Gibberellins in Suspensors of Phaseolus coccineus L. Seeds

Analysis of extracts from 6300 (1.2 grams fresh weight) Phaseolus coccineus suspensors by combined gas chromatography-mass spectrometry has demonstrated the presence of five C₁₉-gibberellins, GA₁, GA₄, GA₅, GA₆, GA₈, and one C₂₀-GA, GA₄₄. The major GAs present were GA₁ and GA₈. Data are discussed in relation to previous results obtained in P. coccineus seed as well as in the embryo-suspensor system.     

Gibberellins in suspensor,embryo and integument from very young seeds of Phaseolus coccineus L.

Endogenous gibberellins (GAs) were extracted from suspensor, embryo and integument of very young seeds of Phaseolus coccineus L. and detected by combined gas chromatography-mass spectrometry (GC-MS). Results show the presence of one C₂₀-GA, GA₄₄ and five C₁₉-GAs in the suspensor: GA₁, GA₄, GA₅, GA₆ and GA₈, and four C₁₉-GAs in the integument: GA₁, GA₅, GA₆ and GA₈. Only traces of GA₁ and GA₅ were identified in the embryo. A compound structurally related to GAs was identified as tetrahydroxy-Kauranoic acid in suspensor, integument and, only in trace amounts, in the embryo.     

Partitioning of 14C-photoassimllates within seeds of Phaseolus coccineus (Lam.) and Phaseolus coccineus x Phaseolus vulgaris L.

The objective of this study was to determine partitioning within seeds of ¹⁴C-photoassimilates at three stages of seed development in two Phaseolus crosses — P. coccineus Lam. selfed, and P. coccineus x P. vulgaris L. Abortion of the interspecific embryos occurred when the seed reached 10 mm seed length. When expressed as sink strength (% dpm) or sink activity (% dmp/d.wt.) there were no differences in partitioning of ¹⁴C-photoassimilates when whole seeds were analyzed. If the seed was divided into seed coat, liquid endosperm, and embryo, the sink activity of the interspecific embryo was higher than that of the embryo in the selfed seed. Therefore, abortion of these interspecific Phaselus embryos appeared not to be caused by a lack of photoassimilates.Assistant Professor, Professors, respectively.Contribution from the Agr. Expt. Station, University of Minnesota, St. Paul, MN 55108. Paper No. 13,548, Scientific Journal Series. This research was supported in part by the Science and Education Administration of the United States Department of Agriculture under Grant 59-2271-9-2-020-0 and in part by a grant from the Minnesota Soybean Research and Promotion Council.     

Mechanics of circadian pulvini movements in Phaseolus coccineus L.

The circadian movement of the lamina of primary leaves of Phaseolus coccineus L. is mediated by antagonistic changes in the length of the extensor and flexor cells of the laminar pulvinus. The cortex of the pulvinus is a concentric structure composed of hexagonal disc-like cells, arranged in longitudinal rows around the central stele. Observations with polarization optics indicate that the cellulose microfibrils are oriented in a hoop-like fashion in the longitudinal walls of the motor cells. This micellation is the structural basis of the anisotropic properties of the cells: tangential sections of the extensor and flexor placed in hypotonic mannitol solutions showed changes only in length. As a consequence a linear correlation between length and volume was found in these sections. Based on the relationship between the water potential (which is changed by different concentrations of mannitol) and the relative volume of the sections and on the osmotic pressure at 50% incipient plasmolysis, osmotic diagrams were constructed for extensor and flexor tissues (cut during night position of the pulvinus). The bulk moduli of extensibility, , were estimated from these diagrams. Under physiological conditions the values were rather low (in extensor tissue below 10 bar, in flexor tissue between 10 to 15 bar), indicating a high extensibility of the longitudinal walls of the motor cells. They are strongly dependent on the turgor pressure at the limits of the physiological pressure range.In well-watered plants, the water potentials of the extensor and flexor tissues were surprisingly low,-12 bar and-8 bar, respectively. This means that the cells in situ are by no means fully turgid. On the contrary, the cell volume in situ is similar to the volume at the point of incipient plasmolysis: the cell volumes of extensor and flexor cells in situ were only 1.01 times and 1.1 times larger, respectively, than at the point of incipient plasmolysis, whereas at full turgidity (cells in water) the corresponding factors were 1.8 and 1.5. It is suggested that the high elasticity of the longitudinal walls, the anisotropy of the cell walls, and the low water potential of the sections which is correlated with slightly stretched cell walls in situ, are favourable and effective for converting osmotic work in changes in length of the pulvinus cells, and thus for the up and down movement of the leaf.Symbols volumetric elastic modulus - _i instantaneous volumetric elastic modulus - _i stationary volumetric elastic modulus - weight-averaged stationary bulk modulus of extensibility - ₀ osmotic pressure of the vacuole of a cell at the point of incipient plasmolysis - weight-averaged osmotic pressure of the vacuoles of the tissue at 50% incipient plasmolysis - water potential     

