Expression of a Functional Recombinant Human Glycogen Debranching Enzyme (hGDE) in N. benthamiana Plants and in Hairy Root Cultures.

BACKGROUND: Glycogen storage disease type III (GSDIII, Cori/Forbes disease) is a metabolic disorder due to the deficiency of the Glycogen Debranching Enzyme (GDE), a large monomeric protein (about 176 kDa) with two distinct enzymatic activities: 4-α-glucantransferase and amylo-α-1,6-glucosidase. Several mutations along the amylo-alpha-1,6-glucosidase,4-alphaglucanotransferase (Agl) gene are associated with loss of enzymatic activity. The unique treatment for GSDIII, at the moment, is based on diet. The potential of plants to manufacture exogenous engineered compounds for pharmaceutical purposes, from small to complex protein molecules such as vaccines, antibodies and other therapeutic/prophylactic entities, was shown by modern biotechnology through "Plant Molecular Farming". OBJECTIVE AND METHODS: In an attempt to develop novel protein-based therapeutics for GSDIII, the Agl gene, encoding for the human GDE (hGDE) was engineered for expression as a histidinetagged GDE protein both in Nicotiana benthamiana plants by a transient expression approach, and in axenic hairy root in vitro cultures (HR) from Lycopersicum esculentum and Beta vulgaris. RESULTS: In both plant-based expression formats, the hGDE protein accumulated in the soluble fraction of extracts. The plant-derived protein was purified by affinity chromatography in native conditions showing glycogen debranching activity. CONCLUSION: These investigations will be useful for the design of a new generation of biopharmaceuticals based on recombinant GDE protein that might represent, in the future, a possible therapeutic option for GSDIII.

Comparative analyses of glutathione system of vacuoles and leucoplasts isolated from the storage parenchyma cells of dormant red beetroots (Beta vulgaris L.).

The role of glutathione in the plant vacuole is still being debated. In the present paper, the redox state of glutathione and the activity of glutathione S-transferase (GST, E 2.5.1.18) in the vacuole compared to those in leucoplast have been studied. Organelles were isolated from dormant red beet (Beta vulgaris L.) taproots. Two generally used approaches have been applied to quantitatively assess the content of glutathione. Initially, levels of glutathione were measured in isolated organelles after labeling with monochlorobimane (MCB) and imaging with the use of confocal laser scanning microscopy. However, there are factors limiting the specificity of this method, because of which the resulting concentrations of vacuolar GSH have been underestimated. Another approach used was HPLC, which allows to simultaneously quantify the reduced glutathione (GSH) and glutathione disulfide (GSSG). The concentration of the total glutathione (GSHt) and GSSG in vacuoles determined with the aid of HPLC-UV was higher in comparison to that in the leucoplasts. The reduction potential (Eh) for the glutathione couple in the vacuoles was more positive (-163 mV), than that in plastids (-282 mV). The relatively rapid increase in fluorescence in the isolated vacuoles and plastids during MCB-labeling has indicated to the contribution of GSTs, since the conjugation of GSH to bimane is catalysed by these enzymes. The GST activity in the vacuoles has been assessed to be quite high compared to that of leucoplasts. The number of isoforms of GSTs also differed markedly in vacuoles and plastids. Collectively, our findings suggest the idea that the glutathione accumulated by central vacuole seems to contribute to the redox processes and to the detoxification, which can take place in this compartment.

Branched Pectic Galactan in Phloem-Sieve-Element Cell Walls: Implications for Cell Mechanics.

