Unusual sequence of an abscisic acid-inducible mRNA which accumulates late in Brassica napus seed development

We have analyzed the nucleotide sequence and accumulation of an mRNA which is prevalent in seeds of Brassica napus L. During normal development, the mRNA begins to accumulate during late embryogeny, is stored in dry seeds, and becomes undetectable in seedlings within 24 hours after imbibition. Moreover, abscisic acid treatment of embryos precociously induces or enhances accumulation of the mRNA. Nucleotide sequencing studies show that the deduced 30 kDa polypeptide has an unusual primary structure; the polypeptide possesses direct amino acid sequence repeats and is virtually entirely hydrophilic with the exception of a hydrophobic carboxyl-terminal region. Based upon the expression pattern and predicted polypeptide sequence, we conclude that the mRNA is encoded by a late embryogenesis-abundant (Lea) gene in B. napus.Abbreviations ABA abscisic acid - bp base pairs - DAF days after flowering - HAI hours after the start of imbibition - kb kilobase (pairs)     

Isolation of prolegumin from developing pea seeds: its binding to endomembranes and assembly into prolegumin hexamers in the protein storage vacuole

A fraction enriched in endoplasmic reticulum and Golgi membranesfrom developing cotyledons of Pisum sativum L. has proved tobe a convenient source for the isolation of prolegumin, theprecursor of the major 11S storage globulin of pea seeds. Twopro-proteins were isolated with molecular masses of 60 kDa and75 kDa, respectively. A monoclonal antibody, designated 2B1,against prolegumin was raised using the in vitro immunizationtechnique. This antibody recognizes the 60 kDa precursor polypeptide,but only the 20 kDa ß-subunit of mature legumin. Prolegumin,like the ß-subunit of the mature legumin, is a hydrophobicprotein. After import into the protein storage vacuole, andafter formation of the protein bodies trimeric 9S proleguminassembles into 12S hexamers without prior processing of theprecursor. Since prolegumin in vitro does not oligomerize intomore than 9S tnmers these results suggest that a protein-mediatedassembly of 9S prolegumin trimers into 12S prolegumin hexamersprobably occurs in the lumen of the protein storage vacuole.Prolegumin, but not mature legumin, binds very tightly to membranes.This property points to a possible way of identifying a putativeprolegumin receptor. Key words: Calcium, Endoplasmic reticulum, Golgi apparatus, legumim, monoclonal antibody, pea cotyledons     

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)     

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.     

Poly(A) polymerase in Dormant Embryos of Triticum durum

Triticum durum‘Cappelli’ has a ‘relative’dormancy which can be broken by dry after-ripening at room temperature.The breakage of dormancy in the embryos of T. durum , is accompaniedby a decline in content and a different degree of synthesisof poly(A)⁺RNA. This work studies the activity of poly(A) polymerase(E.C. 2.7.7.19), the enzyme which permits polyadenylation. Anincrease in the activity of this enzyme in parallel with theenhanced rate of germination is revealed. Since poly(A) polymeraseactivity is the same in dormant and non-dormant dry embryos,it seems that the activity of the enzyme is not involved inthe breakage of dormancy. The use of cycloheximide and cordycepinshows the presence of enzymes with different origins: a storedenzyme and one bound to a long lived mRNA, present in dormantand non-dormant embryos, plus an enzyme bound to newly synthesizedmRNA which is mainly active in non-dormant embryos. Since dormancycould be the result of an interaction between hormones, thiswork analyses the effects of GA₃and ABA on poly(A) polymerase.GA₃enhanced poly(A) polymerase activity only in dormant embryoswhile ABA inhibited this activity only in non-dormant embryos.Cycloheximide applied to excised wheat embryos represses thestimulatory and inhibitory effects of GA₃and ABA, respectively.The hormone action on poly(A) polymerase activity is thus dependenton de novo protein synthesis. Results using cordycepin suggestthe presence of a stored mRNA for poly(A) polymerase, togetherwith hormonal regulation of enzyme activity at a translationallevel. Copyright 1999 Annals of Botany Company Triticum durum , wheat, dormancy breakage, poly(A) polymerase, GA₃, ABA, germination.     

Biochemical Characteristics Associated with the Development of the Desiccation-sensitive Seeds of Machilus thunbergii Sieb. & Zucc.

