Hormonal regulation of gene expression in barley aleurone layers

As part of our investigation of the mode of action of plant hormones in barley (Hordeum vulgare L.) aleurone layers, we have studied the expression of five identified and three unidentified mRNA species in the presence of exogenous gibberellic acid (GA₃) and abscisic acid. Three of the mRNAs are GA₃-inducible, three are suppressed by GA₃, and two are constitutive. The extent of the GA₃ effect differs considerably for both inducible and suppressible mRNAs. For example, a ten-fold higher concentration of GA₃ (10^-8 M) is required for full induction of the high-pl group -amylase mRNA than is required for the low-pI -amylase mRNA (10^-9 M). Temporal regulation of mRNA abundance also varies between the two -amylase isoenzyme groups. The three GA₃-suppressible mRNA species studied, alcohol dehydrogenase (ADH1), a probable amylase and protease inhibitor, and an unidentified barley mRNA species also varied in response to GA₃. The ADH1 mRNA decreased drastically within 8 h of GA₃ treatment, whereas the other two began to decrease in abundance only after 12–16 h of GA₃ treatment. Abscisic-acid treatment counteracted the GA₃ effects for both the inducible and suppressible mRNA species. Comparison of -amylase-mRNA levels and -amylase-synthesis rates showed a strong correlation between the two parameters, the only exception being a lack of -amylase synthesis in the presence of -amylase mRNA at low GA₃ concentrations. Therefore, the expression of -amylase seems to be regulated primarily by its mRNA levels.Abbreviations ABA abscisic acid - ADH1 alcohol dehydrogenase 1 - cDNA copy DNA - GA₃ gibberellic acid - PAPI probable amylase/protease inhibitor     

Expression sites and developmental regulation of genes encoding (1→3,1→4)-β-glucanases in germinated barley

Expression sites of genes encoding (13,14)--glucan 4-glucanohydrolase (EC 3.2.1.73) have been mapped in germinated barley grains (Hordeum vulgare L.) by hybridization histochemistry. A³²P-labelled cDNA (copy DNA) probe was hybridized to cryosections of intact barley grains to localize complementary mRNAs. No mRNA encoding (13,14)--glucanase is detected in ungerminated grain. Expression of (13,14)--glucanase genes is first detected in the scutellum after 1 d and is confined to the epithelial layer. At this stage, no expression is apparent in the aleurone. After 2 d, levels of (13,14)--glucanase mRNA decrease in the scutellar epithelium but increase in the aleurone. In the aleurone layer, induction of (13,14)--glucanase gene expression, as measured by mRNA accumulation, progresses from the proximal to distal end of the grain as a front moving away from, and parallel to, the face of the scutellum.Abbreviations cDNA copy DNA - RNase ribonuclease     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

Inorganic-carbon uptake by a small-celled strain of Stichococcus bacillaris

Air-grown cells of a marine, small-celled (2 m diameter) strain of Stichococcus bacillaris contained appreciable carbonic-anhydrase activity but this was repressed when cells were grown on air enriched with 5% (v/v) CO₂. Assay of carbonic-anhydrase activity using intact cells and cell extracts showed all activity was intracellular in this Stichococcus strain. Measurement of inorganic-carbon-dependent photosynthetic O₂ evolution at pH 5.0, where CO₂ is the predominant form of inorganic carbon, showed that the concentration of inorganic carbon required for half-maximal rate of photosynthetic O₂ evolution [K_0.5(CO₂)] was 4.0 M for both air- and CO₂-grown cells. At pH 8.3 the K_0.5(CO₂) was 0.3 mM for air-grown and 0.6 mM for CO₂-grown cells. Sodium ions did not enhance bicarbonate utilization. Measurement of the internal inorganic-carbon pool (HCO ₃ ^– +CO₂) by the silicone-oil-layer centrifugal filtering technique showed that air- and CO₂-grown cells were able to concentrate inorganic carbon up to 20-fold in relation to the external medium at pH 5.0 but not at pH 8.3. In this alga the high affinity for CO₂ and inorganic-carbon accumulation in CO₂- and air-grown cells results from active CO₂ transport that is not dependent on carbonic-anhydrase activity.Abbreviation Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid     

Regulation of malic-acid metabolism in Crassulacean-acid-metabolism plants in the dark and light: In-vivo evidence from 13C-labeling patterns after 13CO2 fixation

The labeling patterns in malic acid from dark ¹³CO₂ fixation in seven species of succulent plants with Crassulacean acid metabolism were analysed by gas chromatography-mass spectrometry and ¹³C-nuclear magnetic resonance spectrometry. Only singly labeled malic-acid molecules were detected and on the average, after 12–14 h dark ¹³CO₂ fixation the ratio of [4-¹³C] to [1-¹³C] label was 2:1. However the 4-C carboxyl contained from 72 to 50% of the label depending on species and temperature. The ¹³C enrichment of malate and fumarate was similar. These data confirm those of W. Cockburn and A. McAuley (1975, Plant Physiol. 55, 87–89) and indicate fumarase randomization is responsible for movement of label to 1-C malic acid following carboxylation of phosphoenolpyruvate. The extent of randomization may depend on time and on the balance of malic-acid fluxes between mitochondria and vacuoles. The ratio of labeling in 4-C to 1-C of malic acid which accumulated following ¹³CO₂ fixation in the dark did not change during deacidification in the light and no doubly-labeled molecules of malic acid were detected. These results indicate that further fumarase randomization does not occur in the light, and futile cycling of decarboxylation products of [¹³C] malic acid (¹³CO₂ or [1-¹³C]pyruvate) through phosphoenolpyruvate carboxylase does not occur, presumably because malic acid inhibits this enzyme in the light in vivo. Short-term exposure to ¹³CO₂ in the light after deacidification leads to the synthesis of singly and multiply labeled malic acid in these species, as observed by E.W. Ritz et al. (1986, Planta 167, 284–291). In the shortest times, only singly-labeled [4-¹³C]malate was detected but this may be a consequence of the higher intensity and better detection statistics of this ion cluster during mass spectrometry. We conclude that both phosphoenolpyruvate carboxylase (EC 4.1.1.32) and ribulose-1,5-biphosphate carboxylase (EC 4.1.1.39) are active at this time.Abbreviations CAM Crassulacean acid metabolism - GCMS gas chromatography-mass spectrometry - MS mass spectrometry - NMR nuclear magnetic resonance spectrometry - PEP phosphoenolpyruvate - RuBP ribulose 1,5-bisphosphate     

