Analyzing plant signaling phospholipids through 32Pi-labeling and TLC.

Lipidomic analyses through LC-, GC-, and ESI-MS/MS can detect numerous lipid species based on headgroup and fatty acid compositions but usually miss the minor phospholipids involved in cell signaling because of their low chemical abundancy. Due to their high turnover, these signaling lipids are, however, readily picked up by labeling plant material with (32)P-orthophosphate and subsequent analysis of the lipid extracts by thin layer chromatography. Here, protocols are described for suspension-cultured tobacco BY-2 cells, young Arabidopsis seedlings, Vicia faba roots, and Arabidopsis leaf disks, which can easily be modified for other plant species and tissues.

Microtubules are essential for guard-cell function in Vicia and Arabidopsis.

Radially arranged cortical microtubules are a prominent feature of guard cells. Guard cells expressing GFP-tubulin showed consistent changes in the appearance of microtubules when stomata opened or closed. Guard cells showed fewer microtubule structures as stomata closed, whether induced by transfer to darkness, ABA, hydrogen peroxide, or sodium hydrogen carbonate. Guard cells kept in the dark (closed stomata) showed increases in microtubule structures and stomatal aperture on light treatment. GFP-EB1, marking microtubule growing plus ends, showed no change in number of plus ends or velocity of assembly on stomatal closure. Since the number of growing plus ends and the rate of plus-end growth did not change when microtubule structure numbers declined, microtubule instability and/or rearrangement must be responsible for the apparent loss of microtubules. Guard cells with closed stomata showed more cytosolic GFP-fluorescence than those with open stomata as cortical microtubules became disassembled, although with a large net loss in total fluorescence. Microtubule-targeted drugs blocked guard-cell function in Vicia and Arabidopsis. Oryzalin disrupted guard-cell microtubules and prevented stomatal opening and taxol stabilized guard-cell microtubules and delayed stomatal closure. Gas exchange measurements indicated that the transgenes for fluorescent-labeled proteins did not disrupt normal stomatal function. These dynamic changes in guard-cell microtubules combined with our inhibitor studies provide evidence for an active role of microtubules in guard-cell function.

Cytological and molecular characterization of Vicia barbazitae Ten. & Guss.

Vicia barbazitae, a taxon belonging to section Vicia of subgenus Vicia, was recovered and analysed by cytological, karyological and molecular methods with the aim of both proposing a general characterisation of this species and studying the relationships among the species of section Vicia . Phylogenetic relationships among the species of the section Vicia and those of the sections Microcarinae, Wiggersia and Atossa were also analysed. Automated karyotype analysis has been determined after Feulgen's reaction; chromosome banding was performed by sequence-specific fluorochrome staining. Fluorescent chromosome banding showed CMA(+)/DAPI(-) NOR-associated heterochromatin in the satellite pair. Karyomorphological parameters, based on symmetry indices, the dendrogram of linkage distance constructed on 37 chromosome parameters, as well as the molecular data based on internal transcribed spacer sequences provided information about phylogenetic position of this species inside the section Vicia and among the species belonging to the sections Microcarinae, Wiggersia, Atossa and Vicia. From our karyological and molecular results, it emerges that V. barbazitae can be considered a natural member of section Vicia.

Cytology of Vicia species. X. Karyotype evolution and phylogenetic implication in Vicia species of the sections Atossa, Microcarinae, Wiggersia and Vicia.

Automated karyotype analyses and sequence of rDNA spacers have been analysed for the species belonging to sections Atossa, Microcarinae, Wiggersia and Vicia. Karyomorphological parameters, based on Rec, Syi and TF% indices, have been determined and evidenced that, in term of symmetry, the karyotype of Vicia lathyroides was the most asymmetric one. A multivariate analysis using 34 karyological parameters, in addition to the symmetry indices, has been carried out and the corresponding dendrogram of linkage distances showed six different groups. Molecular investigations on the inclusive group in study by employing ITS DNA sequences indicated a different pattern of relationships. The cladistic analysis combining the molecular data set with karyological parameters evidenced that the species of sections Vicia and Atossa join closely to each other in a paraphyletic group, which includes the monophyletic section Wiggersia. Therefore, our karyological and molecular data provide information about the phylogenetic position of the analysed species inside the subgenus Vicia and are discussed in relation to previous results obtained by morphology, isozymes and ribosomal genes analyses.

Cytological characterization of Vicia oroboides Wulfen in Jacq.

Vicia oroboides, a rare taxon belonging to section Atossa of subgenus Vicia, was recovered and analysed by means of cytological and karyological methods with the aim of both characterising this species and integrating our knowledge on phylogeny of subgenus Vicia. Automated karyotype analysis and nuclear DNA content have been determined after Feulgen's reaction; chromosome banding was performed by fluorochrome staining to evidence heterochromatic blocks along the chromosome complement. The chromosome number is in line with the values of the species of section Atossa; the GC- and AT-rich sites were identified by CMA and DAPI staining. Karyomorphological parameters, based on symmetry indices, provide information about the phylogenetic position of this species inside the subgenus Vicia. DNA content is reported for the first time.

Red cabbage anthocyanin extract alleviates copper-induced cytological disturbances in plant meristematic tissue and human lymphocytes.