A major question in plant biology concerns the specification and functional differentiation of cell types. This is in the context of constraints imposed by networks of cell walls that both adhere cells and contribute to the form and function of developing organs. Here, we report the identification of a glycan epitope that is specific to phloem sieve element cell walls in several systems. A monoclonal antibody, designated LM26, binds to the cell wall of phloem sieve elements in stems of Arabidopsis (Arabidopsis thaliana), Miscanthus x giganteus, and notably sugar beet (Beta vulgaris) roots where phloem identification is an important factor for the study of phloem unloading of Suc. Using microarrays of synthetic oligosaccharides, the LM26 epitope has been identified as a ß-1,6-galactosyl substitution of ß-1,4-galactan requiring more than three backbone residues for optimized recognition. This branched galactan structure has previously been identified in garlic (Allium sativum) bulbs in which the LM26 epitope is widespread throughout most cell walls including those of phloem cells. Garlic bulb cell wall material has been used to confirm the association of the LM26 epitope with cell wall pectic rhamnogalacturonan-I polysaccharides. In the phloem tissues of grass stems, the LM26 epitope has a complementary pattern to that of the LM5 linear ß-1,4-galactan epitope, which is detected only in companion cell walls. Mechanical probing of transverse sections of M x giganteus stems and leaves by atomic force microscopy indicates that phloem sieve element cell walls have a lower indentation modulus (indicative of higher elasticity) than companion cell walls.

Characterization of microRNAs of Beta macrocarpa and their responses to Beet necrotic yellow vein virus infection.

Plant microRNAs (miRNAs) are a class of non-coding RNAs that play important roles in plant development, defense, and symptom development. Here, 547 known miRNAs representing 129 miRNA families, and 282 potential novel miRNAs were identified in Beta macrocarpa using small RNA deep sequencing. A phylogenetic analysis was performed, and 8 Beta lineage-specific miRNAs were identified. Through a differential expression analysis, miRNAs associated with Beet necrotic yellow vein virus (BNYVV) infection were identified and confirmed using a microarray analysis and stem-loop RT-qPCR. In total, 103 known miRNAs representing 38 miRNA families, and 45 potential novel miRNAs were differentially regulated, with at least a two-fold change, in BNYVV-infected plants compared with that of the mock-inoculated control. Targets of these differentially expressed miRNAs were also predicted by degradome sequencing. These differentially expressed miRNAs were involved in hormone biosynthesis and signal transduction pathways, and enhanced axillary bud development and plant defenses. This work is the first to describe miRNAs of the plant genus Beta and may offer a reference for miRNA research in other species in the genus. It provides valuable information on the pathogenicity mechanisms of BNYVV.

[PECULIAR PROPERTIES OF SOME COMPONENTS OF MORPHOLOGICAL STRUCTURE IN A PLANT CELL VACUOLE REVEALED BY CONFOCAL MICROSCOPY].

Results of investigations of peculiar properties related to the structure of plant cell vacuolar membranes are discussed. The study was carried out using confocal microscopy, which allowed us in the process of scanning to identify membrane tubes and vesicules in the preparations of isolated vacuoles. Such membrane tubes were found both inside and outside the vacuoles, and, in the case of scanning intermittently at equal time intervals, transition of vesicles with the membrane tube was observed. Furthermore, scanning of isolated vacuoles was conducted at various distances from the glass substrate. Each time, in the upper area of the isolated vacuole lying on the substrate, we observed a large segment of vacuolar membrane and registered the effect of highly intensive fluorescing of some of membrane segments. The distributions of laurdan fluorescence generalized polarization (GP) values for the vacuolar membrane on the whole and for the intensively fluorescing membrane segments have been obtained. We have found that the microviscosity of the intensively fluorescing membrane segments essentially differs from that of the rest part of the membrane.

Choline but not its derivative betaine blocks slow vacuolar channels in the halophyte Chenopodium quinoa: implications for salinity stress responses.

Activity of tonoplast slow vacuolar (SV, or TPC1) channels has to be under a tight control, to avoid undesirable leak of cations stored in the vacuole. This is particularly important for salt-grown plants, to ensure efficient vacuolar Na(+) sequestration. In this study we show that choline, a cationic precursor of glycine betaine, efficiently blocks SV channels in leaf and root vacuoles of the two chenopods, Chenopodium quinoa (halophyte) and Beta vulgaris (glycophyte). At the same time, betaine and proline, two major cytosolic organic osmolytes, have no significant effect on SV channel activity. Physiological implications of these findings are discussed.

Starch biosynthetic genes and enzymes are expressed and active in the absence of starch accumulation in sugar beet tap-root.

