Visualization of Membrane Calcium and Calmodulin in Embryo Sacs in situ and Isolated from Petunia hybrida L. and Nicotiana tabacum L.

We have used chlorotetracycline (CTC) and fluphenazine (FPZ)as fluorescent probes to visualize the distributional patternsof membrane calcium (mCa²⁺) and the Ca²⁺-receptor protein calmodulin(CaM) in various cell types of unfixed living isolated and unisolatedembryo sacs of Petunia hydrida L. and Nicotiana tabacum L. Ourresults indicate that in the young embryo sacs of Petunia, bothsynergids and the central cell sequester relatively higher amountsof mCa²⁺ and CaM than the egg cell and the antipodals. Much of the mCa²⁺ in the synergids is polarized in its distributionin that the mCa²⁺ is higher towards the micropylar end of thesynergids. Interestingly, in the mature embryo sacs of Petuniaonly one of the two synergids and the egg cell proper manifesta higher level of mCa²⁺. In vivo only one of the synergids inthe young as well as in the mature embryo sacs if Nicotianaconsistently show higher mCa²⁺. In the mature embryo sacs of Petunia the level of CaM is almostuniform in all the cell types except that one of the synergidsand the three antipodal cells show a slightly higher level ofCaM. The possible implications of these findings in the late eventsof vectorial orientation of pollen tube tip, pollen-tube-synergidinteractions and sperm delivery mechanism are discussed.Copyright1993, 1999 Academic Press Membrane-Ca²⁺, calmodulin, living embryo sacs, Petunia, Nicotiana, pollen tube-synergid interaction     

The spontaneous growth response of maize coleoptile segments and the change in tissue 8

Decapitated segments from maize (Zea mays L.) coleoptiles orientedvertically in an upright position show a strong spontaneousgrowth response (SGR) 3 h after decapitation. The latent periodof the SGR is markedly reduced when these segments are orientedin an inverted position. Coleoptile segments with intact tipsexhibit a weak and transient SGR in the vertical upright orientation.However, in the inverted orientation, these segments show atypical SGR. The data are inconsistent with the current hypothesisthat the SGR is caused by a time-dependent increase in tissuesensitivity to auxin. The parallel increase in membrane potentialdifference and growth rate during the time-course of the SGRindicates a possible role for PM H⁺-ATPase in the establishmentof the SGR in maize coleoptile segments. Key words: Auxin, spontaneous growth response, membrane potential, plasma membrane H⁺-ATPase, Zea mays L.     

Phenotypic Characterization and Molecular Mapping of an acaulis2 Mutant of Arabidopsis thaliana with Flower Stalks of Much Reduced Length

An acaulis2-1 (acl2-1) mutant of Arabidopsis thaliana (L.) Heynh.was isolated and characterized. The mutant had inflorescencesof much reduced length. The defect was severer in lower internodesand was visible preferentially in type 2 metamers. The defectin the elongation of internodal cells of inflorescences wasattributable to a defect in the mechanism for elongation ofthese cells. The cortical microtubules (MTs) were analyzed inthe acl2 mutant, in a comparison with those of an acl1 mutantwhich was described earlier, but no changes were detected inamounts of MTs. In the type 2 and type 3 metamers, the defectin elongation in acl mutants was loosely correlated with a changein the orientation of MTs. Thus, the abnormal arrangement ofMTs appears to be one of the pleiotropic effects of acaulismutations. Genetic mapping was performed using molecular markers and theACL2 gene was located at 100 map units on chromosome 1. ¹Present address: Institute of Molecular and Cellular Biosciences,The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, 113 Japan ²Present address: Division of Biological Sciences, GraduateSchool of Science, Hokkaido University, Kita-ku, Sapporo, 060Japan     

Sorbitol Synthesis in Transgenic Tobacco with Apple cDNA Encoding NADP-Dependent Sorbitol-6-Phosphate Dehydrogenase

