Identification of 4-Chloroindole-3-acetic Acid and Its Methyl Ester in Immature Seeds of Vicia amurensis (the Tribe Vicieae), and Their Absence from Three Species of Phaseoleae

The natural chlorinated auxins 4-chloroindole-3-acetic acid(4-Cl-IAA) and its methyl ester (4-Cl-IAA Me ester) were found,in addition to IAA and its Me ester, by gas chromatography-massspectrometry in immature seeds of Vicia amurensis, a Vicieaespecies. In contrast, only non-chlorinated, IAA and IAA Me esterwere present in immature seeds of three Phaseoleae species.These results are further evidence of the wide distributionof 4-Cl-IAA and its Me ester in various Vicieae. (Received October 3, 1986; Accepted December 22, 1986)     

Protein Bodies of Developing Seeds of Vicia faba

Changes in fine structure and starch, nitrogen, and solublesugar content were followed through to maturation in developingcotyledons of Vicia faba. Various ultrastructural changes wereobserved in the developing cotyledons, notably an increase inthe number of membrane-bound ribosomes which corresponded withthe onset of storage protein deposition. The build-up of storageprotein was shown to occur in the cytoplasm within membrane-boundvacuoles which subsequently became the protein bodies of themature seed, retaining the original tonoplast as the boundingmembrane of the protein body. Nuclei became lobed during thelater phases of maturation; phytoferritin was observed in plastidsof mature seeds. The deposition of reserves in the cotyledonswas complete by 85–90 days after flowering, followingwhich water was lost until the seed became hard and ‘ripe’by no days after flowering.     

蚕豆未成熟子叶原生质体再生植株

近年来,豆科植物原生质体诱导再生植株的研究越来越受到国内外学者关注。但在籽粒型食用豆科植物中,迄今成功的种类仍然不多,在文献记载中仅见有大豆、赤豆、豇豆、豌豆和刀豆,说明籽粒型食用豆科植物原生质体再生植株仍有较大困难,要取得禾谷类作物那样的重大进展,尚需作出巨大努力。蚕豆原生质体培养仅见有从预培养的叶肉原生质体再生细胞团、从茎尖和叶肉原生质体再生愈伤组织和从     

Isolation of a Novel Auxin,Methyl 4-Chloroindoleacetate from Immature Seeds of Pisum sativum

Human casein was separated by gel filtration on a column of Sephadex G–200 with 0.1 m Tris buffer (pH 8.5) containing 1.0 m NaCl. The effluent which increased in turbidity at 25°C was centrifuged at 25,000 × g for 30 min and the precipitate was obtained as Fraction 6. After centrifugation, the effluent was separated into 5 elution fractions.

Disc gel electrophoretic patterns of each fraction showed occurrence of secondary bands other than major bands especially in Fractions 3, 4 and 5. The casein solutions unheated and heated at 100°C for 5 and 10 min were kept at 5°C for 5 days. No marked changes of electrophoretic pattern were observed among these casein solutions. However, when a casein solution heated at 100°C for 5 min was chroma to graphed under the same condition, secondary bands also appeared.     

Metabolic Conversion of Castasterone and Brassinolide into Their Glucosides in Higher Plants

Castasterone (CS) and brassinolide (BL) were administered to mung bean (Vigna radiata) explants, Arabidopsis thaliana seedlings, and cultured Catharanthus roseus cells, and the glucosylated metabolites were analyzed using LC/MS/MS. In mung bean and C. roseus, CS-2-O-glucoside (CS-2G), -3-O-glucoside (CS-3G), -22-O-glucoside (CS-22G), and -23-O-glucoside (CS-23G) were identified as metabolites of CS, whereas BL-2G, BL-3G, and BL-23G were identified as metabolites of BL. In A. thaliana, CS and BL were converted into their respective 2-O- and 23-O-glucosides. Of the metabolites identified with BL and CS administration, BL-23G was the predominant metabolite in mung bean and A. thaliana, whereas the 3-O-glucoside of BL was abundant in C. roseus. This is the first report of the metabolic conversion of CS into CS-2G, CS-3G, CS-22G, and CS-23G, and of BL into BL-2G and BL-3G. Our results indicate that the glucosylation profiles of BL and CS vary with plant species, and that the glucosylation of CS is rather limited quantitatively, compared with that of BL.     