Phaseolus coccineus L. Storage Proteins. Extraction and Characterization

Phaseolus coccineus storage globulins were extracted from mature cotyledons, purified and characterized. Three major proteins were separated. A component showing erythroagglutinating activity was thoroughly purified by thyroglobulin-Sepharose chromatography. The relative molecular masses of the three fractions are Mr = 330, 178, and 500 kDa as determined by polyacrylamide gel electrophoresis (PAGE). They correspond to the proteins found in other systems and classified as phytohaemagglutinin (PHA), vicilin and legumin, respectively. Electrophoretic analyses under denaturating conditions (SDS-PAGE) evidenced the major subunits for the three proteins. Isoelectrofocusing of the isolated proteins indicated a large heterogeneity for vicilin. Part I.     

Abscisic acid and apical dominance in Phaseolus coccineus L.

Abscisic acid (ABA) in lanolin, applied to the internode of decapitated runner bean plants enhances the outgrowth of lateral buds. The optimum concentration of the paste is 10^-5 M. The effect of ABA is counteracted by indoleacetic acid (IAA) but not by gibberellic acid (GA₃). There is no effect when ABA is applied to the apical bud or lateral buds of intact plants. However, 13.2 ng given to the lateral buds of decapitated plants stimulate their growth, whereas higher concentrations are inhibitory. Consequently, ABA enhances growth of lateral buds directly, but only when apical dominance is already weakened. The growth of the decapitated 2^nd internode was not affected by ABA. Radioactivity from [2-¹⁴C] ABA, applied to nonelongating 2^nd internode stumps of decapitated runner bean plants moves to the lateral buds, whereas [1-¹⁴C]IAA-and [³H]GA₁-translocation is much weaker. ABA transport is inhibited if IAA or [³H]GA₁ is applied simultaneously. In elongating internodes [¹⁴C]ABA is almost completely immobile. [¹⁴C]IAA-and [³H]GA₁-translocation is not affected by ABA. The amount of radioactivity from labelled ABA, translocated to the lateral buds, is highest during the early stages of bud outgrowth.Abbreviations ABA 2,4-cis, trans-(+)-abscisic acid - GA gibberellic acid - IAA indoleacetic acid - p.l. plain lanolin     

Gibberellins in Embryo-Suspensor of Phaseolus coccineus Seeds at the Heart Stage of Embryo Development

Gibberellins (GAs) in suspensors and embryos of Phaseolus coccineus seeds at the heart stage of embryo development were analyzed by combined gas chromatography-mass spectrometry (GC-MS). From the suspensor four C₁₉-GAs, GA₁, GA₄, GA₅, GA₆, and one C₂₀ GA, GA₄₄, were identified. From the embryo, five C₁₉-GAs GA₁, GA₄, GA₅, GA₆, GA₆₀ and two C₂₀ GAs, GA₁₉ and GA₄₄ were identified. The data, in relation to previous results, suggest a dependence of the embryo on the suspensor during early stages of development.     