The apple (Malus domestica) cDNA encoding NADP-dependent sorbitol-6-phosphatedehydrogenase (S6PDH) was stably integrated and expressed intransgenic tobacco (Nicotiana tabacum cv. SR1). Expression ofthe cDNA in either a sense or antisense orientation was accomplishedusing cauliflower mosaic virus regulatory sequences (CaMV35S).Sorbitol synthesis was confirmed by gas-chromatography-mass-spectroscopy(GC-MS). Sorbitol concentration in the leaves of the transgenicplants expressing the sense orientation varied from 186 to 446nmol (g fr wt)^-1. The concentration positively correlates withS6PDH activity in leaves. Neither sorbitol nor S6PDH activitywas detected in the extracts of nontransformed tobacco or transgenictobacco expressing the antisense orientation. These resultsprovide key genetic evidence that S6PDH expression is sufficientfor the synthesis of sorbitol in tobacco, implicating it asa key enzyme in the sorbitol biosynthetic pathway in apple andperhaps other members of the woody Rosaceae. ¹Present address: Laboratory of Pomology, Faculty of Agriculture,Kyoto University, Sakyo, Kyoto, 606-01 Japan     

Inhibition of Phototaxis and Motility by UV-B Irradiation in Dictyostelium discoideum Slugs

The effect of monochromatic irradiation in and near the UV-Bregion (280–320 nm) on motility (speed of movement) andwhite light-induced phototactic orientation was studied in slugsof the cellular slime mold Dictyostelium discoideum (NC-4).Motility decreased to about 50% in UV-B fluences (about 100Jm^–2 at 280 nm) well below those required to inhibit slugand sorocarp development or to impair viability. At wavelengthsof 270–300 nm, the phototactic orientation was almosttotally eliminated by an exposure to U V (about 0.1 Wm^–2)for as short a time as half an hour when administered at thebeginning of the 24 h exposure to white light. (Received July 22, 1983; Accepted September 27, 1983)     

Microheterogeneity of N-glycosylation on a stylar self-incompatibility glycoprotein of Nicotiana alata

Gametophytic self-incompatibility, a mechanism that preventsinbreeding in some families of flowering plants, is mediatedby the products of a single genetic locus, the S-locus. Theproducts of the S-gene in the female sexual tissues of Nicotianaalata are an allelic series of glycoproteins with RNase activity.In this study, we report on the microheterogeneity of N-linkedglycosylation at the four potential N-glycosylation sites ofthe S²-glycoprotein. The S-glycoproteins from N.alata containfrom one to five potential N-glycosylation sites based on theconsensus sequence Asn-Xaa-Ser/Thr. The S²-glycoprotein containsfour potential N-glycosylation sites at Asn²⁷, Asn³⁷, Asn¹³⁸and Asn¹⁵⁰, designated sites I, n, IV and V, respectively. SiteIII is absent from the S₂-glycoprotein. Analysis of glycopeptidesgenerated from the S₂-glycoprotein by trypsin and chymotrypsindigestions revealed the types of glycans and the degree of microheterogeneitypresent at each site. Sites I (Asn²⁷) and IV (Asn¹³⁸) displaymicroheterogeneity, site II (Asn³⁷) contains only a single typeof N-glycan, and site V (Asn¹⁵⁰) is not glycosylated. The microheterogeneityobserved at site I on the S₂-glycoprotein is the same as thatobserved at the only site, site I, on the Srglycoprotein (Woodwardet al., Glycobiology, 2, 241-250, 1992). Since the N-glycosylationconsensus sequence at site I is conserved in all S-glycoproteinsfrom other species of self-incompatible solanaceous plants,glycosylation at this site may be important to their function.No other post-translational modifications (e.g. O-glycosylation,phosphorylation) were detected on the S₂-glycoprotein. fertilization microheterogeneity N-glycans plants RNase     

DENSITY FLUCTUATIONS, GROWTH AND DRY TISSUE PRODUCTION OF HYDROCOCCUS BRAZIERI (TENISON WOODS, 1876) AND ARTHRITICA SEMEN (MENKE, 1843) IN PEEL INLET, WESTERN AUSTRALIA

Hydrococcus brazieri and Arthritica semen on a sandflat at Coodanup,Peel Inlet, Western Australia, accounted for 89.5% of totalmollusc numbers. The mean density of H. brazieri was 9487 m^–2and A. semen averaged 8105 m^–2. H. brazieri grew at 0.5mm month^–1 and reached maturity in 4 months; A. semengrew at 0.3 mm month^–1 and reached maturity in 6 months.Somatic production in the Peel-Harvey estuarine system was estimatedto be 0.5 g m^–2yr^–1for H. brazieri and 4.1 g m^–2yr^–1for A. semen. The positions of these two species in the estuarinefoodweb is discussed. (Received 26 June 1981;     

Characterization and Translation of Poly(A)+RNA from Wounded and Crown Gall Tissues of Potato Tubers

