Effect of Abscisic Acid, Osmoticum, and Desiccation on Synthesis of Storage Proteins during the Development of White Spruce Somatic Embryos

The effect of abscisic acid (ABA), non-permeating osmoticumand desiccation treatment on storage protein synthesis duringmaturation of somatic embryos of Picea glauca (Moench) Voss.was examined. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot analysis demonstrated that someof the major crystalloid and matrix polypeptides were absentfrom somatic embryos maturing on medium containing ABA and lowosmoticum. However, treatment with polyethylene glycol-4000(PEG) in combination with ABA resulted in the synthesis of aspectrum of storage polypeptides resembling that of mature zygoticembryos. These storage proteins accumulated throughout an 8-weekculture period, resulting in a threefold higher protein contentthan somatic embryos maturing for the same time in the absenceof PEG. The structure and distribution of protein bodies incells of these osmotically treated somatic embryos was similarto that in cells of mature zygotic embryos. Treatment with 5·0-7·5%PEG prevented catabolism of the accumulated storage polypeptidesduring desiccation. The optimal culture conditions for somaticembryo maturation and storage protein deposition was 16 µMABA and 7·5% PEG for 8 weeks followed by desiccation.Analysis of mRNAs by in vitro translation and immunoprecipitationof translated products showed that the crystalloid protein mRNAprofiles of zygotic and those of somatic embryos maturing on16 µM ABA in the absence of PEG were similar. The differencesobserved in the pattern of accumulated polypeptides in thesesomatic embryos and those of mature zygotic embryos, therefore,indicates that storage-protein synthesis in response to osmoticumis in part regulated at the translational level. During regenerationof somatic embryos to plantlets the storage polypeptides wererapidly utilized in a manner similar to that in zygotic seedlings.Copyright1993, 1999 Academic Press Desiccation, osmotic stress, storage proteins, Picea, embryogenesis—somatic, mRNA (crystalloid protein)     

Normalizing Development of Cultured Triticum aestivum L. Embryos: I. LOW OXYGEN TENSIONS AND EXOGENOUS ABA

Wheat (Triticum aestivum L.) embryos form in dynamically-regulatedovular environments. Our objectives were to improve developmentof cultured immature wheat embryos by simulating, in vitro,abscisic acid (ABA) levels and O₂ tensions as found in wheatovules during zygotic embryogenesis. We characterized from intactwheat kernels embryo respiration, embryo morphology and embryoand endosperm + ABA levels at 13, 19 and 25 d post-anthesis(DPA). Young (13 DPA) embryos were then excised and culturedin vitro, where they were exposed to 0·2 or 2·Ommol m^–3 ±ABA and 2.·1, 2·5 or 7·4mol m^–3 (6, 7 and 21%, respectively) gaseous O₂. At 6and 12 d in culture, + ABA levels, embryo respiration and embryomorphology were characterized by treatment. Thirteen-day-oldembryos from two different plant populations differed by 17-foldin initial ABA content. However, this difference did not affectprecocious germination in vitro, nor did it affect the amountof exogenous ABA required to reduce precocious germination by40%. In this respect, embryos from both populations were equallysensitive to exogenous ABA. Cavity sap O₂ levels (2·1to 2·5 mol m^–3) were much more effective in preventingprecocious germination of cultured embryos than were cavitysap levels of ABA (0·2 to 2·0 mmol m^–3).The combination of physiological levels of both ABA and O₂ largelynormalized DW accumulation and embryo morphology without alteringendogenous + ABA levels. Residual respiration of cultured embryoswas higher than that of embryos grown in situ, and was not influencedby the exogenous O₂ and ABA treatments Key words: Abscisic acid, embryo development, oxygen tensions, respiration, wheat     

Identification and Characterization of the Seed Storage Proteins from Alfalfa (Medicago sativa)