Biochemical properties, i.e. endogenous abscisic acid, proline,sugars, respiration, adenosine phosphates and adenylate energycharge, and growth and moisture content were measured duringthe development of seeds of Machilus thunbergii. As dry matteraccumulated in the embryo during development, moisture content,ABA, proline, respiration and sugars all declined. At maturity,the dry mass of the seeds failed to attain a plateau beforethe period of natural seed shedding; the axis and cotyledonsreached moisture contents of 58 and 45%, respectively. Dryingof immature seeds at 73% relative humidity and 25 °C for30 d resulted in a complete loss of viability at all developmentalstages tested with the exception of mature seeds that were ableto tolerate a 5% decrease in moisture content before germinationdeclined. ABA was detected in all embryos tested, with a maximum value16.·16 µg g^-1 d. wt about midway through development.Although the presence of ABA induced no tolerance to desiccationof mature seeds, it did coincide with decreased content of waterin the developing seeds and decreased respiration. Desiccationdamage of M. thunbergii seeds occurred when moisture contentwas still high (45%) and this damage was not related to theabsence of oligosaccharides in the mature seeds. We concludethat developing embryos and mature seeds of M. thunbergii haveproperties common to many recalcitrant seeds, with seeds beingsensitive to desiccation at all stages, having a prominent ABApeak, little proline, lacking oligosaccharides, and specifically,little dormancy and a moderate rate of respiration of matureseeds (0·9 µmol O₂ min^-1 g^-1 f. wt). Adenosinetriphosphate content and energy charge decreased from stagefour to stage eight of seed development, then increased againto 103 nmol g^-1 d. wt and 0·73, respectively, in matureseeds. The moderate energy charge observed in mature seeds indicatesthat continuous metabolism is also a characteristic of recalcitrantseeds.Copyright 1995, 1999 Academic Press Machilus thunbergii, seed development, recalcitrant seed, abscisic acid, energy charge     

Accumulation of 19-kDa Plasma Membrane Polypeptide during Induction of Freezing Tolerance in Wheat Suspension-Cultured Cells by Abscisic Acid

Suspension-cultured cells derived from immature embryos of winterwheat (Triticum aestivum L. cv. Chihoku) were used in experimentsdesigned to obtain clues to the mechanism of the ABA-induceddevelopment of freezing tolerance. Cultured cells treated with50 µM ABA for 5 d at 23°C acquired the maximum levelof freezing tolerance (LT50; -21.6°C). The increased freezingtolerance of ABA-treated cells was closely associated with theremarkable accumulation of 19-kDa polypeptides in the plasmamembrane. The 19-kDa polypeptide components were isolated bypreparative gel electrophoresis and were further separated intoone major (AWPM-19) and other minor polypeptide components byTricine-SDS-PAGE. N-terminal ami no acid sequence of AWPM-19was determined, and a cDNA clone encoding AWPM-19 was isolatedby PCR from the library prepared from the ABA-treated culturedcells. The cDNA clone (WPM-J) encoded a 18.9 kDa hydrophobicpolypeptide with four putative membrane spanning domains andwith a high pi value (10.2). Expression of WPM-1 mRNA was dramaticallyinduced by 50 µM ABA within a few hours. These resultssuggest that the AWPM-19 might be closely associated with theABA-induced increase in freezing tolerance in wheat culturedcells. (Received January 20, 1997; Accepted March 31, 1997)     

Effects of abscisic acid on somatic embryo maturation of hybrid larch

Somatic embryos of hybrid larch (Larixleptoeuropaea) whichhad been matured for 4 weeks on maturation medium, developednormally on medium supplemented with 60 µM ABA, but abnormallyon medium with no ABA. A comparative structural and histochemicalinvestigation was carried out on these two types of mature embryos.At the light microscope level, differences between both treatmentswere visible only after 2–3 weeks of maturation. At aroundthis time, abnormal development becomes evident macroscopically:ABA-minus embryos remain rather stubby as opposed to the morecylindrically shaped ABA-plus embryos. Whereas somatic embryosmatured with ABA consist of densely cytoplasmic cells showinga high rate of cell division, ABA-minus embryos are largelymade up of expanded and highly vacuolate cells, indicating thatgrowth in the latter is mainly due to cell expansion and notdivision. After 4 weeks of maturation, ABA-minus embryos beginto elongate in the hypocotyl region, and precocious germinationwas observed frequently. Again, these morphogenetic events werelargely due to abnormal timing of cell expansion. Histochemically,storage proteins were found only in somatic embryos maturedfor 4 weeks with ABA. This observation is in line with resultsobtained by total protein analysis, yielding significantly lowertotal protein contents in ABA-minus embryos both on a freshweight and a per embryo basis after 4–5 weeks of maturation.Deposition of starch grains mainly in the cortex tissue of thehypocotyl region was observed within 2 weeks of maturation invarying amounts regardless of ABA supply. Polyphenols, in particularcatechins and proanthocyanidins, were present in all embryosfrom the very onset of development. They were localized preferentiallyin the proximal suspensor cells and the basal region of theembryo. However, accumulation of polyphenols was generally muchmore pronounced in embryos matured without ABA, indicating alack of biochemical regulatory competence in those embryos. Key words: Abscisic acid, embryonal development, somatic embryo, storage protein, polyphenols     

