Arabinoxylan, ß-glucan and pectin in barley and malt endosperm cell walls: a microstructure study using CLSM and cryo-SEM.

The architecture of endosperm cell walls in Hordeum vulgare (barley) differs remarkably from that of other grass species and is affected by germination or malting. Here, the cell wall microstructure is investigated using (bio)chemical analyses, cryogenic scanning electron microscopy (cryo-SEM) and confocal laser scanning microscopy (CLSM) as the main techniques. The relative proportions of ß-glucan, arabinoxylan and pectin in cell walls were 61, 34 and 5%, respectively. The average thickness of a single endosperm cell wall was 0.30 µm, as estimated by the cryo-SEM analysis of barley seeds, which was reduced to 0.16 µm after malting. After fluorescent staining, 3D confocal multiphoton microscopy (multiphoton CLSM) imaging revealed the complex cell wall architecture. The endosperm cell wall is composed of a structure in which arabinoxylan and pectin are colocalized on the outside, with ß-glucan depositions on the inside. During germination, arabinoxylan and ß-glucan are hydrolysed, but unlike ß-glucan, arabinoxylan remains present in defined cell walls in malt. Integrating the results, an enhanced model for the endosperm cell walls in barley is proposed.

Aluminum Alters the Histology and Pectin Cell Wall Composition of Barley Roots.

Aluminum (Al) is one of the most important crust elements causing reduced plant production in acidic soils. Barley (Hordeum vulgare L.) is considered to be one of the crops that is most sensitive to Al, and the root cell wall is the primary target of Al toxicity. In this study, we evaluate the possible involvement of specific pectic epitopes in the cells of barley roots in response to aluminum exposure. We targeted four different pectic epitopes recognized by LM5, LM6, LM19, and LM20 antibodies using an immunocytochemical approach. Since Al becomes available and toxic to plants in acidic soils, we performed our analyses on barley roots that had been grown in acidic conditions (pH 4.0) with and without Al and in control conditions (pH 6.0). Differences connected with the presence and distribution of the pectic epitopes between the control and Al-treated roots were observed. In the Al-treated roots, pectins with galactan sidechains were detected with a visually lower fluorescence intensity than in the control roots while pectins with arabinan sidechains were abundantly present. Furthermore, esterified homogalacturonans (HGs) were present with a visually higher fluorescence intensity compared to the control, while methyl-esterified HGs were present in a similar amount. Based on the presented results, it was concluded that methyl-esterified HG can be a marker for newly arising cell walls. Additionally, histological changes were detected in the roots grown under Al exposure. Among them, an increase in root diameter, shortening of root cap, and increase in the size of rhizodermal cells and divisions of exodermal and cortex cells were observed. The presented data extend upon the knowledge on the chemical composition of the cell wall of barley root cells under stress conditions. The response of cells to Al can be expressed by the specific distribution of pectins in the cell wall and, thus, enables the knowledge on Al toxicity to be extended by explaining the mechanism by which Al inhibits root elongation.

Barley metallothioneins: MT3 and MT4 are localized in the grain aleurone layer and show differential zinc binding.

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins believed to play a role in cytosolic zinc (Zn) and copper (Cu) homeostasis. However, evidence for the functional properties of MTs has been hampered by methodological problems in the isolation and characterization of the proteins. Here, we document that barley (Hordeum vulgare) MT3 and MT4 proteins exist in planta and that they differ in tissue localization as well as in metal coordination chemistry. Combined transcriptional and histological analyses showed temporal and spatial correlations between transcript levels and protein abundance during grain development. MT3 was present in tissues of both maternal and filial origin throughout grain filling. In contrast, MT4 was confined to the embryo and aleurone layer, where it appeared during tissue specialization and remained until maturity. Using state-of-the-art speciation analysis by size-exclusion chromatography inductively coupled plasma mass spectrometry and electrospray ionization time-of-flight mass spectrometry on recombinant MT3 and MT4, their specificity and capacity for metal ion binding were quantified, showing a strong preferential Zn binding relative to Cu and cadmium (Cd) in MT4, which was not the case for MT3. When complementary DNAs from barley MTs were expressed in Cu- or Cd-sensitive yeast mutants, MT3 provided a much stronger complementation than did MT4. We conclude that MT3 may play a housekeeping role in metal homeostasis, while MT4 may function in Zn storage in developing and mature grains. The localization of MT4 and its discrimination against Cd make it an ideal candidate for future biofortification strategies directed toward increasing food and feed Zn concentrations.

NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley.

Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores.

The use of cyanobacteria as filler in nitrocellulose capillaries improves ultrastructural preservation of immature barley pollen upon high pressure freezing.

The high pressure freezing (HPF) followed by freeze substitution technique has advantages over chemical fixation in the context of preserving sample ultrastructure. However, when HPF is applied to cultured pollen grains, the large intercellular spaces present lead to a poor level of ultrastructure preservation. We report here that the mixing of cyanobacteria with immature barley pollen grains succeeded in greatly reducing the volume of liquid present between the large pollen grains, and so improved the loading of the sample into a nitrocellulose capillary. The use of yeast or cyanobacteria paste to surround the filled capillaries was beneficial in speeding the transfer of heat during the freezing process. This modification of the HPF method resulted in a greatly improved level of ultrastructure preservation.

[Antimitotic activity of new 2,6-dinitroaniline derivatives and their synergistic activity in compositions with graminicides].

Shown antimicrotrubules activity of new 2,6-dinitroaniline compounds. Investigated their ability on apoptotic processes in a plant cell when used as a composition with inhibitors of acetyl-CoA carboxylase.

[Pharmacognostical study on Fructus Hordei germinatus, Fructus Oryzae germinatus and Fructus Setariae germinatus].

OBJECTIVE: To establish the standard for quality control of Fructus Hordei germinatus, Fructus Oryzae germinatus and Fructus Setariae germinatus. METHODS: The digital microscope and infrared spectroscopy were used in the pharmacognostical study. RESULTS: Distinguished differences were found on morphological and microscopical features of these three crude drugs. Whereas, their infrared spectrums were basically all the same. CONCLUSION: The study provides a convenient, effect method for the identification of these three medicinal materials.

Influence of Fe concentration in the medium on multicellular pollen grains and haploid plants induced by mannitol pretreatment in barley (Hordeum vulgare L.).

This study aims to clarify the short- and long-term effects of the iron concentration in the medium on androgenesis induced in barley by isolated microspore culture. The ultrastructural features and pectin composition of the intine wall were studied in the initial stages of androgenesis. The evolution of electron-dense iron deposits on the intine was analysed in multicellular pollen grains obtained by isolated microspore culture performed for 3, 6, and 9 days using various concentrations of FeNa(2) EDTA. Finally, the number of embryo-like structures and green plants obtained by microspore culture using different Fe concentrations was evaluated in order to estimate the optimum concentration for isolated microspore culture.

Apoptosis in developing anthers and the role of ABA in this process during androgenesis in Hordeum vulgare L.

Intra-nucleosomal cleavage of DNA into fragments of about 200 bp was demonstrated to occur in developing anthers, in which microspores had developed into the mid-late to late uni-nucleate stage in situ, i.e. at the verge of mitosis. The same was observed, but to a much larger extent, if these anthers were pre-treated by a hyper-osmotic shock. Pretreatment of anthers before the actual culture of microspores was required for optimal androgenesis of microspores. The use of the TUNEL reaction, which specifically labels 3' ends of DNA breaks, after intra-nucleosomal cleavage of DNA, revealed that DNA fragmentation mainly occurred in the loculus wall cells, tapetum cells and filament cells. TUNEL staining was absent or infrequently observed in the microspores of developing anthers in situ. Electron microscopy studies showed condensed chromatin in nuclei of loculus wall cells in the developing anthers. These observations at the chromatin and DNA level are known characteristics of programmed cell death, also known as apoptosis. Features of apoptosis were infrequently found in microspores from freshly isolated mature anthers. However, most tapetum cells had disappeared in these anthers and the remaining cell structures showed loss of cellular content. The viability of microspores in pre-treated anthers was comparable to those in freshly isolated anthers and almost four times higher than in anthers from control experiments. This observation was correlated with three to four times less microspores showing TUNEL staining and a two times higher level of ABA in the anther plus medium samples than in controls. Addition of ABA to the controls enhanced the viability and lowered the occurrence of apoptosis linked characteristics in the microspores. These data suggest that pre-treatment is effective in stimulating androgenesis because it leads to an increase in ABA levels which protects microspores from dying by apoptosis.