A method for long-term micropropagation of Phaseolus coccineus L.

A method for long-term plant regeneration of Phaseolus coccineus L, is described. Shoot-tips and cotyledonary nodes cultured on a Murashige and Skoog medium supplemented with N⁶-benzylaminopurine, 10 M, and -naphthaleneacetic acid, 1M, formed multiple bud-shoots. These shoots were transferred to medium containing BAP 1 M, NAA 0.1 M, and gibberellic acid 3 M to promote shoot growth and further shoot multiplication. Rooting was achieved in medium with 11 M indole-3-acetic acid. Rooted plants grew to maturity and were fertile. Cultures have maintained their ability to regenerate plants for more than two years. A sample of 30 regenerated plants (R₀) was tested for chromosome number, all of them being diploid; seven isozymatic systems were electrophpretically analyzed in 82 R₀ regenerated plants. No differences were observed in their electrophoretic patterns in comparison with those shown by seedlings. Histological studies revealed the origin of buds from calluses via organogenesis.Abbreviations BAP N⁶-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - ADH alcohol dehydrogenase - GOT glutamic-oxaloacetic transaminase - MDH malate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase - PGI Phosphoglucose isomerase - PGM phosphoglucose mutase - SK shikimate dehydrogenase     

Factors affecting morphogenesis from immature cotyledons of Phaseolus coccineus L.

Direct somatic embryogenesis as well as somatic embryogenesis and organogenesis mediated by small glossy calluses were obtained from immature cotyledon explants of bean (P. coccineus) cv Streamline 770 on a modified half-strength MS medium (Murashige & Skoog 1962) containing various concentrations of (2-isopentenyl)adenine and 2-naphthoxyacetic acid. Substitution of sucrose with glucose gave, in the range of concentrations tested, the strongest enhancement of the morphogenic process. Further improvement regarding the number of morphogenic cotyledons, the number of regenerations per cotyledon and the quality of the embryos was observed when 2,3,5-triiodobenzoic acid or abscisic acid were added to the medium. After cycles of micropropagation on MS medium plus 4.4 M 6-benzyladenine and rooting in the absence of growth factors, plantlets were adapted to ex vitro conditions and grown to maturity.Abbreviations ABA abscisic acid - AC activated charcoal - BA 6-benzyladenine - 2,4-D (2,4-dichlorophenoxy)acetic acid - DHZ dl-dihydrozeatin - IAA indole-3-acetic acid - 2iP (2-isopentenyl)-adenine - NAA 1-naphthaleneacetic acid - NOA 2-naphthoxyacetic acid - PBA N-benzyl-9-(2-tetrahydropyranyl)adenine - PIC picloram - PVP polyvinylpyrrolidone - TDZ thidiazuron - TIBA 2,3,5-triiodobenzoic acid - ZEA zeatin     

Diurnal Changes in the Malic Acid Concentration in Phaseolus coccineus L. Pulvini

Concentration of malic acid was determined in pulvini and petiolesand in isolated parts of the pulvinus, i.e. extensor and flexorregions, in Phaseolus coccineus. In the light period of thecircadian cycle, the concentration of malic acid in whole pulvinireached the highest value of 35.1 mmole CW while in the darkphase the respective value was 21.0 mmole CW. In the petiole,the highest concentration of malic acid was only 15.3% of themaximum concentration in the whole pulvinus. In isolated regions of motor cells, a cyclic alternation inthe concentration of malic acid was observed. In the light phase,the maximum acid concentration of 43.7 mmole CW in the extensorzone corresponds with the lowest concentration of 15.5 mmoleCW in the flexor region. The lowest value of acid concentrationof 30.8 mmole CW in the extensor part corresponds with the highestacid concentration of 31.1 mmole CW in the flexor part in thedark phase. About 22% of the total concentration of malic acid was transportedbetween the two opposite parts of the pulvinus as dependingupon the phases of leaf movement. (Received February 28, 1986; Accepted May 23, 1986)     