The major seed storage proteins in alfalfa are medicagin (alegumin-like globulin), alfin (a vicilin-like globulin) anda family of Lower Molecular Weight albumins (LMW₁₃). These comprise30%, 10% and 20%, respectively, of the total extractable proteinfrom cotyledons of mature seeds. Alfin is a heterogeneous oligomericprotein (Mr 150 kD) composed of polypeptides ranging in sizefrom Mr 50 to 14 kD (₁,-₆; 50, 38, 32, 20, 16 and 14 kD, respectively).Medicagin is also a high molecular weight oligomeric protein,but requires high concentrations of salt for solubilization.It is comprised of a family of individually distinct subunits,each composed of an acidic polypeptide (A₁–A₉; Mr 49 to39 kD) linked via disulphide bond(s) to a basic polypeptide(B₁, B₂, B₃; Mr 24, 23 and 20 kD, respectively). This pairingis highly specific and two families are recognizable on thebasis of the B polypeptide (B₃ or B₁/B₂). Subunits (M_r 50–65kD) are assembled as trimers (8S) or larger oligomers (12S–15S)in mature seeds. The lower molecular weight albumins (LMW₁–₃)are acidic (pl<6), and consist of sets of disulphide-bondedpolypeptides (Mr 15 and 11 kD). Key words: Medicago sativa, seed storage proteins, alfin, medicagin     

Purification and Characterization of Arginine Decarboxylase from Soybean (Glycine max) Hypocotyls

Arginine decarboxylase (EC 4.1.1.19 [EC] ) was purified from soybean,Glycine max, hypocotyls by a procedure which includes ammoniumsulfate fractionation, acetone precipitation, gel filtrationchromatography, and affinity chromatography. Using this procedure,ADC was purified to one band in non-denaturing PAGE. The purifiedADC has an M_r of 240 kDa based on gel filtration chromatographyand is a trimer of identical subunits which has an estimatedM_r of 74 kDa based on SDS-PAGE. ADC is active between 30 and50°C and has a K_m value of 46.1 µM. ADC is very sensitiveto agmatine or putrescine but not to spermidine or spermine.In the presence of 0.5 mM agmatine (or putrescine), the enzymeactivity was inhibited by 70%. However, at the same concentrationof spermidine (or spermine), the enzyme activity was inhibitedby only 10–20%. (Received April 2, 1997; Accepted August 18, 1997)     

Genotypic Variability in Differential Expression of lea2 and lea3 Genes and Proteins in Response to Salinity Stress in Fingermillet (Eleusine coracanaGaertn) and Rice (Oryza sativaL.) Seedlings

Some late embryogeny abundant (LEA) proteins, which are developmentallyregulated in embryos, are also known to be expressed in meophytictissues in response to osmotic stress. Here we report the extentof genetic variability in the level of expression of lea2 andlea3, under stress, in fingermillet and rice seedlings. In bothspecies, the expression of lea genes was seen in the mesophytictissue in response to salinity, partial dehydration and abscisicacid. Tolerant genotypes exhibited higher expression of rab16Aand M3 that code for LEA2 proteins, than susceptible genotypes.A novel approach, that of raising antibodies against the conservedpeptides of these proteins was used to study genetic variabilityin LEA protein levels. Since stress proteins are known to beexpressed in response to mild, non-lethal induction-stress (Uma,Prasad and Udayakumar,Annals of Botany76: 43–49, 1995),we developed an optimum induction protocol for salinity stressin rice and fingermillet. We studied the quantitative differencesin expression of these proteins by western blot and ELISA techniquesin different genotypes. A positive correlation was found betweenLEA2 and LEA3 protein levels and the growth of seedlings duringstress and recovery in both rice and fingermillet, indicatinga possible relevance of these proteins in stress tolerance.Copyright1998 Annals of Botany Company LEA proteins, ABA responsive proteins, induction response, ELISA, fingermillet, rice, salinity-stress.     