Localization of a myosin-like protein to plasmodesmata.

Myosin has been localized to plasmodesmata in root tissues of Allium cepa, Zea mays and Hordeum vulgare using a polyclonal antibody to animal myosin in both fluorescence and electron microscopy. Labelling was also observed throughout the cytoplasm, mainly associated with the endoplasmic reticulum and plasma membrane. On Western blots, bands of 180 and 110 kDa were consistently labelled in all three species. These bands were also labelled when the blot was incubated in actin prior to staining with antibodies to actin, raising the possibility that either of these proteins (180 kDa or 110 kDa) may be present in plasmodesmata. Pre-treatment of the tissue with 2,3-butanedione monoxime (BDM), an inhibitor of actin-myosin motility, resulted in a strong constriction of the neck region of plasmodesmata. These results indicate that a myosin-like protein may be present in plasmodesmata and may also play a role in the regulation of transport at the neck region.

Purification of highly intact plastids from various heterotrophic plant tissues: analysis of enzymic equipment and precursor dependency for starch biosynthesis.

Starting with a protocol originally developed for the purification of intact plastids from cauliflower buds [Journet and Douce (1985) Plant Physiol. 79, 458-467] we have modified this method to obtain intact heterotrophic plastids from etiolated barley leaves (Hordeum vulgare) and pea (Pisum sativum) and maize (Zea mays) endosperm. Two subsequent centrifugation steps on Percoll gradients were performed, the first as an isopycnic, the second as zonal, centrifugation step in a swing-out rotor. Percoll density and centrifugation time were adjusted for the various tissues. The obtained plastid preparations are characterized by a low degree of contamination with other cellular components and an intactness of at least 90%. In isolated maize endosperm amyloplasts, starch synthesis is driven by exogenously applied hexose phosphates (glucose 6-phosphate and glucose 1-phosphate) rather than by dihydroxyacetone phosphate. The hexose-phosphate-dependent starch synthesis is strictly dependent upon the intactness of the plastids and is increased up to 9-fold when ATP and 3-phosphoglyceric acid are added to the incubation medium. The occurrence of fructose-1,6-bisphosphatase and malate dehydrogenases in some plastid types is discussed in relation to their possible role in starch synthesis.

Precursors of one integral and five lumenal thylakoid proteins are imported by isolated pea and barley thylakoids: optimisation of in vitro assays.

In vitro assays for the import of proteins by isolated pea thylakoids have been refined and optimised with respect to (a) the method of thylakoid preparation, (b) the concentration of thylakoids in the import assay, and (c) the pH and temperature of the import assay. As a result, the 23 kDa and 16 kDa proteins of the photosynthetic oxygen-evolving complex are imported with efficiencies approaching 100%; import of the third oxygen-evolving complex protein is also observed, albeit with lower efficiencies. We have also demonstrated import of three further thylakoid proteins: plastocyanin, the CFoII subunit of the ATP synthase, and the photosystem I subunit, PSI-N, using this import assay. Import of plastocyanin, PSI-N and the 33 kDa oxygen-evolving complex protein subunit requires the presence of stromal extract whereas the other three proteins are efficiently imported in the absence of added soluble proteins. Import into isolated barley thylakoids was achieved under identical assay conditions, although with somewhat lower efficiency than into pea thylakoids.