction. Further, the isozyme pattern of SOD revealed increased activity of the MnSOD, which could provide hydrogen peroxide as a substrate for in vivo peroxidase reactions (including ascorbate peroxidase). Together, these data reveal the role of enzymes controlling oxidative stress during induction of maize androgenesis.

ER disruption and GFP degradation during non-regenerable transformation of flax with Agrobacterium tumefaciens.

Flax is considered as plant species susceptible to Agrobacterium-mediated genetic transformation. In this study, stability of flax transformation by Agrobacterium rhizogenes versus Agrobacterium tumefaciens was tested by using combined selection for antibiotic resistance and visual selection of green fluorescent protein (GFP)-fusion reporter targeted to the endoplasmic reticulum (ER). Transformation with A. rhizogenes was stable for over 2 years, whereas transformation by A. tumefaciens resulted in non-regenerable stable transformation which was restricted solely to transgenic callus and lasted only 6-8 weeks. However, shoots regenerated from this callus appeared to be non-transgenic. Importantly, callus and root cells stably transformed with A. rhizogenes showed typical regular organization and dynamics of ER as visualized by GFP-ER marker. On the other hand, callus cells transformed with A. tumefaciens showed disintegrated ER structure and impaired dynamics which was accompanied with developmental degradation of GFP. Consequently, shoots which regenerated from such callus were all non-transgenic. Possible reasons for this non-regenerable flax transformation by A. tumefaciens are discussed.

Stable transformation of Mesembryanthemum crystallinum (L.) with Agrobacterium rhizogenes harboring the green fluorescent protein targeted to the endoplasmic reticulum.

Stable transformation of Mesembryanthemum crystallinum L. (common ice plant) with a green fluorescent protein (GFP) construct targeted to the endoplasmic reticulum was obtained. Seven and fourteen days after germination seedlings were infected with Agrobacterium rhizogenes strain ARqua1 either by direct coating of the cut radicles with bacteria growing on solid medium or by immersion of the cut surface in bacterial suspension at different optical densities. Both methods of infection resulted in production of GFP-positive roots with a frequency ranging from 6 to 20% according to the age of the explants and the application procedure. The green fluorescing roots displayed the typical hairy root phenotype and were easily maintained in liquid medium without growth regulators for over 2 years. Stable expression of the transgene in the roots was confirmed by polymerase chain reaction (PCR), immunoblotting and the capacity of roots to grow and produce callus on kanamycin-enriched medium. Nineteen endogenous cytokinins were determined in transgenic and non-transformed roots. The results revealed significantly lower levels of the free bases of isopentenyladenine, dihydrozeatin, cis- and trans-zeatin, as well as a conspicuous decline in concentrations of the corresponding nucleosides and most nucleotides in transgenic roots compared to the wild type. Comparison of the cytokinin profiles in transgenic and non-transformed roots suggested that transformation by A. rhizogenes disturbed cytokinin metabolism during the early steps of biosynthesis. Calli obtained from transformed roots were GFP-positive and remained non-regenerative or displayed high rhizogenic potential depending on the auxin/cytokinin ratio in the medium. Calli and callus-derived roots showed a strong GFP signal for over 2 years.

Using proteomics to study sexual reproduction in angiosperms.

While a relative latecomer to the postgenomics era of functional biology, the application of mass spectrometry-based proteomic analysis has increased exponentially over the past 10 years. Some of this increase is the result of transition of chemists, physicists, and mathematicians to the study of biology, and some is due to improved methods, increased instrument sensitivity, and better techniques of bioinformatics-based data analysis. Proteomic Biological processes are typically studied in isolation, and seldom are efforts made to coordinate results obtained using structural, biochemical, and molecular-genetic strategies. Mass spectrometry-based proteomic analysis can serve as a platform to bridge these disparate results and to additionally incorporate both temporal and anatomical considerations. Recently, proteomic analyses have transcended their initial purely descriptive applications and are being employed extensively in studies of posttranslational protein modifications, protein interactions, and control of metabolic networks. Herein, we provide a brief introduction to sample preparation, comparison of gel-based versus gel-free methods, and explanation of data analysis emphasizing plant reproductive applications. We critically review the results from the relatively small number of extant proteomics-based analyses of angiosperm reproduction, from flowers to seedlings, and speculate on the utility of this strategy for future developments and directions.