BACKGROUND: Starch is the predominant storage compound in underground plant tissues like roots and tubers. An exception is sugar beet tap-root (Beta vulgaris ssp altissima) which exclusively stores sucrose. The underlying mechanism behind this divergent storage accumulation in sugar beet is currently not fully known. From the general presence of starch in roots and tubers it could be speculated that the lack in sugar beet tap-roots would originate from deficiency in pathways leading to starch. Therefore with emphasis on starch accumulation, we studied tap-roots of sugar beet using parsnip (Pastinaca sativa) as a comparator. RESULTS: Metabolic and structural analyses of sugar beet tap-root confirmed sucrose as the exclusive storage component. No starch granules could be detected in tap-roots of sugar beet or the wild ancestor sea beet (Beta vulgaris ssp. maritima). Analyses of parsnip showed that the main storage component was starch but tap-root tissue was also found to contain significant levels of sugars. Surprisingly, activities of four main starch biosynthetic enzymes, phosphoglucomutase, ADP-glucose pyrophosphorylase, starch synthase and starch branching enzyme, were similar in sugar beet and parsnip tap-roots. Transcriptional analysis confirmed expression of corresponding genes. Additionally, expression of genes involved in starch accumulation such as for plastidial hexose transportation and starch tuning functions could be determined in tap-roots of both plant species. CONCLUSION: Considering underground storage organs, sugar beet tap-root upholds a unique property in exclusively storing sucrose. Lack of starch also in the ancestor sea beet indicates an evolved trait of biological importance.Our findings in this study show that gene expression and enzymatic activity of main starch biosynthetic functions are present in sugar beet tap-root during storage accumulation. In view of this, the complete lack of starch in sugar beet tap-roots is enigmatic.

A protocol for sonication-assisted Agrobacterium rhizogenes-mediated transformation of haploid and diploid sugar beet (Beta vulgaris L.) explants.

Hairy root cultures obtained after Agrobacterium rhizogenes-mediated genetic transformation can serve as a model system for studying plant metabolism and physiology, or can be utilized for the production of secondary metabolites. So far no efficient protocol of hairy root development in sugar beet has been publically released. In this work, two A. rhizogenes strains (A4T and LBA1334) carrying a binary vector pBIN-m-gfp5-ER or pCAMBIA1301 possessing gfp and uidA reporter genes were used to transform petiole explants of haploid and diploid sugar beet genotypes. Five treatment combinations of sonicated-assisted Agrobacterium-mediated transformation were compared. Hairy roots appeared on 0% to 54% of explants depending on the treatment combination used. The highest frequency was achieved when explants of a diploid genotype were sonicated for 15 s in the inoculum containing A. rhizogenes of OD600=0.5 and then co-cultured for three days. Using the same treatment combinations the explants of haploid genotypes developed hairy roots with the frequency ranging from 10% to 36%. Transformation efficiency was independent on the bacterial strain used. The results indicate that haploid sugar beet explants are amenable to transformation using A. rhizogenes, and that the efficiency of that process can be increased by applying short ultrasound treatment.

Modeling hairy root tissue growth in in vitro environments using an agent-based, structured growth model.

An agent-based model for simulating the in vitro growth of Beta vulgaris hairy root cultures is described. The model fitting is based on experimental results and can be used as a virtual experimentator for root networks. It is implemented in the JAVA language and is designed to be easily modified to describe the growth of diverse biological root networks. The basic principles of the model are outlined, with descriptions of all of the relevant algorithms using the ODD protocol, and a case study is presented in which it is used to simulate the development of hairy root cultures of beetroot (Beta vulgaris) in a Petri dish. The model can predict various properties of the developing network, including the total root length, branching point distribution, segment distribution and secondary metabolite accumulation. It thus provides valuable information that can be used when optimizing cultivation parameters (e.g., medium composition) and the cultivation environment (e.g., the cultivation temperature) as well as how constructional parameters change the morphology of the root network. An image recognition solution was used to acquire experimental data that were used when fitting the model and to evaluate the agreement between the simulated results and practical experiments. Overall, the case study simulation closely reproduced experimental results for the cultures grown under equivalent conditions to those assumed in the simulation. A 3D-visualization solution was created to display the simulated results relating to the state of the root network and its environment (e.g., oxygen and nutrient levels).