Poly(A)⁺ and poly(A)^–RNA from wounded potato tuber tissuesand crown gall tumors were separated from total RNA by oligodeoxythymidylicacid-cellulose affinity chromatography. The poly(A)⁺RNA wascharacterized by sucrose density gradient centrifugation, hybridizationwith ³(H)polyuridylic acid [Poly(U)] and in vitro translationin a rabbit reticulocyte lysate system. The tumor poly(A)⁺RNAwas a heterodisperse mixture from 3.5S to 35S. Upon poly(U)hybridization of the gradient fractions two major hybridizationpeaks at 7S and 21S and two peaks at 11S and 16S appeared. Inan in vitro translation system the poly(A)⁺RNA programmed thesynthesis of 23 different polypeptides of 9,000 to 79,800 daltonsmolecular weight as determined by SDS-polyacrylamide gel electrophoresis.The 21S poly(A)+RNA was about 5 times more active in in vitroprotein synthesis than the 7S poly(A)⁺RNA. The poly(A)⁺RNA from wounded tissues was also heterodisperse(from 4.5S to 31S) with a modal peak at 18S. This RNA codedfor at least 28 polypeptides, which were different from thoseof crown gall tumor tissues. On a per unit poly(A)⁺RNA basis the tumor RNA was slightly moreactive in translation than that from wounded tissues. The translationof tumor poly(A)⁺RNA was completely blocked by 0.5 mM 7-methylguanosine5'-phosphate, but not by 7-methylguanosine, suggesting the presenceof a 5'-cap structure. (Received May 15, 1982; Accepted June 30, 1982)     

Predation impact of Cyclops vicinus on the rotifer community in Lake Constance in spring

The predation impact of Cyclops vicinus on rotifers was studiedunder near-natural conditions in small enclosures to evaluatewhether copepod predation is responsible for the decline ofrotifers in Lake Constance in spring. Cyclops vicinus fed selectivelyon Synchaeta spp.; Keratella and Polyarthra spp. were not selectedfor. Predation rates increased with prey density up to a maximumof 37 Synchueta day^–1 at a density of 1.6 x 10⁶ Synchaetam^–2, i.e. at -1200 Synchaeta l^–1. Calculation ofcropping rates suggests that Cyclops alone can control the abundanceof Synchaeta in spring, i.e. that mainly Cyclops is responsiblefor the decline of Synchaeta species in Lake Constance in May.     

Salt Tolerance in the HalophyteHalosarcia pergranulatasubsp.pergranulata

Halosarcia pergranulata(P. G. Wilson) subsp.pergranulatais amember of the Salicornioideae and is native to Australia. Salttolerance inH. pergranulatasubsp.pergranulatawas assessed bygrowing plants for 83 d at seven NaCl concentrations from 10to 800 mol m^-3. Shoot biomass was greatest for plants grownat 10 to 200 mol m^-3NaCl, while at salinities of 300 mol m^-3orhigher it was inhibited. There was little increase in succulencein response to NaCl, and it even declined at the highest salinities.The K⁺[ratio]Na⁺molar ratio in succulent shoot tissues decreasedfrom 0.30[ratio]1 in plants grown at 10 mol m^-3NaCl to 0.02[ratio]1in plants at 600 mol m^-3, due to a three-fold increase in tissueNa⁺concentration and a five-fold decline in tissue K⁺. The osmoticpotential of sap (     

The effect of UV-B radiation and humic substances on growth and motility of the flagellate, Euglena gracilis

The effects of ultraviolet-B radiation (UV-B, 250–315nm) were determined on Euglena gracilis with respect to speed,phototactic orientation, specific growth rate, and in the presenceof humic substances. Humic substances had a protective effectwhen studying the speed and specific growth rate. However, thedegree of phototactic orientation decreased in UV-B radiationboth with and without humic substances. The inhibition of O₂evolution and speed was most pronounced when using cutoff filtersWG280 and WG295. The photosynthetic inhibitor DCMU (10^–6M) did not have any effect on the speed, but the 0₂ evolutiondecreased to zero. The effect of different wavelengths in theUV-B region on the speed of E.gracilis showed the maximum sensitivityat 280 and 290 nm.     