Enhanced Maturation and Desiccation Tolerance of White Spruce [Picea glauca (Moench) Voss] Somatic Embryos: Effects of a Non-plasmolysing Water Stress and Abscisic Acid

A non-plasmolysing moisture stress effected by polyethyleneglycol (PEG) was beneficial when applied to maturing white spruce(Picea glauca) somatic embryos for the following reasons. Anosmotic treatment of 5.0–7.5% PEG stimulated a threefoldincrease in the maturation frequency. The osmotically treatedsomatic embryos displayed higher dry weights and lower moisturecontents than the controls, indicating a greater accumulationof storage reserves. Moisture contents of mature, osmotically-treated,hydrated somatic embryos were 40–45%, in contrast to 57%for the non-osmotically treated controls. Desiccation was achievedby placing the somatic embryos in a range of relative-humidityenvironments. No clear trend for the effect of PEG on survivalof desiccated somatic embryos was observed; mean survival valuesranged from 34 to 62% when somatic embryos from all osmotictreatments were desiccated for 14 d at 81% relative humidity.Following this desiccation treatment, somatic embryos from allosmotic concentrations had moisture contents of 26–31%,similar to the 32% recorded for unimbibed zygotic embryos. Afterimbibition, moisture contents for these zygotic and somaticembryos were in the order of 60%. Somatic embryos matured withPEG remained quiescent during desiccation due to their low initialmoisture contents, and gave rise to plantlets of normal appearance.Gradual desiccation of the somatic embryos directly followingmaturation with abscisic acid (ABA) was crucial to survivalduring desiccation. A plasmolysing water stress effected bysucrose at osmotic potentials similar to PEG was detrimentalto somatic embryo maturation, thereby emphasizing the importanceof the choice of osmoticum. Desiccation, maturation, osmotic potential, Picea glauca, polyethylene glycol, somatic embryo, water stress, white spruce     

Characterization of a novel voltage-dependent outwardly rectifying anion current in Caenorhabditis elegans oocytes

An inwardly rectifying swelling- and meiotic cell cycle-regulated anion current carried by the ClC channel splice variant CLH-3b dominates the whole cell conductance of the Caenorhabditis elegans oocyte. Oocytes also express a novel outwardly rectifying anion current termed I_Cl,OR. We recently identified a worm strain carrying a null allele of the clh-3 gene and utilized oocytes from these animals to characterize I_Cl,OR biophysical properties. The I_Cl,OR channel is strongly voltage dependent. Outward rectification is due to voltage-dependent current activation at depolarized voltages and rapid inactivation at voltages more hyperpolarized than approximately +20 mV. Apparent channel open probability is zero at voltages less than +20 mV. The channel has a 4:1 selectivity for Cl^– over Na⁺ and an anion selectivity sequence of SCN^– > I^– > Br^– > Cl^– > F^–. I_Cl,OR is relatively insensitive to most conventional anion channel inhibitors including DIDS, 4,4'-dinitrostilbene-2,2'-disulfonic acid, 9-anthracenecarboxylic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid. However, the current is rapidly inhibited by niflumic acid, metal cations including Gd³⁺, Cd²⁺, and Zn²⁺, and bath acidification. The combined biophysical properties of I_Cl,OR are distinct from those of other anion currents that have been described. During oocyte meiotic maturation, I_Cl,OR activity is rapidly downregulated, suggesting that the channel may play a role in oocyte Cl^– homeostasis, development, cell cycle control, and/or ovulation. chloride channel; ovulation; cell cycle; meiotic maturation     

Effects of Desiccation, Medium Osmolarity and Abscisic acid on the Maturation of Hevea brasiliensis Somatic Embryos

The effects of desiccation of Hevea somatic embryos and of sucroseand ABA concentrations in the maturation medium on their germinabilitywere investigated. Conversion into plant, water and histochemicalstatus of somatic embryos were compared systematically to thoseof the zygotic embryos used as reference. Slow desiccation ormaturation on 351 mol m^–3 sucrose supplemented with 1mmol m^–3 ABA strongly improved germinability and conversionof embryos into plants. The combination of the two treatmentswas the most effective, increasing the germination frequencyby 3·7 and plant conversion by 6·6 in clone PR107. Each of these two treatments increased the vigour of somaticembryos, stimulated the formation of root and shoot meristemsand the synthesis and accumulation of starch and protein reserves.At the end of maturation, the Hevea somatic embryos bore ananatomical and histochemical resemblance to mature zygotic embryos.Likewise, the two treatments brought the water status of somaticembryos closer to that of the mature zygotic embryos, but withoutachieving a perfect match. Optimization of the successful conversioninto plants may require full acquisition of this water status. Key words: ABA, embryo maturation, Hevea, somatic embryogenesis, water status     