In vitro dual culture of Polymyxa betae in Agrobacterium rhizogenes transformed sugar beet hairy roots in liquid media.

Polymyxa betae is a soil-borne protist and an obligate parasite of sugar beet that transmits the beet necrotic yellow vein virus. Sugar beet hairy roots, transformed by Agrobacterium rhizogenes, were inoculated with surface-sterilized root fragments infected by P. betae. After 10 wk in a liquid medium, typical structures of P. betae were observed in this in vitro system. This first in vitro culture of P. betae in liquid medium will contribute to a better understanding of this protist's biology through providing a way to conserve and produce purified isolates of the protist.

Stomatal and mesophyll conductances to CO2 are the main limitations to photosynthesis in sugar beet (Beta vulgaris) plants grown with excess zinc.

*The effects of zinc (Zn) toxicity on photosynthesis and respiration were investigated in sugar beet (Beta vulgaris) plants grown hydroponically with 1.2, 100 and 300 microM Zn. *A photosynthesis limitation analysis was used to assess the stomatal, mesophyll, photochemical and biochemical contributions to the reduced photosynthesis observed under Zn toxicity. *The main limitation to photosynthesis was attributable to stomata, with stomatal conductances decreasing by 76% under Zn excess and stomata being unable to respond to physiological and chemical stimuli. The effects of excess Zn on photochemistry were minor. Scanning electron microscopy showed morphological changes in stomata and mesophyll tissue. Stomatal size and density were smaller, and stomatal slits were sealed in plants grown under high Zn. Moreover, the mesophyll conductance to CO(2) decreased by 48% under Zn excess, despite a marked increase in carbonic anhydrase activity. Respiration, including that through both cytochrome and alternative pathways, was doubled by high Zn. *It can be concluded that, in sugar beet plants grown in the presence of excess Zn, photosynthesis is impaired due to a depletion of CO(2) at the Rubisco carboxylation site, as a consequence of major decreases in stomatal and mesophyll conductances to CO(2).

Pollen dispersal in sugar beet production fields.

Pollen-mediated gene flow has important implications for biodiversity conservation and for breeders and farmers' activities. In sugar beet production fields, a few sugar beet bolters can produce pollen as well as be fertilized by wild and weed beet. Since the crop, the wild beets, and the weed beets are the same species and intercross freely, the question of pollen flow is an important issue to determine the potential dispersal of transgenes from field to field and to wild habitats. We report here an experiment to describe pollen dispersal from a small herbicide-resistant sugar beet source towards male sterile target plants located along radiating lines up to 1,200 m away. Individual dispersal functions were inferred from statistical analyses and compared. Pollen limitation, as expected in root-production fields, was confirmed at all the distances from the pollen source. The number of resistant seeds produced by bait plants best fitted a fat-tailed probability distribution curve of pollen grains (power-law) dependent on the distance from the pollen source. A literature survey confirmed that power-law function could fit in most cases. The b coefficient was lower than 2. The number of fertilized flowers by background (herbicide-susceptible) pollen grains was uniform across the whole field. Airborne pollen had a fertilization impact equivalent to that of one adjacent bolter. The individual dispersal function from different pollen sources can be integrated to provide the pollen cloud composition for a given target plant, thus allowing modeling of gene flow in a field, inter-fields in a small region, and also in seed-production area. Long-distance pollen flow is not negligible and could play an important role in rapid transgene dispersal from crop to wild and weed beets in the landscape. The removing of any bolting, herbicide-resistant sugar beet should be compulsory to prevent the occurrence of herbicide-resistant weed beet, thus preventing gene flow to wild populations and preserving the sustainable utility of the resistant varieties. Whether such a goal is attainable remains an open question and certainly would be worth a large scale experimental study.

WtsE, an AvrE-family type III effector protein of Pantoea stewartii subsp. stewartii, causes cell death in non-host plants.