Na+ fluxes in Chara under salt stress

The influx and efflux of Na⁺ across the plasma membrane of Characorallina and Chara longifolia were examined under mild saltstress conditions. Na⁺ influx was found to be rapid in bothspecies with the freely exchangeable cytoplasmic Na⁺ cominginto isotopic equilibrium with external ²²Na⁺ within 1 h ofexposure to isotope. Cytoplasmlc Na⁺ concentration and Na⁺ influxwere greater in C. corallina than in C. longifolla under thesame conditions. Na⁺ influx across the tonoplast was much lowerthan the flux across the plasma membrane. Na⁺ efflux was stimulatedat pH 5 relative to pH 7 by 218% in C. coralllna and 320% inC. longifolia. In both species externally applied Li⁺ inhibitedNa⁺ efflux at pH 5 but not at pH 7. Na⁺ etflux was not significantlyinhibited by amiloride. Key words: Na⁺ influx, Na⁺ efflux, Na⁺/H⁺ antiport, Chara     

Temporal Changes in Swimming Direction during the Phototactic Orientation of Individual Cells in Cryptomonas sp.

The response of individual Cryptomonas cells to continuous lightwas recorded using infrared video-micrography. Swimming directionsand temporal shifts in swimming direction of each cell weremeasured. White light of 0.1–1 W m^–2 elicited apositive phototactic orientation, but did not induce any photophobicresponse. Light of 100 W m^–2 induced a photophobic responseat the onset of actinic irradiation, but did not induce positivephototactic orientation. No correlation between positive phototacticorientation and photophobic response was found in this species.The direction toward the light source was defined as 0°,and the direction away from the source as 180°. Within 2s after the onset of lateral monochromatic light of 570 nm at0.1 W m^–2, cells which were swimming in a direction ofless than 120° predominantly shifted their course towardthe light source. Cells swimming in directions of larger than120° shifted their course as randomly as those in the dark.Thus, for phototactic orientation, the cells must perceive thelight from their anterior side. (Received July 29, 1985; Accepted November 4, 1985)     

Irradiance and daylength effects on growth and chemical composition of Gyrodinium aureolum Hulburt in culture

For Gyrodinium aureolum significant irradiance and daylengtheffects were found on the division rate and on the growth-relevantChla-normalized photosynthetic rate (^gP^B). Optimum conditionsof irradiance and daylength were found at 230 µmol m^–2s^–1 and 14 h for the division rate, and at >260 µmolm^–2 s^–1 and <6 h for ^gP^B.^gP^B showed no photoinhibition,while the division rate decreased markedly at irradiances abovesaturation. This difference and the difference in optimum irradiancebetween the division rate and ^gP^B are explained by a decreasein cellular Chla/carbon ratio with increasing irradiance. Thecellular content of carbon and nitrogen decreased significantlywith increasing irradiance. Total phosphorus was independentof irradiance and daylength. Below the saturation irradiancefor ^gP^B the daily Chla-normalized carbon yield may be describedas an exponential function of the daily irradiance (irradiancex daylength).     

Carbonic anhydrase 2-like a and 15a are involved in acid-base regulation and Na+ uptake in zebrafish H+-ATPase-rich cells

H⁺-ATPase-rich (HR) cells in zebrafish gills/skin were found to carry out Na⁺ uptake and acid-base regulation through a mechanism similar to that which occurs in mammalian proximal tubular cells. However, the roles of carbonic anhydrases (CAs) in this mechanism in zebrafish HR cells are still unclear. The present study used a functional genomic approach to identify 20 CA isoforms in zebrafish. By screening with whole mount in situ hybridization, only zca2-like a and zca15a were found to be expressed in specific groups of cells in zebrafish gills/skin, and further analyses by triple in situ hybridization and immunocytochemistry demonstrated specific colocalizations of the two zca isoforms in HR cells. Knockdown of zca2-like a caused no change in and knockdown of zca15a caused an increase in H⁺ activity at the apical surface of HR cells at 24 h postfertilization (hpf). Later, at 96 hpf, both the zca2-like a and zca15a morphants showed decreased H⁺ activity and increased Na⁺ uptake, with concomitant upregulation of znhe3b and downregulation of zatp6v1a (H⁺-ATPase A-subunit) expressions. Acclimation to both acidic and low-Na⁺ fresh water caused upregulation of zca15a expression but did not change the zca2-like a mRNA level in zebrafish gills. These results provide molecular physiological evidence to support the roles of these two zCA isoforms in Na⁺ uptake and acid-base regulation mechanisms in zebrafish HR cells. ionocytes; Na⁺/H⁺ exchanger; skin; gill; embryo     

Changes of action potentials and force at lowered [Na+]o in mouse skeletal muscle: implications for fatigue