Proteomic analyses of somatic and zygotic embryos of Cyclamen persicum Mill. reveal new insights into seed and germination physiology

In the horticulturally important ornamental species Cyclamen persicum Mill., somatic embryogenesis is an efficient vegetative propagation method and the development of artificial seeds is an ultimate aim. This study aims at a systematic comparison of the proteomes of zygotic embryos, somatic embryos grown in liquid medium containing 30 or 60 g l⁻¹ sucrose, germinating embryos of both types and endosperm in order to obtain novel insights into seed and germination physiology. Using high resolution two-dimensional isoelectric focussing/sodium dodecylsulfate polyacrylamide gel electrophoresis (2D IEF/SDS PAGE), 74% of the proteins expressed in zygotic embryos were found in similar abundance in somatic embryos grown in 60 g l⁻¹ sucrose. Somatic embryos grown in 30 g l⁻¹ sucrose accumulated fewer protein species than those grown in 60 g l⁻¹. Selected proteins were identified following mass spectrometry (nano-LC-MS/MS). Four enzymes involved in glycolysis (UDP-glucose pyrophosphorylase, fructose bisphosphate aldolase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase GAPDH) were specifically induced in somatic embryos. 11S globulin proteins identified by MS were present in high levels in somatic embryos, zygotic embryos and endosperm, whereas 7S globulins were detected mainly in endosperm and zygotic embryos. These are the first storage proteins identified in C. persicum. Xyloglucans are known to be another group of seed storage compounds in C. persicum. Interestingly, xyloglucan endotransglycosylases were found to be highly expressed in endosperm tissue. We discuss the physiological implications of these observations.     

Proteins of the Crustacean Exoskeleton

We describe here some of the components of the exoskeleton ofthe decapod crustacean with emphasis on the constituent proteins,including both structural and enzymatic. All four layers, butparticularly the inner three, of the exoskeletons of four brachyuranscontain high concentrations of proteins 31 kDa; the innermostmembranous layer is especially rich in such proteins. A numberof crab exoskeletal proteins resemble insect cuticle proteinsin size (M_r) and isoelectric point (pI). A further similarityis the cross reactivity of crab exoskeletal proteins with fourdifferent antibodies against cuticular proteins of two speciesof insects. One of the small M_r exoskeletal proteins in theBermuda land crab Gecarcinus lateralis has a similar distributionas a protein of similar size in the cuticle of the tobacco hornworm Manduca sexta. The partial dissolution of an old exoskeletonand formation of the two outer layers of a new exoskeleton aremajor events in readying a crustacean for the increase in sizethat occurs at each molt. Expressing both parallel and sequentialactivation of a number of genes, a single layer of epidermalcells that bounds a crustacean such as G. lateralis synthesizesspecific proteins at different stages of the intermolt cycleas the outermost epicuticle and exocuticle are formed duringproecdysis and as the endocuticle and membranous layer are formedduring metecdysis. Finally, two sets of proteinases isolatedfrom integumentary tissues of land crabs degrade the same exoskeletalproteins in vitro as are degraded in vivo during proecdysis.     

Identification, Purification and Properties of the Precursor of Conglutin {beta}, The 7S Storage Globulin of Lupinus albus L. Seeds

Lupinus albus L. developing cotyledons 35 d after floweringcontained a major polypeptide of-average M_r 64000, immunologicallyrelated to conglutin ß, the 7S storage globulin ofthis seed. This polypeptide decreased during seed maturation,without completely disappearing in the mature seed. This dropwas accompanied by the formation of polypeptide fragments typicalof the mature conglutin ß. The 64000 polypeptide hasbeen identified as the precursor polypeptide of conglutin ß. Undenatured conglutin ß precursor, purified by ionexchange chromatography and size exclusion chromatography, showedsurface and association properties identical to the mature conglutinß molecule. The precursor oligomer, of M_r 190000,consisted of an association of three 64000 subunits. They stronglyreacted with concanavalin A indicating the presence of covalentlylinked carbohydrate. Tryptic treatment of the undenatured conglutin ß precursorled to the accumulation of a relatively stable 59000 polypeptidewhich was cleaved later on and produced three large polypeptidefragments differing from the mature conglutin ß polypeptides. Key words: Conglutin ß, precursor, developing seeds, Lupinus albus L.     