Nucleic Acids of Developing Seeds of Vicia faba L.

Changes have been followed in the base composition of RNA andthe amounts of RNA and DNA during the development of Vicia fabaseeds. Both acids were synthesized most rapidly during days36–46 of the 72-day developmental period. Changes foundwere related to the development of the embryo and to the synthesisof storage protein.     

Variability and inheritance of histone genes H3 and H4 in Vicia faba

Summary We have compared copy numbers and blothybridization patterns of histone genes (H3 plus H4) between and within individuals of broad bean (Vicia faba). Copy number differences among individuals in the population of 200 individuals were as great as 27 fold, and as much as 3.2 fold among separate leaves of the same plant. Among F₂ progeny from genetic crosses, up to a 5.4-fold range was seen (mean=3.5 fold), and among F₁ progeny of self-pollinated plants, up to a 5.9-fold range was observed (mean=2.3 fold). Histone gene blot-hybridization patterns for EcoRI and HindIII were also variable among individuals and indicated that the genes are probably clustered in only a few chromosomal loci. The degree of variation in histone gene copy number per haploid genome (2–55 copies, or 27 fold) was similar to that found previously for ribosomal RNA genes (230–22000, or 95 fold) of V. faba. However, the two gene families change independently, since individuals with a high or low copy number for one gene can have either a high or low copy number for the other. The mechanisms(s) for rapid gene copy number change may be similar for these gene families.     

The Effect of Temperature on Development and Dry-matter Accumulation of Vicia faba Seeds

Vicia faba plants were grown at 16 °C and the temperatureraised to 21 or to 26 °C, 51 d post anthesis (p.a.). Temperatureincrease accelerated pod and seed development, stimulated dry-matteraccumulation, starch and protein synthesis. However, the durationof dry-matter accumulation of seeds was shortened, resultingin a decrease of final seed weight. The effect of temperature on storage capacity (determined bycell number and cell size) was also investigated. Formationof new cells in the layer under the epidermis continued fora much longer period than previously has been described in theliterature. At all stages of development cotyledons containedyoung and small cells with small nuclei at the outside and theybecame larger and older towards the centre of the cotyledon.Cells at the centre were only able to divide at an early stageof seed development up to day 59 p.a. Thereafter cell divisionoccurred mainly in the first cell layer under the epidermisup to 63–67 d p.a. Nuclear counts of macerated cotyledoncells did not show an effect of temperature on the number ofcells. It has, however, been questioned if this method was suitableto measure accurately the number of cells at the last stagesof seed development. The rate of cell expansion was stimulated by higher temperaturesbut the duration of expansion was shortened and the final sizeof cells was not affected by the different temperature treatments.Senescence of the pod wall was accelerated at higher temperaturesand it is possible that transport of sucrose from the pod wallto the seeds terminated earlier than at lower temperatures.This may have resulted in an inhibition of cell formation atthe periphery of the cotyledons and in an inhibition of starchand protein synthesis. Vicia faba, protein, starch, cell size, cell number, temperature     

The Metabolism of Raffinose and Sucrose in Germinating Broad-Bean (Vicia faba) Seeds

Enzyme preparations from germinating broad-bean seeds can produceUDPG from sucrose or raffinose when incubated with UDP; in thelatter case D-galactose is also formed. This observation suggeststhat raffinose can be metabolized in two stages: (1) the hydrolyticcleavage of the trisaccharide by a-galactosidase with the releaseof galactose and sucrose; (2) a sucrose: UDP glucosyltransferase(reversed sucrose synthetase)-catalysed transfer of D-glucosefrom sucrose to UDP. The galactose liberated in stage (I) inVicia faba seeds can probably be converted to a-D-galactose-1-phosphateby galactokinase. The levels of the glucosyltransferase at variousstages of germination have been measured, the enzyme has beenpartially purified, and an estimate has been made of its apparentmolecular weight. The glucosyltransferase is specifically inhibitedby D-glucose and D-fructose and the role of this enzyme in catabolicprocesses is discussed in general terms.     