mRNAs for two ribosomal proteins are preferentially translated in the chloroplast of Chlamydomonas reinhardtii under conditions of reduced protein synthesis

Previous studies have identified a set of highly phosphorylated proteins of 23–25 kDa accumulated during normal embryogenesis of Zea mays L. and which disappear in early germination. They can be induced precociously in embryos by abscisic acid (ABA) treatment. Here the synthesis and accumulation of this group of proteins and their corresponding mRNAs were examined in ABA-deficient viviparous embryos at different developmental stages whether treated or not with ABA, and in water-stressed leaves of both wild-type and viviparous mutants.During embryogenesis and precocious germination of viviparous embryos the pattern of expression of the 23–25 kDa proteins and mRNAs closely resembles that found in non-mutant embryo development. They are also induced in young viviparous embryos by ABA treatment. In contrast, leaves of ABA-deficient mutants fail to accumulate mRNA in water stress, yet do respond to applied ABA. In water-stressed leaves of wild type plants the mRNAs are induced and translated into 4 proteins with a molecular weight and isoelectric point identical to those found in embryos.These results indicate that the 23–25 kDa protein set is a new member of the recently described class or proteins involved in generalized plant ABA responses.The different pattern of expression for the ABA-regulated 23–25 kDa proteins and mRNAs found in embryo and in vegetative tissues of viviparous mutants is discussed.     

C-ABI3, the Carrot Homologue of the Arabidopsis ABI3, is Expressed during Both Zygotic and Somatic Embryogenesis and Functions in the Regulation of Embryo-Specific ABA-Inducible Genes

A carrot gene homologous to the ABI3 gene of Arabidopsis wasisolated from a carrot somatic embryo cDNA library and designatedC-ABI3. The sequence of C-ABI3 was very similar to those ofABI3 of Arabidopsis and VP1 of maize in certain conserved regions.The expression of C-ABI3 was detected specifically in embryogeniccells, somatic embryos and developing seeds. Thus, expressionof C-ABI3 was limited to tissues that acquired desiccation tolerancein response to endogenous or exogenous abscisic acid (ABA).Endogenous levels of ABA in seeds increased transiently andthen desiccation of seeds started. The expression of C-ABI3in developing seeds was observed prior to the increase in levelsof endogenous ABA that was followed by desiccation of seeds.In transgenic mature leaves in which C-ABI3 was ectopicallyexpressed, expression of ECP31, ECP63 and ECP40 was inducedby treatment with ABA, which indicates that the expression ofECP genes was controlled by the pathway(s) that involved C-ABI3and ABA. This suggests that C-ABI3 has the same function asVP1/ABI3 factor in carrot somatic embryos. (Received March 4, 1998; Accepted September 4, 1998)     

Sequencing and Expression of the Gene that Encodes a 20-kDa Polypeptide of the PSI Complex from Cucumber Cotyledon

A cDNA clone encoding the polypeptide from cucumber PS I thatmigrates with an apparent molecular weight of 20 kDa on SDS-polyacrylamidegels has been isolated. The 907-bp sequence of this clone hasbeen determined and contains one large open reading frame thatencodes a 22,720-Da precursor polypeptide (207 amino acid residues).The molecular weight of the mature polypeptide was predictedto be 17,037-Da (153 amino acid residues). The deduced aminoacid sequence of this protein indicates that it is routed towardsthe stromal side of the thylakoid membrane and has no membrane-spanningregions. The sequence also confirmed the identity of the proteinas the product of the psa D gene. Chemical cross-linking offerredoxin to the PS I complex identified the 20-kDa subunitas the ferredoxin-binding protein. Northern hybridization experimentsrevealed that the mRNA of approximately 1,100 nucleotides forthe 20-kDa polypeptide was present in etiolated cucumber cotyledons,and its level increased about 5-fold during greening. The 20-kDapolypeptide was not detected by immunoblotting in etiolatedcotyledons, and it accumulated only after illumination. Labelingexperiments in vivo showed the absence of incorporation of [³⁵S]Metinto the polypeptide in etiolated cotyledons. These resultssuggest that the expression of the psa D gene is controlledat the translational level. (Received April 5, 1990; Accepted June 28, 1990)     