Embryogeny of gymnosperms: advances in synthetic seed technology of conifers

Synthetic seed technology requires the inexpensive production of large numbers of high-quality somatic embryos. Proliferating embryogenic cultures from conifers consist of immature embryos, which undergo synchronous maturation in the presence of abscisic acid and elevated osmoticum. Improvements in conifer somatic embryo quality have been achieved by identifying the conditions in vitro that resemble the conditions during in ovulo development of zygotic embryos. One normal aspect of zygotic embryo development for conifers is maturation drying, which allows seeds to be stored and promotes normal germination. Conditions of culture are described that yield mature conifer somatic embryos that possess normal storage proteins and fatty acids and which survive either partial drying, or full drying to moisture contents similar to those achieved by mature dehydrated zygotic embryos. Large numbers of quiescent somatic embryos can be produced throughout the year and stored for germination in the spring, which simplifies production and provides plants of uniform size. This review focuses on recent advances in conifer somatic embryogenesis and synthetic seed technology, particularly in areas of embryo development, maturation drying, encapsulation and germination. Comparisons of conifer embryogeny are made with other gymnosperms and angiosperms.Abbreviations ABA abscisic acid - LEA late embryogenesis abundant - PEG polyethylene glycol - PGR plant growth regulator - RH relative humidity - TAG triacylglycerol     

An Analysis of Seed Development in Pisum sativum III. The Relationship Between the r Locus, the Water Content and the Osmotic Potential of Seed Tissues in vivo and in vitro

The water content and osmotic potential (₂) of the differentparts of the pea fruit have been measured during developmentof the seed in two lines near-isogenic except for the r locus.During the early development of both genotypes, the testa possesseda more negative ₂ than either embryo, endosperm or pod while,at stages when liquid endosperm was present, the embryo alwaysmaintained ₂, more negative than the endosperm. A clear effectof the r locus on water content and ₂ was only observed in embryotissue — wrinkled (rr) embryos possessing more water andmaintaining a more negative ₂ than round (RR) for most of thedevelopmental period studied. The more negative ₂ of wrinkledembryos correlated with their greater uptake of water in vivo. When cultured in vitro, the embryos of both genotypes showedmaximum growth (fresh or dry weight) if 10 per cent sucrosewas added to the medium (equivalent to about — 1.2 MPa).Round embryos, however, increased in weight more than wrinkledat all sucrose concentrations examined. Cultured embryos maintaineda similar or more negative ₂ than their surrounding medium;wrinkled more negative than round. Embryo culture, osmotic potential, Pisum sativum, pea, r locus, seed development, tissue culture, water content     