Order of fertilization within the ovary in Phaseolus coccineus L. (Leguminosae)

Summary Phaseolus coccineus typically has six linearly arranged ovules per ovary. The three ovules near the stylar end of the fruit (positions one, two, and three) are more likely to produce mature seeds, to produce heavier seeds, and to produce more vigorous progeny than the ovules in positions near the peduncular/basal end of the fruit (ovule positions four, five, and six). We conducted a series of field experiments designed to supplement our understanding of the mechanisms determining these position effects. We found that approximately 98% of the ovules in 752 fruits were fertilized — about 0.6% of the stylar ovules were not fertilized, whereas 3.2% of the basal ovules were unfertilized. Moreover, we found that only about 49% of the ovules in these 752 fruits produced mature seeds. Over 60% of the stylar ovules produced mature seeds, whereas only 37% of the basal ovules produced mature seeds. Consequently, the proportion of fertilized ovules cannot explain the differences in seed maturation among the ovule positions. We found that after 6.5 h most of the fertilized ovules were located in the stylar ovule positions, and that there were no fertilized ovules in ovule positions five and six, indicating that the stylar ovules are fertilized first. When only the fastest growing pollen tubes were permitted to enter the ovary (due to exision of the style), only the ovules at the stylar end were fertilized, indicating that the ovule positions that are fertilized first are indeed fertilized by the fastest growing pollen tubes.On leave from the Escuela de Biologia, Universidad de Costa Rica, Cuidad Universitaria Rodrigo Facio, San Jose, Costa Rica, Central America     

Embryo development in reciprocal crosses of Phaseolus vulgaris L. and P. coccineus Lam.

Summary Embryo development was examined in reciprocal crosses of Phaseolus vulgaris cv. Great Northern and P. coccineus cv. Scarlet Runner. The formation of abnormal (shrunken and underdeveloped) embryos constituted the primary crossing barrier between the two species when P. coccineus was the female parent. Plants of P. coccineus X P. vulgaris were obtained by embryo culture. Although the P. vulgaris X P. coccineus cross resulted in normal seed development, the fertility of the resulting hybrids was much lower (27%) than that of the reciprocal hybrids (81%). Three classes of F₂ embryos, normal, shrunken, and underdeveloped were formed on reciprocal F₁s and the frequencies did not differ between reciprocal populations. Thus, the interactions between embryo and endosperm and/or maternal parent rather than cytoplasmic-nuclear effects seem to be important in the determination of the extent of embryo growth. The examination of pollen fertility of F₂ plants and the development of F₂ and F₃ embryos suggests that the formation of abnormal embryos and reduced male fertility are independent events. The P. vulgaris — P. coccineus crosses may be useful in studying the possible involvement of interspecific differences in hormonal metabolism in the development of hybrid embryos.     

Identification of gibberellin A1 in the embryo suspensor of Phaseolus coccineus

By combined gas chromatography-mass spectrometry the gibberellin present in suspensors of heart-shaped embryos of Phaseolus coccineus has been identified as Gibberellin A₁ (GA₁). The amount of GA₁ in 2000 suspensors (452 mg), as estimated by gas chromatography. was 4g. The presence of GA₁ in suspensors of P. coccineus is discussed in relation to our present knowledge of the occurrence of many gibberellins in developing seeds and immature fruits of the same species.Abbreviations FID flame ionization detector - GA gibberellin - GC gas chromatography - MS mass spectrometry - PGC preparative gas chromatography - Stage A heart-shaped embryo - Stage B cotytedonary embryo - TMS trimethylsilyl     

Protoplasts from Phaseolus coccineus L. Pulvinar Motor Cells Show Circadian Volume Oscillations

