Ca2+ signalling in plant cells: the big network!

Significant advances in Ca2+ and calmodulin signalling in whole plants and individual cells have been recently reported. Particular relevant contributions have been made to the study of the modification of gene expression by osmotic, light and gravity signals and the growth of root hairs and pollen tubes.     

Different effects of an oligonucleotide uptake stimulating protein on leukemic cells in their primitive and differentiated state

The single stranded [³H]oligonucleotide uptake by HL-60 human promyelocyte and K562 human erythroleukemia cells was stimulated 20–45-fold by DUSF (DNA uptake stimulating protein), and this effect was drastically reduced (to 1.6–13×) if the cells were induced to differentiate. The oligonucleotide uptake stimulating effect of DUSF was not altered in HL-60 and K562 cells, if the proliferation of the cells was inhibited by hydroxyurea (HU) treatment. The oligonucleotide uptake by separated granulocytes and mononuclear cells from healthy donors was not stimulated by DUSF, while the uptake of oligonucleotides by myeloid and lymphoid leukemic cells was greatly stimulated (10–15×). The uptake of oligonucleotides by differentiated mononuclear cells of healthy donors could not be stimulated by DUSF, but the oligonucleotide uptake was greatly increased (11×) by DUSF if the cells were subjected to blast transformation.     

H2O2 feeding enables LPMO-assisted cellulose saccharification during simultaneous fermentative production of lactic acid

Simultaneous saccharification and fermentation (SSF) is a well-known strategy for valorization of lignocellulosic biomass. Because the fermentation process typically is anaerobic, oxidative enzymes found in modern commercial cellulase cocktails, such as lytic polysaccharide monooxygenases (LPMOs), may be inhibited, limiting the overall efficiency of the enzymatic saccharification. Recent discoveries, however, have shown that LPMOs are active under anoxic conditions if they are provided with H₂O₂ at low concentrations. In this study, we build on this concept and investigate the potential of using externally added H₂O₂ to sustain oxidative cellulose depolymerization by LPMOs during an SSF process for lactic acid production. The results of bioreactor experiments with 100 g/L cellulose clearly show that continuous addition of small amounts of H₂O₂ (at a rate of 80 µM/h) during SSF enables LPMO activity and improves lactic acid production. While further process optimization is needed, the present proof-of-concept results show that modern LPMO-containing cellulase cocktails such as Cellic CTec2 can be used in SSF setups, without sacrificing the LPMO activity in these cocktails.     

Fungal Elicitor-Mediated Induction of Innate Immunity in Catharanthus roseus Against Leaf Blight Disease Caused by Alternaria alternata

Journal of Plant Growth Regulation - Catharanthus roseus (L.) G. Don has a broad range of medicinal importance besides its ornamental exterior. In the present work, biotic (fungal) elicitor was...     

Somatic embryogenesis and plant regeneration from hypocotyl protoplasts of sunflower (Helianthus annum L.)

A sunflower genotype (Helianthus annuus L. cv. Florom-328) able to regenerate plants from in vitro cultures was identified by screening hybrids and inbred lines. Protoplasts of this genotype were isolated from dark grown hypocotyls and were cultured in droplets of agarose-solidified V-KM medium covered by liquid V-KM supplemented with naphthaleneacetic acid (NAA) and benzylaminopurine (BAP). One week later colonies were subjected to 2,4-dichlorophenoxyaceticacid for a one week period. Further culture in V-KM with reduced concentrations of NAA and BAP resulted in the appearence of somatic embryos. Maturation of embryos was achieved by culture on MS medium supplemented with NAA, BAP, gibberellic acid A₃ and the ethylene inhibitor AgNO₃. Embryos were then transferred onto hormone free MS medium for germination. The frequency of shoot formation in the best case was 9.6 percent of viable colonies (1.3 percent of protoplasts plated). Some of the shoots with roots could be transplanted into soil, others were grafted on hypocotyls of in vivo germinated seedlings. Eighty percent of grafted shoots and over 95 percent of rooted shoots survived. The plants flowered and produced 5 to 10 seeds each. Factors affecting the frequency of embryo formation and plant regeneration are discussed.Abbreviations BAP 6-benzylaminopurine - GA3 gibberellic acid - MES morpholinoethanesulfonic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid - V-KM protoplast culture medium of Binding and Nehls - 2,4D 2,4-dichlorophenoxyacetic acid     

Inactivation of photosynthetic oxygen evolution by UV-B irradiation: A thermoluminescence study

The influence of UV-B irradiation on photosynthetic oxygen evolution by isolated spinach thylakoids has been investigated using thermoluminescence measurements. The thermoluminescence bands arising from the S₂Q_B ^- (B band) and S₂Q_A ^– (Q band) charge recombination disappeared with increasing UV-B irradiation time. In contrast, the C band at 50°C, arising from the recombination of Q_A ^- with an accessory donor of Photosystem II, was transiently enhanced by the UV-B irradiation. The efficiency of DCMU to block Q_A to Q_B electron transfer decreased after irradiation as detected by the incomplete suppression of the B band by DCMU. The flash-induced oscillatory pattern of the B band was modified in the UV-B irradiated samples, indicating a decrease in the number of centers with reduced Q_B. Based on the results of this study, UV-B irradiation is suggested to damage both the donor and acceptor sides of Photosystem II. The damage of the water-oxidizing complex does not affect a specific S-state transition. Instead, charge stabilization is enhanced on an accessory donor. The acceptor-side modifications decrease the affinity of DCMU binding. This effect is assumed to reflect a structural change in the Q_B/DCMU binding site. The preferential loss of dark stable Q_B ^- may be related to the same structural change or could be caused by the specific destruction of reduced quinones by the UV-B light.Abbreviations Chl chlorophyll - DCMU 3-(3,4,-dichlorophenyl)-1,1-dimethylurea - PS II Photosystem II - Q_A first quinone electron acceptor of PS II - Q_B second quinone electron acceptor of PS II - Tyr-D accessory electron donor of PS II - S₀-S₄ charge storage states of the water-oxidizing complex     

Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast-derived cells and transgenic plants in maize

A reproducible and efficient transformation system has been developed for maize that is based on direct DNA uptake into embryogenic protoplasts and regeneration of fertile plants from protoplast-derived transgenic callus tissues. Plasmid DNA, containing the -glucuronidase (GUS) gene, under the control of the doubled enhancer element (the –208 to –46 bp upstream fragment) from CaMV 35S promoter, linked to the truncated (up to –389 bp from ATG) promoter of wheat, -amylase gene was introduced into protoplasts from suspension culture of HE/89 genotype. The constructed transformation vectors carried either the neomycin phosphotransferase (NPTII) or phosphinothricin acetyltransferase (PAT) gene as selective marker. The applied DNA uptake protocol has resulted at least in 10–20 resistant calli, or GUS-expressing colonies after treatment of 10⁶ protoplasts. Vital GUS staining of microcalli has made possible the shoot regeneration from the GUS-stained tissues. 80–90% of kanamycin or PPT resistant calli showed GUS activity, and transgenic plants were regenerated from more than 140 clones. Both Southern hybridization and PCR analysis showed the presence of introduced foreign genes in the genomic DNA of the transformants. The chimeric promoter, composed of a tissue specific monocot promoter, and the viral enhancer element specified similar expression pattern in maize plants, as it was determined by the full CaMV 35S promoter in dicot and other monocot plants. The highest GUS specific activity was found in older leaves with progressively less activity in young leaves, stem and root. Histochemical localization of GUS revealed promoter function in leaf epidermis, mesophyll and vascular bundles, in the cortex and vascular cylinder of the root. In roots, the meristematic tip region and vascular tissues stained intensively. Selected transformants were grown up to maturity, and second-generation seedlings with segregation for GUS activity were obtained after outcrossing. The GUS-expressing segregants carried also the NPTII gene as shown by Southern hybridization.     

Modulation of the effect of glibenclamide on KATP channels by ATP and ADP

The inside-out configuration of the patch-clamp technique was used to study the effect of glibenclamide on the ATP-sensitive K⁺ channel current in isolated guinea-pig ventricular myocytes. The inhibitory effect of glibenclamide was tested in the bath solution containing two different concentrations of ATP (100 M and 200 M). It was found that the effect of the drug on the K_ATP current was stronger in the presence of the higher concentration of ATP. The blocking effect of glibenclamide on the channels was weaker if, in addition to ATP, ADP was applied in the intracellular solution. Similarly, the inhibitory effect of the drug was not pronounced for the channels reactivated by ADP after run-down. As application of the drug in the presence and absence of Mg²⁺ did not show different effects on the channel inhibition, we concluded that the effect of glibenclamide may not depend on the phosphorylation of the channel protein. These results suggest that in addition of the previously described effect of ADP, ATP also has some modulatory effect on inhibition of the K_ATP channel by glibenclamide.     

Stilbene disulfonates block ATP-sensitive K+ channels in guinea pig ventricular myocytes

Effects of stilbene disulfonates on single K_ATP channel currents were investigated in inside-out and outside-out membrane patches from guinea pig ventricular myocytes. All drugs tested, 4,4′-diisothiocyanatostilbene, 2,2′-disulfonic acid (DIDS), 4-acetamido0-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS), and 4,4′-diaminostilbene-2,2′-disulfonic acid (DADS), inhibited the K_ATP channel when they were applied to the intracellular, but not extracellular side of the membrane patch. Inhibitory actions of DIDS and SITS were irreversible, whereas those induced by DNDS and DADS were reversible. K_ATP channel inhibition was concentration dependent with an order of potency of DIDS>SITS ≈ DNDS > DADS; the Hill coefficient was close to unity for each drug. No change in channel conductance was observed during exposure to DIDS or DNDS; however, channel kinetics was altered. Distribution of the open time within bursts and that between bursts could be described by a single exponential relation in the absence and presence of DIDS or DNDS. The time constant of the open time within bursts was not altered, but that between bursts was decreased by DIDS (from 40.0±8.1 to 29.8±6.7 msec, P< 0.05) and by DNDS (from 43.1±9.3 to 31.9±7.1 msec, P<0.05). Distributions of closed time within bursts were also fitted to a single exponential function both in the absence and presence of drugs, while those of the closed time between bursts were fitted to a single exponential function in the absence of drugs, but a double exponential function was required in the presence of drugs. The rates of onset and development of channel inhibition by DIDS and DNDS appeared to be concentration dependent; a longer time was required to reach a new steady-state of channel activity as drug concentration was decreased. Inhibition by DIDS or DNDS was regulated by intracellular pH; inhibition was greater during acidic conditions. For DIDS (0.1 mm), the open probability (P _o) expressed as a fraction of the value before drug application was 42.9±8.3% at pH 7.4 and 8.2±6.6% at pH 6.5 (P<0.01); corresponding values for DNDS (1 mm) were 39.6±17.6 and 8.9 ±5.8%, respectively (P<0.01). From these data, we conclude that stilbene disulfonates block the K_ATP channel by binding to their target site with one-to-one stoichiometry. Similar to glibenclamide, the binding of stilbene disulfonates may reflect interpolation in an “intermediate lipid compartment” between the cytosolic drug and the site of drug action.