Allocation of Organ Biomass in Perfect and Imperfect Flowers

Perianth M_P, gynoecium M_G, and androecium M_A dry-weight biomass(in g) of 39 species of perfect flowers was measured. Thesedata were pooled with published data from an additional 51 speciesand used to determine size-dependent variations in (M_G and M_A)in terms of the hypothesis that the quotient of M_G and M_A exceeds1·0 for out-breeding (xenogamous) species and less than1·0 for in-breeding (autogamous) species. Ordinary leastsquare regression of the pooled data (n = 90) showed M_G = 0·118M^0·916_P (r² = 0·884) and M_A = 0·186 M^0·975_P(r² = 0·865), indicating that the biomass of the gynoeciumproportionally decrease as floral size increases. The exponentsof these regressions indicate that the ratio of gynoecial toandroecial biomass decreased with increasing floral size suchthat comparatively small flowers (M_P < 0·0021 g) hadM_G/M_A > 1·0 (predicted for 'out-breeders') while comparativelylarger flowers (M_P > 0·0021 g) had M_G /M_A < 1·0(predicted for 'in-breeders'). Thus, on average, the type ofbreeding system was a size-dependent phenomenon. To test whether the biomass of a floral organ-type is a legitimateindicator of gender reproductive effort, the biomass (in g)of stamen filaments M_m and anther sacs M_AS of 39 species wasdetermined. Least square regression of these data showed M_AS= 0·188 M^0·854_fil (r² = 0·967), indicatingthat species with larger stamen filaments, on the average, boreproportionally smaller anther sacs and thereby cautioning againstthe uncritical use of the allocation of biomass to floral organ-typeas a strict gauge of gender-function investment. To determine whether the loss of one gender-function resultsin proportional reallocation of biomass to the remaining gender-function,the size-dependency of androecial and gynoecial biomass wasdetermined for a total of 33 perfect and imperfect flowers ofCucumis melo. Regression of the data obtained from perfect flowersyielded M_A = 0·402 M^1·47_P (r² = 0·898)and M_G = 4·63 M^1·36_P (r² = 0·842). SinceM_G/M_A M^0·11_P , the biomass allocation to the gynoeciumrelative to the androecium decreased with increasing floralsize. This result was consistent with the broad interpecificcomparison based on 90 species with perfect flowers . Regressionof the data for imperfect flowers yielded M_A = 0·151M^1·02_P (r² = 0·675) and M_G = 4·68 M^1·47_P(r² = 0·996), indicating a near allometric relation forthe androecium and a strong positive anisometry for the gynoecium.Thus, for flowers of comparable size, a loss of female genderobtains a modest to significant again in androecial biomasswhereas the loss of male gender yields only a slight increasein gynoecial biomass. Collectively, the results of these studies indicate that biomassallocation patterns are size-dependent phenomena whose complexitieshave been largely ignored in the literature.Copyright 1993,1999 Academic Press Allometry, floral biomass, reproduction     

Extracellular acidification elicits a chloride current that shares characteristics with ICl(swell)

A Cl^– current activated by extracellular acidification, I_Cl(pHac), has been characterized in various mammalian cell types. Many of the properties of I_Cl(pHac) are similar to those of the cell swelling-activated Cl^– current I_Cl(swell): ion selectivity (I^– > Br^– > Cl^– > F^–), pharmacology [I_Cl(pHac) is inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 1,9-dideoxyforskolin (DDFSK), diphenylamine-2-carboxylic acid (DPC), and niflumic acid], lack of dependence on intra- or extracellular Ca²⁺, and presence in all cell types tested. I_Cl(pHac) differs from I_Cl(swell) in three aspects: 1) its rate of activation and inactivation is very much more rapid, currents reaching a maximum in seconds rather than minutes; 2) it exhibits a slow voltage-dependent activation in contrast to the fast voltage-dependent activation and time- and voltage-dependent inactivation observed for I_Cl(swell); and 3) it shows a more pronounced outward rectification. Despite these differences, study of the transition between the two currents strongly suggests that I_Cl(swell) and I_Cl(pHac) are related and that extracellular acidification reflects a novel stimulus for activating I_Cl(swell) that, additionally, alters the biophysical properties of the channel. cell swelling-activated chloride current; patch clamp; pH     

A Protein from Immature Raspberry Fruits which Inhibits Endopolygalacturonases from Botrytis cinerea and other Micro-organisms

A polygalacturonase-inhibiting protein (PGIP) was purified fromimmature raspberry fruits using ion exchange chromatography.The protein was composed of a single polypeptide chain withM_r of 38·5 kDa and a pI residing above pH 10. Kineticstudies suggested that the inhibition was of a non-competitivenature. The PGIP inhibited two endopolygalacturonases (endo-PG)purified from Botrytis cinerea and an endo-PG produced by Aspergillusniger to varying degrees but did not inhibit two exo-PGs purifiedfrom B. cinerea, bacterial endopectate lyases and bacterialendo-PGs. The concentration of PGIP at various stages of flowerand fruit development was determined. The inhibitor was notdetected in the flower, but reached a maximum of 69 units g^–1in the immature green fruit decreasing to 9 units g^–1as fruits matured. The N-terminal amino-acid sequence was determined. Key words: Polygalacturonase-inhibiting protein, Rubus idaeus, red raspberry, Botrytis cinerea, pectinases     