The circadian movement of the lamina of the primary leaf of Phaseolus coccineus is mediated by circadian volume changes of the extensor and flexor cells in the upper and lower half of the laminar pulvinus. Isolated protoplasts from the extensor, flexor, and flank cells of the pulvinus showed a circadian volume rhythm with a period longer than 24 h. In the case of the flexor protoplasts, we found a period length of 28 h, which is similar to that of the pulvinar cells in situ. In the extensor protoplasts, the volume rhythm was synchronized with 14-h light/10-h dark cycles. The larger volume was correlated with the early hours in the light period and the smaller volume with the dark period, as would be expected from the behavior of the extensor cells in situ.     

Protoplasts from Phaseolus coccineus L. Pulvinar Motor Cells Show Circadian Volume Oscillations

The circadian movement of the lamina of the primary leaf of Phaseolus coccineus is mediated by circadian volume changes of the extensor and flexor cells in the upper and lower half of the laminar pulvinus. Isolated protoplasts from the extensor, flexor, and flank cells of the pulvinus showed a circadian volume rhythm with a period longer than 24 h. In the case of the flexor protoplasts, we found a period length of 28 h, which is similar to that of the pulvinar cells in situ. In the extensor protoplasts, the volume rhythm was synchronized with 14-h light/10-h dark cycles. The larger volume was correlated with the early hours in the light period and the smaller volume with the dark period, as would be expected from the behavior of the extensor cells in situ.     

Ribosomal RNA genes of Phaseolus coccineus. I

rDNA fragments, including the whole intergenic spacer (IGS) region of P. coccineus, were cloned into dephosphorylated pUC 13 plasmid. Four clones of different insert size were analysed. Restriction patterns and physical maps of these length variants (pPH1, pPH2, pPH5, pPH6) were performed through complete Eco RI cleavage and partial digestion with Hpa II, Hae III, Sau 3AI, Sma I. Evidence was obtained that the length heterogeneity of the four genes was mainly due to a differing number of about 170 bp sub-repeating elements in the IGS. Indeed, there are 16 of these in pPH1, about 34 in pPH2, 10 in pPH5 and about 60 in pPH6. The sequence analysis of pPH6 sub-repeats revealed that there are two types of sub-repeats: short ones (S) of 162 bp and long ones (L) of 176 bp. The homology between S and L is high (93.8%). S and L elements are present in at least three of the four genes investigated, as shown by a restriction pattern obtained with Hae III digestion to completion. The relative frequency of S and L types, however, differs among the four genes. The possible functional meaning of the subrepeat structure is discussed on the basis of the homology between the S and L sequences on the one hand and on the other the ribosomal sequences of: i) Xenopus promoter(s); ii) wheat block A sub-repeats; iii) presumptive promoter(s) of wheat.     

Aluminum effects on embryo suspensor polytene chromosomes of Phaseolus coccineus L

Aluminum (Al) represents a widespread environmental pollutant, with severe toxic impacts on plants. In this study, we documented for the first time the structural and functional responses induced by two concentrations of AlCl₃ (10^?2 M and 10^?1 M) in the polytene chromosomes that characterize the chromatin organization in the embryo suspensor cells of Phaseolus coccineus. Polytene chromosomes showed signs of dose-dependent genotoxicity following AlCl₃ treatments with a significant increase in both chromatin stickiness and chromatin fragmentation. Polytene chromosomes specifically reacted to AlCl₃ also in terms of DNA and RNA puffing activity: with respect to the control, the treatments promoted ex-novo and/or inhibited puff formation along chromosome arms, suggesting a fine modulation of the differential genome activity in response to the treatments. The nuclei of suspensors from control and treated seeds showed nucleoli mainly arranged by more than one NOR-bearing chromosome. In addition, AlCl₃ treatments affected the frequency of nucleoli organized by singular organizer chromosomes, with an increase in the frequencies of nucleoli organized by chromosome II and a reduction in the frequencies of those organized by chromosomes I or V. These results confirm that, also in our system, nucleolus may react as stress response organelle.     