A Comparison of Leek (Allium porrum) and Onion (Allium cepa) Seed Development

Leek and onion seed dry weight increased exponentially for thefirst 31 days after flowering (DAF) but thereafter the increasein dry weight was slower. Before maximum seed dry weight wasreached at 45 DAF in onion and 59 to 66 DAF in leek, seed moisturecontent, seed oxygen uptake and conductivity of the seed steepwater fell from initially high levels. Although some seeds germinated31 DAF in both species, full germination in both was not reacheduntil 66–80 DAF. Tolerance of the seed to artificial dryingimmediately after harvesting occurred 45 DAF in onion and 74DAF in leek. Free nuclear division continued in the endospermuntil 17–22 DAF in onion and until 31–35 DAF inleek but it was not until 45 DAF in onion and 66 DAF in leekthat the embryo and endosperm filled the cavity formed by thepericarp. After formation of cell walls in the endosperm thepattern of change in cell number in both species was similar.The shrunken appearance of the seed coat in leek, which occurredearly in seed development, was associated with the period offree nuclear division in the endosperm and, in addition, thepericarp was thinner than in onion. There was no evidence thatthe shrunken seed coat early in development was associated withself as opposed to open-pollination. Allium porrum, Allium cepa, seed development, endosperm, embryo, cell number, germination, respiration, seed leachates     

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     

Dormancy in somatic embryos and seeds ofVitis: changes in endogenous abscisic acid during embryogeny and germination

Abscisic acid (ABA) in extracts of somatic embryos and seeds of Gloryvine (Vitis vinifera L.xV. rupestris Scheele) was measured by gas chromatography-mass spectrometry-selected ion monitoring using deuterated ABA, (±)-[C-3Me-²H₃]ABA, ([²H₃]ABA) as internal standard. The ABA content increased rapidly during embryogeny (0.035 ng/embryo at the globular stage to 0.22 ng/embryo at the mature stage). The level of ABA in the tissues of somatic embryos, expressed in ng/mg dry weight, decreased from the globular stage (0.76 ng/mg) to the mature stage (0.25 ng/mg). Chilling (4° C) induced normal germination of seeds and mature somatic embryos and precocious germination of globular, heart-shaped and torpedoshaped somatic embryos. In all cases chilling led to a marked reduction in endogenous ABA. Exogenous (±)-ABA inhibited the germination of chilled somatic embryos.Abbreviations ABA abscisic acid - [²H₃]ABA (±)-[C-3Me-²H₃]-abscisic acid - BHT 2,6-di-t-butyl-4-methylphenol - GC-MS gas chromatography-mass spectrometry - Me-ABA and Me-[²H₃]ABA methyl esters of ABA and [²H₃]ABA, respectively - SIM selected ion monitoring     

Abscisic Acid and Precocious Germination in Soybeans

Immature embryos of soybeans (Glycine max L. Merr.) can be inducedto germinate precociously by depleting the endogenous pool ofabscisic acid (ABA). Washing the embryos or allowing the embryosto dry slowly within or out of detached pods causes a gradualdecline in ABA content. The extent of germination is correlatedwith the length of washing or drying treatments, which in turn,affects the level of endogenous ABA. Providing embryos are threeweeks of age or older, maturation and precocious germinationcan be induced by treating embryos in a way that causes a reductionin embryo ABA content. Drying is not an obligatory requirementfor soybean seed maturation or germination. Key words: ABA, Germination, Embryos     

Regulation of Soybean Embryogenesis by Abscisic Acid

Abscisic Acid (ABA) stimulates growth and protein accumulationin soybean (Glycine max. L. Merr.) embryos during the earlyphases of embryogenesis. Growth of mid-stage embryos is suppressedby ABA, but protein accumulation is not impaired. Metabolitedistribution studies indicate that ABA alters partitioning ofsucrose in older embryos such that protein accumulation is sustainedat the expense of lipid accumulation. The responses of in vitrocultured embryos to ABA is consistent with the normal patternof ABA accumulation and disappearance that occurs during embryogenesisin situ. A close correlation exists between ABA levels and embryogrowth rates in situ in three cultivars of soybeans. Dependingon the age or stage of the developing embryo, ABA either servesto promote or inhibit embryo growth. Key words: Embryogenesis, ABA, Seeds, Soybean.