We examined 1) whether the effects of lowered trans-sarcolemmal Na⁺ gradient on force differed between nonfatigued fast- and slow-twitch muscles of mice and 2) whether effects on action potentials could explain the decrease of force. The Na⁺ gradient was reduced by lowering the extracellular [Na⁺] ([Na⁺]_o). The peak force-[Na⁺]_o relationships for the twitch and tetanus were the same in nonfatigued extensor digitorum longus and soleus muscles: force was maintained over a large range of [Na⁺]_o and then decreased abruptly over a much smaller range. However, fatigue was significantly exacerbated at a lowered [Na⁺]_o that had little effect in nonfatigued soleus muscle. This finding suggests that substantial differences exist in the Na⁺ effect on force between nonfatigued and fatigued muscle. The reduced contractility in nonfatigued muscles at lowered [Na⁺]_o was largely due to 1) an increased number of inexcitable fibers and threshold for action potentials, 2) a reduction of action potential amplitude, and 3) a reduced capacity to generate action potentials throughout trains. sodium gradient; muscle contraction; action potential train; extensor digitorum longus; soleus     

Response to Waterlogging and Differing Sensitivity to Divalent Iron and Manganese in Clones of Dactylis glomerata L. derived from Well-drained and Poorly-drained Soils

Clonally propagated plants of Dactylis glomerata derived froma well-drained, heavily grazed cliff habitat (clone L) and froman undergrazed poorly-drained soil (clone A) were tested forwaterlogging tolerance in soil-culture. Water-logging did notaffect the two clones differentially, a result, which contrastedstrongly with that of a previous experiment in which simulatedgrazing (clipping to 20 cm) unexpectedly caused clone A to beless tolerant of waterlogging than clone L. Maximum leaf andleaf sheath length was reduced more by water-logging in cloneL than in clone A (P < 0.05). In solution-culture when providedwith factorial combinations of 0.5, 5 and 50 mg dm^–2 ofFe²⁺ and Mn²⁺ the shoot dry weight yield of the dry-soil clonewas reduced more than that of the wet-soil clone by 50 mg Fedm^–3 irrespective of Mn²⁺ concentration (P < 0.01)but the reduction of growth was less at higher Mn²⁺ concentrations.Fifty milligrams of Mn²⁺ dm^–3 reduced the growth of thedry soil clone but increased the growth of the wet soil clonewith Fe²⁺ at 5 mg dm^–2 (P < 0.05). Iron at 0.5 mg dm^–2was suboptimal for shoot growth of both clones at any levelof Mn²⁺ and caused more severe leaf chlorosis in the wet soilclone. Leaf tissue of clone L contained more iron than thatof clone A after waterlogging (P < 0.01) but in solutionculture, though increasing iron from 0.5 to 50 mg dm^–3almost doubled leaf iron content (P < 0.001), the interactionClones x Mn x Fe just failed to reach significance at P <0.05. The manganese content of leaf tissue from the two clonesvaried differently in response to solution manganese (Clonesx Mn P < 0.01), clone A showing a slightly greater increaseof manganese content at high solution concentration. Iron at50 mg dm^–3 suppressed Mn uptake (Mn x Fe, P < 0.001)in both clones. The two clones thus show marked environmentaladaptation to the chemistry of wet and dry soils. Dactylis glomerata, Cocksfoot grass, Orchard grass, waterlogging, iron, manganese, toxicity, deficiency, ecotypes     

Fluxes of diatoms in the Dona Paula Bay, west coast of India

Sediment traps were deployed at a station in the Dona PaulaBay to collect sedimenting particles at weekly intervals fromNovember to May during 1995–1997. Sedimented particleswere analysed for total diatom flux, chlorophyll a (Chl a) andparticulate organic carbon (POC). The highest diatom flux wasrecorded in April–May for both the years. Fluxes of diatomsvaried from0.6 x 10⁴ cells m^–2 day^–1 (November 1995)to 121.47 x 10⁴ cells m^–2 day^–1 (December 1996).In all, 19 diatom genera were identified in the sedimented material.Navicula, Nitzschia, Pleurosigma, Licmophora, Coscinodiscus,Rhizosolenia and Surirella were the most abundant genera inthe sedimented material throughout the sampling period. Meanflux of POC and diatom carbon was 251 and 0.39 mg C m^–2day^–1, respectively. The diatom carbon accounted for 0.15%of the POC flux. Mass flux of diatoms showed significant negativecorrelation with the concentration of nitrate and phosphate.This suggests that the nutrient concentration played an importantrole in influencing the sedimentation of diatoms at this coastalstation.