Abscisic Acid Accumulation in Developing Seeds of Phaseolus vulgaris L

Free and bound abscisic acid (ABA) in the pod, seed coat, and embryo were determined separately throughout seed development of Phaseolus vulgaris L. cv. `Taylor's Horticultural.' An internal standard method of gas-liquid chromatography was used for ABA quantification. In the embryo, two peaks of free ABA occurred at days 22 (1.18 micrograms per gram or 5.5 micromolar) and 28 (1.74 micrograms per gram or 12 micromolar); and a single peak of bound ABA at day 30. In the seed coat, there was one peak of free ABA at day 22 and only small amounts of bound ABA. Very small amounts of ABA were detected in the pod at any stage of development. In cv. PI 226895, in which seed development is more rapid than in `Taylor's Horticultural,' the embryo ABA peaks occur on days 20 and 26. The timing of the ABA peak in the embryo, and the concentration attained, are consistent with previous reports on the natural pattern of RNA synthesis and with ABA inhibition of RNA synthesis in developing bean fruit.     

Functional aspects of Abscisic Acid Metabolism in Cotyledons of Phaseolus vulgaris L. Seedlings

During the early stages of germination and vegetative development,cotyledons of intact bean (Phaseolus vulgaris L.) seedlingsshowed active ABA catabolism causing a low endogenous ABA content.At the end of the substrate mobilizing phase, when the cotyledonsbecame senescent, a drastic increase of the endogenous ABA contentlinked with a decrease of the ABA catabolic activity was observed.This indicates that a close connection exists between senescenceand endogenous ABA content and metabolism in bean cotyledons. Removal of the apical growth region induced in the cotyledonsactivation of the ABA catabolism and the endogenous ABA concentrationdecreased below the detection limit (0.1 ng/g fr wt) at theonset of the outgrowth of the axillary buds. From then, apicaldominance was restored and the cotyledons returned to the senescentstate, which was correlated with a drastic increase of theirendogenous ABA content. ¹ Bevoegd verklaard navorser N. F. W. O. ² Wetenschappelijk medewerker F. K. F. O. (Received November 25, 1980; Accepted February 13, 1981)     

Pattern of Variations in Abscisic Acid Content in Suspensors, Embryos, and Integuments of Developing Phaseolus coccineus Seeds

Free abscisic acid (ABA) content in suspensors, embryos, and integuments was determined during seed development of Phaseolus coccineus. A highly specific and sensitive solid-phase radioimmunoassay based on a monocional antibody raised against free (S)-ABA was used for ABA quantification. Very small amounts of ABA were detected in the suspensor during initial stages of development; later two peaks of ABA occurred. Levels of ABA in the embryo and integument show a coincident triphasic distribution: two maxima in ABA content occurred when the embryo was 11 to 12 and 15 to 16 millimeters in length; later, when the embryo was 19 to 20 millimeters long, a further increase was observed. The role of ABA in runner bean seeds is discussed in relation to the development of the different seed tissues.     

Characteristics of Membrane-Bound Lectin in Developing Phaseolus vulgaris Cotyledons

Cotyledons of developing Phaseolus vulgaris L. cv Greensleeves seeds were labeled for 2 to 3 hours with ³H-amino acids, and newly synthesized phytohemagglutinin (PHA) was isolated by affinity chromatography with thyroglobulin-Sepharose. The presence of 1% Tween in the homogenate increased the yield of radioactive PHA by 50 to 100%. Isopycnic sucrose gradients were used to show that this detergent-released PHA was associated with the endoplasmic reticulum (ER), and pulse-chase experiments showed that the half-life of the PHA in the ER was 90 to 120 minutes. Since PHA is transiently associated with the ER and accumulates in protein bodies, we postulate that this rapidly turning over pool of PHA in the ER represents protein en route to the protein bodies. The PHA in the ER has the same sedimentation constant as mature PHA and is capable of agglutinating red blood cells. The observations substantiate earlier claims that plant cells contain membrane-bound lectins. However, they also indicate that these lectins are not necessarily functional components of the membranes with which they are associated, but may represent transport pools of lectin normally localized in other cellular compartments.     