DCEBIO stimulates Cl- secretion in the mouse jejunum

We investigated the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one(DCEBIO) on the Cl^– secretory response of the mouse jejunum using the Ussing short-circuit current (I_sc) technique. DCEBIO stimulated a concentration-dependent, sustained increase in I_sc (EC₅₀ 41 ± 1 µM). Pretreating tissues with 0.25 µM forskolin reduced the concentration-dependent increase in I_sc by DCEBIO and increased the EC₅₀ (53 ± 5 µM). Bumetanide blocked (82 ± 5%) the DCEBIO-stimulated I_sc consistent with Cl^– secretion. DCEBIO was a more potent stimulator of Cl^– secretion than its parent molecule, 1-ethyl-2-benzimidazolinone. Glibenclamide or NPPB reduced the DCEBIO-stimulated I_sc by >80% indicating the participation of CFTR in the DCEBIO-stimulated I_sc response. Clotrimazole reduced DCEBIO-stimulated I_sc by 67 ± 15%, suggesting the participation of the intermediate conductance Ca²⁺-activated K⁺ channel (IK_Ca) in the DCEBIO-activated I_sc response. In the presence of maximum forskolin (10 µM), the DCEBIO response was reduced and biphasic, reaching a peak response of the change in I_sc of 43 ± 5 µA/cm² and then falling to a steady-state response of 17 ± 10 µA/cm² compared with DCEBIO control tissues (61 ± 6 µA/cm²). The forskolin-stimulated I_sc in the presence of DCEBIO was reduced compared with forskolin control tissues. Similar results were observed with DCEBIO and 8-BrcAMP where adenylate cyclase was bypassed. H89, a PKA inhibitor, reduced the DCEBIO-activated I_sc, providing evidence that DCEBIO increased Cl^– secretion via a cAMP/PKA-dependent manner. These data suggest that DCEBIO stimulates Cl^– secretion of the mouse jejunum and that DCEBIO targets components of the Cl^– secretory mechanism. 1-ethyl-2-benzimidazolinone; forskolin; glibenclamide; clotrimazole; H89     

Proteins arising during the late stages of embryogenesis in Pisum sativum L.

Two abscisic acid (ABA)-responsive seed proteins, ABR17 and ABR18 (ABA-responsive 17000-M_r and 18000-M_r, respectively), previously found to be induced in cultured embryos of pea (Pisum sativum L.) are major components synthesised during normal seed desiccation. The ABR17 and ABR18 proteins showed different patterns of accumulation. The ABR18 protein was abundant in the testa during early seed development but in desiccating seed it was synthesised in the embryo, indicating spacial as well as temporal regulation of expression. The ABR18 protein was undetectable soon after germination but reappeared after adding ABA. The ABR17 protein was not detected in the testa but appeared in the embryo just prior to maximum fresh weight. The ABR17 protein continued to be synthesised during germination and was also present in non-stressed leaves. A high level of endogenous ABA or added ABA increased levels of translatable ABR17 mRNA. The ABR17 and ABR18 proteins were further characterised so as to help determine their structure and function. Neither protein appeared to contain a signal peptide but both proteins appeared to be glycosylated. The proteins had similar amino-acid compositions and limited Nterminal analysis showed 56% sequence identity. Neither protein had any significant N-terminal sequence homology to any of the late embryogenesis-abundant (LEA) proteins or dehydrins. Both proteins, however, show striking homology with a pea disease-resistance-response protein and the major birch pollen allergen, indicating that the ABR17 and ABR18 proteins may be members of a distinct group of stress-induced proteins.Abbreviations ABA (±) cis,trans-abscisic acid - ABR17 M_r-17200 ABA-responsive protein - ABR18 M_r-18 100 ABA-responsive protein - FW fresh weight - I_gG immunoglobulin G - LEA late embryogenesis-abundant - M_r apparent molecularmass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid This work was supported by the Agricultural and Food Research Council via grants-in-aid to Long Ashton Research Station.     