4-Chloroindole-3-acetic acid and plant growth

4-Chloroindole-3-acetic acid (4-Cl-IAA) is a potent auxin in various auxin bioassays. Researchers have used 4-Cl-IAA as well as other halogenated auxins in biological assays to understand the structural features of auxins required to induce auxin mediated growth in plants. 4-Cl-IAA is a naturally occurring auxin in plants from the Vicieae tribe of the Fabaceae family; and 4-Cl-IAA has also been identified in one species outside the Vicieae tribe, Pinus sylvestris. The apparent function of the unique auxin 4-Cl-IAA in normal plant growth and development will be discussed with a focus on Pisum sativum and Vicia faba     

Localization of 4-Chloroindole-3-acetic Acid in Seeds of Pisum sativum and Its Absence from All Other Organs

4-Chloroindole-3-acetic acid (4-Cl-IAA) was shown by GC-MS analysisto be present in immature and mature seeds of Pisum sativum,but not in any other organs of this plant. Its content was maximalat one week after anthesis and decreased as the seeds matured.Only indole-3-acetic acid (IAA) was detected in the other organsof P. sativum, its content being particularly high in the flowersand young pods during anthesis and the early pod set. (Received January 18, 1988; Accepted April 6, 1988)     

Mitochondrial Arginase Activity from Cotyledons of Developing and Germinating Seeds of Vicia faba L

Differential and sucrose density gradient centrifugation established that about 80% of the total arginase activity (EC 3.5.3.1) in cotyledons of germinating broad bean seeds (Vicia faba L.) was present in the mitochondrial fraction. The mitochondrial arginase activity was enhanced considerably by exposure to osmotic shock, by freezing and thawing, or by Triton X-100 treatment. About 10% of the total arginase activity was recovered from the 40,000g supernatant fraction. During seed maturation, arginase activity in the cotyledons decreased to about one-third of its maximal activity, while increasing over 10-fold during subsequent germination. The time courses of mitochondrial arginase, succinate oxidase, and succinate dehydrogenase activities differed considerably during germination.     

Occurrence of heparin inhibitable hemagglutinating activity in leaves of fava bean (Vicia faba)

A hemagglutinating activity which is inhibitable by heparin was extracted from leaves of fava bean. The activity was partially purified by ammonium sulfate fractionation followed by affinity chromatography of heparin-agarose. The purified activity was inhibited only by heparin but not by simple monosaccharides or glycosaminoglycans tested. These results showed that this activity was very different from its well known seed lectin, Vicia faba agglutinin.     

Amino Acid Release from the Seed Coat of Developing Seeds of Vicia faba and Pisum sativum

After removal of the embryo from developing ovules of Viciafaba L. and Pisum sativum L., seed-coat exudates were collectedand the amino acid fraction of the exudate was analyzed. InV. faba, alanine was the most important compound of the aminoacid fraction. In P. sativum, alanine and glutamine were thetwo most important components, whereas only small amounts ofasparagine were present. Comparison with published data suggeststhat seed-coat exudates may differ from phloem sap in the relativeimportance of these amino acids. Pisum sativum, pea, Vicia faba, broad bean, amino acid transport, amino acid unloading, seed-coat exudate, seed development     

Protein Bodies of Germinating Seeds of Vicia faba: CHANGES IN FINE STRUCTURE AND BIOCHEMISTRY

Changes in fine structure and starch, total nitrogen and solublesugar contents were followed during the first 3 weeks of germinationin Vicia faba cotyledons. Following hydration the reserves were mobilized and loss ofprotein from the cotyledons began after 4 days; concomitantfine structural changes included the swelling and coalescenceof protein bodies, the disappearance of their contents, andthe differentiation of the provascular tissue in the cotyledonwithin which ‘transfer cells’ developed. There wassome development of rough endoplasmic reticulum during the earlypart of germination. A pattern of protein-body degradation appeared in the cotyledonsduring germination, those cells nearest to the vascular bundlesand to the epidermis being the first to lose their reserves.After 3 weeks' growth the parenchyma cells of the cotyledonwere very vacuolate and typical of senescent tissue, while thevascular bundle cells still retained their contents and remainedactive-looking.     