Abscisic Acid in Developing Zygotic Embryos of Theobroma cacao

Abscisic acid (ABA) levels were measured by enzyme-linked immunosorbent assay in developing zygotic embryos of Theobroma cacao. ABA was detected in all embryos tested, with a peak of ABA at levels of 1 to 3 micrograms per gram fresh weight during early maturation. This corresponded to embryos of 10 to 30% dry weight and to early stages of anthocyanin and lipid accumulation.     

Synthesis of Lectin-Like Protein in Developing Cotyledons of Normal and Phytohemagglutinin-Deficient Phaseolus vulgaris

The genome of the common bean Phaseolus vulgaris contains a small gene family that encodes lectin and lectin-like proteins (phytohemagglutinin, arcelin, and others). One of these phytohemagglutinin-like genes was cloned by L. M. Hoffman et al. ([1982] Nucleic Acids Res 10: 7819-7828), but its product in bean cells has never been identified. We identified the product of this gene, referred to as lectin-like protein (LLP), as an abundant polypeptide synthesized on the endoplasmic reticulum (ER) of developing bean cotyledons. The gene product was first identified in extracts of Xenopus oocytes injected with either cotyledonary bean RNA or LLP-mRNA obtained by hybrid-selection with an LLP cDNA clone. A tryptic map of this protein was identical with a tryptic map of a polypeptide with the same SDS-PAGE mobility detectable in the ER of bean cotyledons pulse-labeled with either [³H]glucosamine or [³H]amino acids, both in a normal and in a phytohemagglutinin-deficient cultivar (cultivars Greensleeves and Pinto UI 111). Greensleeves LLP has M_r 40,000 and most probably has four asparagine-linked glycans. Pinto UI 111 LLP has M_r 38,500. Unlike phytohemagglutinin which is a tetramer, LLP appears to be a monomer by gel filtration analysis. Incorporation of [³H]amino acids indicates that synthesis of LLP accounts for about 3% of the proteins synthesized on the ER, a level similar to that of phytohemagglutinin.     

Two Kinds of Protein Glycosylation in a Cell-Free Preparation from Developing Cotyledons of Phaseolus vulgaris

Membrane preparations from developing cotyledons of red kidney bean (Phaseolus vulgaris L.) transferred radioactive mannose from GDP-mannose (U-[¹⁴C]mannose) to endogenous acceptor proteins. The transfer was inhibited by the antibiotic tunicamycin, suggesting the involvement of lipidoligosaccharide intermediates typical of the pathway for glycosylation of asparagine residues. This was supported by the similarity of the linkage types of radioactive mannose in lipid-oligosaccharide and glycoprotein products; both contained labeled 2-linked mannose, 3,6-linked and terminal mannose typical of glycoprotein “core” oligosaccharides. As expected for “core” glycosylation, the transfer of labeled N-acetylglucosamine (GlcNAc) from UDP-GlcNAc (6-[³H]GLcNAc) to 4-linkage in endogenous glycoproteins could also be demonstrated. However, most of the radioactive GlcNAc was incorporated into terminal linkage, in a reaction insensitive to tunicamycin. The proteins receiving “core” oligosaccharide in vitro were heterogeneous in size, in contrast to those receiving most of the GlcNAc (which chiefly comprised the seed reserve-proteins phaseolin and phytohemagglutinin). It is suggested that following “core” glycosylation, single GlcNAc residues are attached terminally to the oligosaccharides of these seed proteins, without the involvement of lipid-linked intermediates. Phaseolin from mature seeds does not possess a significant amount of terminal GlcNAc and so it is possible that these residues are subsequently removed in a processing event.     

Abscisic Acid Decreases Leaf Na+ Exclusion in Salt-Treated Phaseolus vulgaris L.