Monensin inhibits the secretion of α-amylase but not polysaccharide slime from seedling tissues ofZea mays

Summary The effect of monensin on polysaccharide slime secretion by root tips of corn (Zea mays) was studied. Various treatment times and ionophore concentrations were tested: none resulted in inhibition of slime secretion. Because monensin changes the pH of the medium, its effect was also monitored in strongly buffered media and at different pH's. Even in such media, monensin did not inhibit slime secretion. We also measured the effect of the drug after a pulse with [³H]fucose or a pulse followed by a chase. The amount of labeled slimed secreted was not altered by the ionophore. However, 10M monensin affected the development of root tips and drastically reduced their growth. We showed that monensin inhibits the secretion of -amylase by the scutellum of the same plantlet. The importance of the nature of the secretory compound in relation to monensin inhibition of its secretion is discussed.Abbrevations Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sul-fonic acid - Mes 2-(N-morpholino)ethane-sulfonic acid     

Characterization of the pyrenoid isolated from unicellular green algaChlamydomonas reinhardtii: Particulate form of RuBisCO protein

Summary The pyrenoid is a protein complex in the chloroplast stroma of eukaryotic algae. After the treatment with mercury chloride, pyrenoids were isolated by sucrose density gradient centrifugation from cell-wall less mutant cells, CW-15, as well as wild type cells, C-9, of unicellular green algaChlamydomonas reinhardtii. Pyrenoids were characterized as a fraction whose protein/chlorophyll ratio was very high, and also examined by Nomarski differential interference microscopy. Most of the components consisted of 55 kDa and 16 kDa polypeptides (11) which were immunologically identified as the large and small subunit of RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) protein, respectively. Some minor polypeptides were also detected. Substantial amount of RuBisCO protein is present as a particulate form in the pyrenoid in addition to the soluble form in algal chloroplast stroma.Abbreviations BPB bromophenol blue - DAB 3,3-diaminobenzidine - DTT dithiothreitol - ELISA enzyme-linked immunosorbent assay - High-CO₂ cells cells grown under air enriched with 4% CO₂ - Low-CO₂ cells cells grown under ordinary air (containing 0.04% CO₂) - NP-40 nonionic detergent (Nonidet) P-40 - PAGE polyacrylamide gel electrophoresis - PAP peroxidase-antiperoxidase conjugate - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SDS sodium dodecylsulfate     

(2'-5')An-dependent endoribonuclease: enzyme levels are regulated by IFN beta, IFN gamma, and cell culture conditions

The levels of a (2'-5')An-dependent endonuclease (RNase L) were determined in extracts prepared from murine L cells and Ehrlich ascites tumor (EAT) cells by measuring specific binding of protein to a labeled derivative of (2'-5')An, (2'-5')A3[32P]pCp. RNase L levels were found to depend both on interferon (IFN) treatment and on cell growth conditions. Treatment of murine L cells and EAT cells with 100-2,000 IRU IFN beta or IFN gamma resulted in a similar 2-4-fold increase in the levels of RNase L when cells were present at low density. The levels of RNase L were also shown to increase 2-3-fold as cells approached saturation density. Serum-starved cells also displayed relatively high levels of RNase L. RNase L levels in cells maintained at high cell density did not change appreciably following treatment with IFN beta or IFN gamma. Regulation of RNase L levels by cell growth conditions as well as by IFN beta or IFN gamma treatment suggests that RNase L may play an important role in regulating the levels of cellular mRNAs as well as acting to degrade viral RNAs.     

Effect of exogenous hormones on the ovulation of primary and secondary oocytes in LT/Sv strain mice

LT/Sv strain mice ovulate both primary and secondary oocytes. These are fertilizable and give rise to digynic triploid and normal diploid conceptuses, respectively. A previous study [Kaufman and Speirs, 1987] had indicated that just over 20% of embryos recovered on the 10th day of gestation from spontaneously ovulating females had a triploid chromosome constitution. This value was considerably lower than might have been expected by extrapolation from earlier studies in which LT/Sv mice had been given exogenous gonadotrophins. In the present study, therefore, cytogenetic analysis of fertilized eggs was performed at the first cleavage mitosis in (1) spontaneously ovulating females mated to F1 hybrid males, and (2) superovulated females mated to similar males. Additional females from group (1) were autopsied on the 10th day of gestation, and the ploidy of embryos isolated at this stage of gestation was determined. Exposure to exogenous gonadotrophins significantly increased the proportion of eggs that were ovulated as primary oocytes (34.4%), compared to the situation observed following spontaneous ovulation (24.4%). All the triploids encountered in both series were of the digynic type and characteristically (for LT/Sv mice) had an oocyte-derived set with 40 chromosomes present, and a sperm-derived set containing 20 chromosomes. Similar numbers of eggs were recovered from spontaneously ovulating females on the 1st and 10th days of gestation, and the incidence of triploidy observed on the 10th day was 22.1%. The influence of exogenous hormones in increasing the “spontaneous” level of triploidy in LT/Sv and in other strains of mice is briefly reviewed.