Pantoea stewartii subsp. stewartii (Pnss) causes Stewart's bacterial wilt of sweet corn and leaf blight of maize. The pathogenicity of Pnss depends on synthesis of extracellular polysaccharide and an Hrp type III secretion system. WtsE, a type III secreted effector protein, is essential for the virulence of Pnss on corn. It belongs to the AvrE family of effectors, which includes DspA/E from Erwinia amylovora and AvrE1 from Pseudomonas syringae. Previously, WtsE was shown to cause disease-associated cell death in its host plant, sweet corn. Here, we examine the biological activity of WtsE in several non-host plants. WtsE induced cell death in Nicotiana benthamiana, tobacco, beet and Arabidopsis thaliana when it was transiently produced in plant cells following agroinfiltration or translocated into plant cells from Pnss, Escherichia coli or Pseudomonas syringae pv. phaseolicola (Pph). WtsE-induced cell death in N. benthamiana, tobacco and beet resembled a hypersensitive response and in N. benthamiana it was delayed by cycloheximide. Interestingly, WtsE strongly promoted the growth of Pnss in N. benthamiana prior to the onset of cell death. Deletion derivatives of WtsE that failed to induce cell death in N. benthamiana and tobacco also did not complement wtsE mutants of Pnss for virulence in sweet corn, indicating a correlation between the two activities. WtsE also induced cell death in A. thaliana, where it suppressed basal defences induced by Pph. Thus, WtsE has growth-promoting, defence-suppressing and cell death-inducing activities in non-host plants. Expression of WtsE also prevented the growth of yeast, possibly due to an innate toxicity to eukaryotic cells.

[The growth and differentiation of root cap columella cells and the proper root grown in the stationary conditions and under clinorotation].

The results of light- and electron-microscopic investigations of root apices of Beta vulgaris 3-day-old seedlings grown in the stationary conditions and under clinorotation are presented. It was shown that ultrastructure and topography of organelles in root cap statocytes (graviperceptive cells) and in the cells of distal elongation zone clearly reflected the different direction in their growth and differentiation in space and time in dependence on specialization and functions. Cell growth and genetically determined differentiation occur similarly to control, although certain differences in ultrastructure are evident on metabolism changes.

Arabinogalactan-proteins stimulate the organogenesis of guard cell protoplasts-derived callus in sugar beet.

Arabinogalactan proteins (AGPs) represent a class of proteoglycans implicated in the development and differentiation of cells and tissues both in planta and in vitro. Here we report that AGP-rich extracts isolated from media of embryogenic and non-embryogenic suspension cultures of sugar beet (Beta vulgaris L.) are able to enhance the organogenesis of guard protoplast-derived callus and to increase the number of shoots formed, in comparison to control cultures. Immunocytochemical detection of carbohydrate antigens in the extracts revealed the presence of epitopes that typify both AGP and pectin, the latter being frequently bound to AGPs or, in some cases, even contributing to the polysaccharide structure of proteoglycan molecules. The most abundant epitopes proved to be those recognized by the JIM13, LM2, and MAC207 antibodies, whereas some others could be found only in relatively small or trace amounts--these included epitopes recognized by JIM16, JIM5, and LM6. Surprisingly, the JIM4- and JIM8-binding epitopes that are expressed in the course of in vitro morphogenetic processes of many species could not be detected at all in sugar beet AGPs. This is the first report of the improvement of sugar beet protoplast-derived callus organogenesis by exogenous AGP-rich extracts, an achievement that will have great impact on the biotechnological applications of protoplast technology in this species.

Beta vulgaris L. suspension cultures permeabilized with triton X-100 retain cell viability and betacyanines production ability: a digital image analysis study.

The influence of Triton X-100 on Beta vulgaris L. permeabilized cell culture viability, regrowth, and ability to produce betacyanines was evaluated in this study. A non-destructive method based on the analysis of images in the RGB (red, green, blue) system was developed to estimate betacyanines content. A treatment for 15 min with 0.7 mM Triton X-100 induced the release of 30% of betacyanines without loss of cell viability (>or=70%). After this permeabilization treatment, B. vulgaris cultures regrew normally, reaching a maximum biomass concentration of 48% higher than non-permeabilized cultures after 14 days of culture. Also, maximum betacyanines concentration was only 25% lower than that of non-permeabilized cultures.