Proteomics on brefeldin A-treated Arabidopsis roots reveals profilin 2 as a new protein involved in the cross-talk between vesicular trafficking and the actin cytoskeleton.

The growing importance of vesicular trafficking and cytoskeleton dynamic reorganization during plant development requires the exploitation of novel experimental approaches. Several genetic and cell biological studies have used diverse pharmaceutical drugs that inhibit vesicular trafficking and secretion to study these phenomena. Here, proteomic and cell biology approaches were applied to study effects of brefeldin A (BFA), an inhibitor of vesicle recycling and secretion, in Arabidopsis roots. The main aim of this study was to obtain an overview of proteins affected by BFA, but especially to identify new proteins involved in the vesicular trafficking and its cross-talk to the actin cytoskeleton. The results showed that BFA altered vesicular trafficking and caused the formation of BFA-compartments which was accompanied by differential expression of several proteins in root cells. Some of the BFA-up-regulated proteins belong to the class of the vesicular trafficking proteins, such as V-ATPase and reversibly glycosylated polypeptide, while others, such as profilin 2 and elongation factor 1 alpha, are rather involved in the remodeling of the actin cytoskeleton. Upregulation of profilin 2 by BFA was verified by immunoblot and live imaging at subcellular level. The latter approach also revealed that profilin 2 accumulated in BFA-compartments which was accompanied by remodeling of the actin cytoskeleton in BFA-treated root cells. Thus, profilin 2 seems to be involved in the cross-talk between vesicular trafficking and the actin cytoskeleton, in a BFA-dependent manner.

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.

Calreticulin mRNA and protein are localized to protein bodies in storage maize callus cells.

Maize callus cells possess numerous protein bodies which develop as sub-compartments of the endoplasmic reticulum. We localized maize calreticulin mRNAs and protein in maize callus cells using in situ hybridization and immunocytochemistry. Calreticulin mRNAs were selectively targeted to the endoplasmic reticulum (ER) subdomains surrounding protein bodies. Profilin mRNAs, used as a positive control for in situ hybridization experiments, showed distinct and rather diffuse localization pattern. Using both, immunofluorescence and immunogold electron microscopy localization techniques, calreticulin was found to be enriched around and within protein bodies in maize callus storage cells. As a positive control for reticuloplasmins, HDEL antibody revealed labelling of protein bodies and of the nuclear envelope. The identity of protein bodies was confirmed by specific binding of an alpha zein antibody. These data suggest that calreticulin mRNA is targeted towards protein body forming subdomains of the ER, and that calreticulin is localized and enriched in these protein bodies. The possibility that calreticulin plays an important role in zein retention within the ER and/or its assembly and packaging into protein bodies during protein body biogenesis in maize callus is discussed.

Genetic transformation of barley microspores using anther bombardment.

Bombardment of intact anthers of commercial barley (Hordeum vulgare) varieties resulted in 0.5-1.0% of transformed microspores of which 20-40% continued in androgenic development (0.2% of all bombarded microspores). Using a system based on bombardment of anthers is therefore likely to be more technically efficient than the use of a microspore isolation, transformation and regeneration system. Bombardment of anthers has a number of technical and scientific advantages over existing systems for gene transfer and can be considered as a alternative method to existing methods for genetic transformation in barley.

Moderation of morphogenetic and oxidative stress responses in flax in vitro cultures by hydroxynonenal and desferrioxamine.

Hypocotyl segments of 7-day old seedlings of flax (Linum usitatissimum L.) cultivars Atalante, Flanders, Jitka, Szegedi 30 and Super were screened for organogenesis (shoot and root induction) and embryo-like structure production. A non-destructive assay for hydroxyl radicals (*OH), utilising DMSO as a radical trap, was used to determine *OH formation during tissue culture and morphogenesis. Desferrioxamine, an inhibitor of Fenton reaction, and 4-hydroxy-2-nonenal, a cytotoxic Lipid peroxidation product, were exogenously applied to flax cultures to determine the effect of antioxidative and prooxidative status on morphogenetic responses induced through the exogenous application of plant growth regulators. Flax genotypes varied in their response to treatments after exposure to different plant hormones. Hydroxyl radical (*OH) formation correlated with morphogenetic responses and this was affected by plant hormones. Desferrioxamine and 4-hydroxy-2-nonenal also moderated morphogenetic responses and influenced hydroxyl radical formation during in vitro propagation.