Proteomic analysis of pathogenesis-related proteins (PRs) induced by compatible and incompatible interactions of pepper mild mottle virus (PMMoV) in Capsicum chinense L3 plants

Resistance conferred by the L³ gene is active against most ofthe tobamoviruses, including the Spanish strain (PMMoV-S), aP_1,2 pathotype, but not against certain strains of pepper mildmottle virus (PMMoV), termed P_1,2,3 pathotype, such as the Italianstrain (PMMoV-I). Both viruses are nearly identical at theirnucleotide sequence level (98%) and were used to challenge Capsicumchinense PI159236 plants harbouring the L³ gene in order tocarry out a comparative proteomic analysis of PR proteins inducedin this host in response to infection by either PMMoV-S or PMMoV-I.PMMoV-S induces a hypersensitive reaction (HR) in C. chinensePI159236 plant leaves with the formation of necrotic local lesionsand restriction of the virus at the primary infection sites.In this paper, C. chinense PR protein isoforms belonging tothe PR-1, β-1,3-glucanases (PR-2), chitinases (PR-3), osmotin-likeprotein (PR-5), peroxidases (PR-9), germin-like protein (PR-16),and PRp27 (PR-17) have been identified. Three of these PR proteinisoforms were specifically induced during PMMoV-S-activationof C. chinense L³ gene-mediated resistance: an acidic β-1,3-glucanaseisoform (PR-2) (M_r 44.6; pI 5.1), an osmotin-like protein (PR-5)(M_r 26.8; pI 7.5), and a basic PR-1 protein isoform (M_r 18;pI 9.4–10.0). In addition, evidence is presented for adifferential accumulation of C. chinense PR proteins and mRNAsin the compatible (PMMoV-I)–C. chinense and incompatible(PMMoV-S)–C. chinense interactions for proteins belongingto all PR proteins detected. Except for an acidic chitinase(PR-3) (M_r 30.2; pI 5.0), an earlier and higher accumulationof PR proteins and mRNAs was detected in plants associated withHR induction. Furthermore, the accumulation rates of PR proteinsand mRNA did not correlate with maximal accumulation levelsof viral RNA, thus indicating that PR protein expression mayreflect the physiological status of the plant. Key words: Capsicum chinense, compatible interaction, incompatible interaction, HR-induction, PMMoV, PR proteins Received 5 December 2007; Revised 21 January 2008 Accepted 22 January 2008     

Dormancy in Dioscorea: Rapid Germination of Detached Embryos from Dormant Seeds of D. tokoro

The intact dormant seeds of Dioscorea tokoro germinate slowlyif at all between 11-23°C; for full and rapid germinationthey require prior chilling treatment [Okagami and Kawai (1982)Bot. Mag. Tokyo 95: 155]. The germination abilities of zygoticembryos detached from dormant seeds of this species were studiedunder various nutritional and temperature regimes. For germinationof embryos, the minimum nutritional components in Murashigeand Skoog's (1962) medium that were required were sucrose andNO^–₃ or SO^2–₄. As the source of carbohydrate forgermination of detached embryos, sucrose, mannose and maltosewere effective; glucose and fructose were less effective; andrhamnose was entirely unable to support germination. Embryos detached from dormant seeds, incubated with the sucroseplus KNO₃, germinated more rapidly with increasing temperatureup to 35°C. However, application of sucrose and KNO₃ didnot induce germination of intact seeds above 26°C. Therefore,it is very possible that the endosperm exerts an inhibitoryfunction on germination at such high temperatures. When seeds were incubated after a cut was made over a smallpart of the edge of the endosperm in which the radicle of theembryo is encased, germination occurred rapidly but the increasein germination percentage was slight. This result suggests thatthe endosperm suppots part of the germination inhibition bymeans of a mechanical barrier or its impermeability to wateror gases. Physiological features of the endosperm alone or interactionsbetween the embryo and endosperm may contribute significantlyto the characteristics of dormancy of intact seeds of this species. (Received May 30, 1988; Accepted January 11, 1989)