Abscisic Acid and Apical Dominance in Phaseolus coccineus L. The Role of Tissue Age

Abscisic acid (ABA) applied to the decapitated second internodeof runner bean plants enhanced outgrowth of lateral buds onlywhen internode stumps were no longer elongating. Applied toelongating internodes of slightly younger plants, ABA causesinhibition of bud outgrowth. Together with 10^–4 M indol-3-ylacetic acid (IAA), ABA stimulated internode elongation and interactedadditively in the inhibition of bud outgrowth. A mixture of10^–5 M ABA and 10^–6 M gibberellic acid (GA₃ ) causedsimilar effects on internode growth as IAA + ABA, but was mutuallyantagonistic in effect on growth of the lateral buds. Abscisic acid, apical dominance, gibberellic acid, indol-3yl acetic acid, Phaseolus coccineus, bean     

Genetic diversity and structure of a worldwide collection of Phaseolus coccineus L

Phaseolus coccineus L. is closely related to P. vulgaris and is the third most important cultivated Phaseolus species. Little is known about the patterns of its diversity. In this work, a representative collection of its worldwide diversity was initially developed. The collection includes 28 wild forms (WFs) and 52 landraces (LRs) from Mesoamerica (the crop domestication area), and 148 LRs from Europe (where the crop was introduced in the sixteenth century). The collection was studied by using 12 SSR molecular markers that were developed for the P. vulgaris genome. They were proved to be effective and reliable in P. coccineus in this work. Fourteen LRs of P. dumosus (previously identified as a subspecies of P. coccineus) were also studied. The genetic diversity, population structure and phylogenetic relationships were investigated. The results indicate that: (a) the European and Mesoamerican gene pools are clearly differentiated, (b) a certain reduction of diversity occurred with introduction into Europe, and (c) the Mesoamerican LRs (P. dumosus included) and WFs are closely related and are connected by a high gene flow. Inferences on the domestication process of P. coccineus are also presented. This study provides a picture of the genetic diversity distribution and outcomes with introduction into the Old World, which was not available before. It also underlines that the genetic diversity of both WFs and LRs is an important source for Phaseolus spp. breeding programs and deserves to be preserved in situ and ex situ.     

Glycoproteins from the cell wall of Phaseolus coccineus.

1. The use of a modified sodium chlorite/acetic acid delignification procedure for the solubilization of a hydroxyproline-rich glycoprotein fraction from the depectinated cell walls of Phaseolus coccineus is described. 2. The crude glycoprotein was associated with some pectic material; hydroxyproline and serine were the most abundant amino acids, and arabinose, galactose and galacturonic acid the predominant monosaccharides. 3. The bulk of the hydroxyproline is O-glycosidically substituted with tetra- and tri-arabinofuranosides. From methylation analysis the linkages in these arabinosides could be inferred. 4. Ion-exchange chromatography of the crude glycoprotein gave one major and two minor hydroxyproline-rich fractions, with similar amino acid but different monosaccharide composition. 5. In the major fraction, serine appears to be O-glycosidically substituted with a single galactopyranoside residue that can be removed by the action of alpha-galactosidase but not beta-galactosidase. Removal of arabinofuranoside residues by partial acid hydrolysis greatly enhanced the action of alpha-galactosidase. 6. Methylation followed by carboxy reduction with LiAl2H4 has shown the presence of (1 leads to 4)-linked galacturonic acid in the crude glycoprotein fraction but not in the major fraction from the ion-exchange column. Hence the bulk of the pectic material is not associated with the major glycoprotein component. It is suggested that the glycoprotein is held in the wall by phenolic cross-links. 7. Similarities with the glycopeptide moiety of potato lectin provides further evidence for a class of hydroxyproline-rich glycoproteins with common features.