Auxin, transport and growth in intact roots of Vicia faba

Transport of IAA applied to the intact root of Vicia seedlingswas investigated in relation to root growth. The root was treatedat 3–4, 4–5 or 7–8 mm from the tip with anarrow ring of lanolin paste containing IAA or IAA-2-¹⁴C ingrowth or transport experiments, respectively. The growth processalong the root axis was examined in every 1-mm part from thetip at 30 min, 1 or 4 hr intervals. The elongation zone of thecontrol root was 1–9 mm from the tip. IAA treatment broughtabout no significant change in the growth of the region apicalto the treated site, whereas distinct inhibition of growth occurredin the region basal to the treated site within 1 hr. The transportof radioactivity was observed in both acropetal and basipetaldirections within 1 hr, but the latter predominated for 8 hror more; the nearer to the tip the treatment site, the longerthe predominance lasted. The velocities of acropetal and basipetaltransport were estimated at about 4 and 8 mm/hr, respectively.Autoradiographs of transverse section of roots showed that basipetaltransport occurred mainly through the outer part of the root,whereas acropetal transport occurred mainly through the innerpart, the central cylinder. It may be concluded that the basipetallytransported IAA which passed through the outer part of the rootinhibited the elongation of the intact root. (Received November 25, 1975; )     

Gibberellins in Immature Seeds of Canavalia

Two novel gibberellins, GA₂₁ (I) and GA₂₂ (II), were isolated from immature seeds of sword bean, Canavalia gladiata DC. The isolation procedure of these substances as well as their growth-promoting effects on dwarf maize mutants d₁ and d₅, rice, cucumber and dwarf peas (Progress No. 9) are described.

The structures of two new gibberellins, GA₂₁ and GA₂₂, isolated from immature seeds of sword bean, were determined as 4a_α, 7_α-dihydroxy-8-methylenegibbane-1, 1, 10β-tricarboxylic acid 1→4a lactone (II) and 4a_α, 7_α-dihydroxy-l β-hydroxymethyl-8-methylenegibb-2-ene-1_α, 10β-dicaboxylic acid 1→4a lactone (IV), respectively, on the basis of chemical and physicochemical studies.     

Linkage among isozyme,RFLP and RAPD markers in Vicia faba

Summary Segregating allozyme and DNA polymorphisms were used to construct a preliminary linkage map for faba bean. Two F₂ populations were analyzed, the most informative of which was segregating for 66 markers. Eleven independently assorting linkage groups were identified in this population. One of the groups contained the 45s ribosomal array and could be assigned to the large metacentric chromosome I on which the nucleolar organizer region is located. This linkage group also contained two isozyme loci, Est and Tpi-p, suggesting that it may share some homology with chromosome 4 of garden pea on which three similar markers are syntenic. Additional aspects of the map and the extent of coverage of the total nuclear genome are discussed.     

'Rinrei', a brassinosteroid-deficient dwarf mutant of faba bean (Vicia faba L.)

A dwarf mutant of broad bean ( Vicia faba L.), the variety Rinrei, has been created by γ -ray irradiation. Rinrei is characterized by dark green leaves and by reduced plant length, internode and petiole length, shoot weight, and number of branches. Genetic analysis of hybrids between Rinrei and two wild-type lines indicated that these characteristics are controlled by a single recessive gene. The phenotype of Rinrei was restored to that of the wild type by application of brassinolide, but not by GA₃. Qualitative and quantitative analysis by gas chromatography–mass spectrometry indicated that 24-methylenecholesterol and isofucosterol accumulated in Rinrei to levels more than 30 times higher than in the wild type. In contrast, Rinrei had lower than wild-type levels of campesterol, sitosterol and brassinosteroids. Therefore, Rinrei is a brassinosteroid-deficient mutant defective in sterol C-24 reduction. The gene was tentatively designated as brassinosteroid deficient dwarf 1 , bdd1 , which seems to be a homologue of Arabidopsis dwf1 ( dim , cbb1 ) and pea lkb .