Red cabbage is a source of health beneficial substances with antioxidant and antigenotoxic properties. HPLC analysis specifying the content of the investigated extract indicated that mainly anthocyanins (ATH) were responsible for its abilities. Cytological research was conducted with two experimental models: plant tissues--meristematic cells of Vicia faba, and animal tissue elements--human lymphocytes. Positive influence of ATH extract on mitotic activity of Vicia cells exposed to Cu(2+) stress, and inhibitory effect of ATH on cytotoxic actions of Cu(2+) on lymphocytes were demonstrated. In all experimental series with ATH application in combinations with Cu(2+), mitotic index (MI) were higher than those obtained for only Cu(2+) stressed tissues. Preincubation in ATH before Cu(2+) stress had the best effect. Similarly, after ATH applications in all tested series decrease in frequency of micronuclei (MN) appearance was noticed in comparison with only Cu(2+) stressed material. In the case of Vicia cells ATH acted effectively even applied after Cu(2+) stress. It suggests that this ATH mixture not only prevents and limits but also heals the cytological injury caused by Cu(2+) stress.

Visualization of symbiotic tissue in intact root nodules of Vicia tetrasperma using GFP-marked Rhizobium leguminosarum bv. viciae.

In rhizobial symbiosis with legume plant hosts, the symbiotic tissue in the root nodules of indeterminate type is localized to the basal part of the nodule where the symbiotic zones contain infected cells (IC) interspersed with uninfected cells (UC) that are devoid of rhizobia. Although IC are easily distinguished in nodule sections using standard histochemical techniques, their observation in intact nodules is hampered by nodule tissue characteristics. Tagging of Rhizobium leguminosarum bv. viciae strain 128C30 with a constitutively expressed gene for green fluorescent protein (nonshifted mutant form cycle3) in combination with the advantages of the tiny nodules formed by Vicia tetrasperma (L.) SCHREB . allowed for vital observation of symbiotic tissue using fluorescence microscopy. Separation of a red-shifted background channel and digital image stacking along z-axis enabled us to construct a nodule image in a classical fluorescence microscopy of nodules exceeding 1 mm in diameter. In parallel, visualization of nodule bacteria inside the symbiotic tissue by confocal microscopy at the excitation wavelength 488 nm clearly distinguished IC/UC pattern in the nodule virtual sections and revealed red-shifted fluorescence of nonrhizobial origin. This signal was located on the periphery of IC and increased with their degradation, thus suggesting accumulation of secondary metabolites, presumably flavonoids. The simultaneous detection of bacteria and secondary metabolites can be used for monitoring changes to intact nodule physiology in the model legumes. The advantage of V. tetrasperma as a suggested laboratory model for pea cross-inoculation group has been demonstrated.

Cytological and molecular characterization of Vicia esdraelonensis Warb. & Eig: a rare taxon.

Vicia esdraelonensis, a rare taxon belonging to section Hypechusa of subgenus Vicia, was recovered and analyzed by cytological, karyological, and molecular methods, with the aim of both characterizing this species and furthering our knowledge of the phylogeny of subgenus Vicia. Automated karyotype analysis, nuclear DNA content, and chromatin organization were determined by the Feulgen reaction, as well as chromosome banding after double staining with 4',6-diamidino-2-phenylindole (DAPI) and chromomycin A3. The chromosome number and the nuclear DNA content were in agreement with the values of the species of section Hypechusa. The GC- and AT-rich preferential sites were determined by chromomycin A3 and DAPI staining. Karyomorphological parameters indicated that V. esdraelonensis is in an intermediate position in the spatial representation of the species of section Hypechusa on the basis of symmetry indices, as well as in the dendrogram of linkage distance constructed on 37 chromosome parameters. Molecular data based on internal transcribed spacer sequences show that V. esdraelonensis can doubtlessly be included in section Hypechusa and document its closeness to V. noeana. A cladistic analysis combining the molecular data set with karyological characters is also reported. Karyological, cytological, and molecular data allow characterization of the V. esdraelonensis genome and provide information about the phylogenetic position of this species within the Hyrcanicae series of section Hypechusa.

Antisense inhibition of the plastidial glucose-6-phosphate/phosphate translocator in Vicia seeds shifts cellular differentiation and promotes protein storage.

The glucose-6-phosphate/phosphate translocator (GPT) acts as an importer of carbon into the plastid. Despite the potential importance of GPT for storage in crop seeds, its regulatory role in biosynthetic pathways that are active during seed development is poorly understood. We have isolated GPT1 from Vicia narbonensis and studied its role in seed development using a transgenic approach based on the seed-specific legumin promoter LeB4. GPT1 is highly expressed in vegetative sink tissues, flowers and young seeds. In the embryo, localized upregulation of GPT1 at the onset of storage coincides with the onset of starch accumulation. Embryos of transgenic plants expressing antisense GPT1 showed a significant reduction (up to 55%) in the specific transport rate of glucose-6-phosphate as determined using proteoliposomes prepared from embryos. Furthermore, amyloplasts developed later and were smaller in size, while the expression of genes encoding plastid-specific translocators and proteins involved in starch biosynthesis was decreased. Metabolite analysis and stable isotope labelling demonstrated that starch biosynthesis was also reduced, although storage protein biosynthesis increased. This metabolic shift was characterized by upregulation of genes related to nitrogen uptake and protein storage, morphological variation of the protein-storing vacuoles, and a crude protein content of mature seeds of transgenics that was up to 30% higher than in wild-type. These findings provide evidence that (1) the prevailing level of GPT1 abundance/activity is rate-limiting for the synthesis of starch in developing seeds, (2) GPT1 exerts a controlling function on assimilate partitioning into storage protein, and (3) GPT1 is essential for the differentiation of embryonic plastids and seed maturation.