Previous results showed that in short-term NaCl-treated beans increased leaf abscisic acid (ABA) concentration was triggered by Na⁺ but not by Cl^-. In this work, the specificity of ABA signaling for Na⁺ homeostasis was studied by comparing the plant’s responses to solutions that modified accumulation of ABA and/or Na⁺ uptake and distribution, such as supplemental Ca²⁺, increased nutrient strength, different isosmotic composition, application of exogenous ABA, fluridone (an ABA inhibitor) and aminooxiacetic acid (AOA, an ethylene inhibitor). After fluridone pretreatment, salt-treated beans had lower Na⁺ uptake and higher leaf Na⁺ exclusion capacity than non-pretreated plants. Moreover, Na⁺ uptake was increased and leaf Na⁺ exclusion was decreased by AOA and ABA. NaCl and KCl similarly increased leaf ABA and decreased transpiration rates, whereas supplemental Ca²⁺ and increased strength nutrient solution decreased leaf ABA and leaf Na⁺. These results show (1) a non-ion-specific increase in ABA that probably signaled the osmotic component of salt, and (2) increased ABA levels that resulted in higher leaf Na⁺ concentrations due to lower Na⁺ exclusion or increased root-shoot Na⁺ translocation.     

Physiological Changes in Phaseolus vulgaris in Response to Long-term Ozone Exposure

The effects of ozone on Phaseolus vulgaris cv. Lit were investigatedusing open-top chambers (OTCs) ventilated with charcoal andPurafil filtered air (CF treatments), ambient air (NF treatments)and ambient air to which low, medium or high concentrationsof ozone were added (NFL, NFM and NFH). Ozone additions of 8,16 and 23 nl l^–1 were made during phase 1 of the experiment(0–44 d after emergence, DAE), and additions of 15, 30and 47 nl l^–1 were made during phase 2 (45–99 DAE).Ozone was added to the chambers between 1100 and 1800 h GMT,for 3 or 4 consecutive days each week. The seasonal 7-h meanozone concentrations were 8, 21, 27, 33 and 38 nl l^–1in the CF, NF, NFL, NFM and NFH treatments, respectively. No visible symptoms of ozone injury or significant physiologicalchanges were detected in P. vulgaris during phase I of the experiment.In phase 2, the photosynthesis (Pn) and stomatal conductance(gs) of NFH-plants were inhibited by 73% and 86%, respectively,during ozone exposure, and recovered to pre-exposure valueson the following day. These observations were made prior tothe appearance, 60 DAE, of bronze lesions on the leaves of NFH-plants.The photosynthetic capacity and gs of NFH-leaves decreased asthe severity of ozone injury increased. Rates of weight lossfrom excised leaves also increased with increasing ozone injury.Microscopic investigations of the bronzed regions revealed extensivecellular breakdown, including tonoplast and chloroplast enveloperupture, and the aggregation of the cytoplasmic contents towardsone end of the cell. Severely damaged leaves abscised from the plants, resultingin premature canopy senescence in the NFM and NFH treatments.This, coupled with the lower photosynthetic capacity of existingleaves led to 25 % lower yield in the NFH than the NF treatment(P < 0.05). Phaseolus vulgaris, green bean, ozone, symptom development, photosynthesis, cell ultrastructure     

Changes in Cell Fine Structure in the Cotyledons of Phaseolus vulgaris L. during Germination

When germination begins, the storage cells of Phaseolus vulgariscotyledons are packed with starch grains and protein bodies.Digestion of these reserves starts in cells furthest away fromthe vascular bundles and is practically completed in eight daysat 25° C. After the reserves are hydrolysed, the storagecells die. The changes in fine structure during the processof digestion and protoplasmic breakdown are described. Vascularbundle and epidermal cells survive till the cotyledons absciss,but in these tissues also profound changes occur in cellularorganization. The observations on fine structure are discussedwith reference to the metabolic activities of the cotyledons.     

Involvement of Abscisic Acid in Regulating Water Status in Phaseolus vulgaris L. during Chilling

During the first hours of chilling, bean (Phaseolus vulgaris L., cv Mondragone) seedlings suffer severe water stress and wilt without any significant increase in leaf abscisic acid (ABA) content (P. Vernieri, A. Pardossi, F. Tognoni [1991] Aust J Plant Physiol 18: 25-35). Plants regain turgor after 30 to 40 h. We hypothesized that inability to rapidly synthesize ABA at low temperatures contributes to chilling-induced water stress and that turgor recovery after 30 to 40 h is mediated by changes in endogenous ABA content. Entire bean seedlings were subjected to long-term (up to 6 d) chilling (3°C, 0.2-0.4 kPa vapor pressure deficit, 100 μmol·m⁻²·s⁻¹ photosynthetic photon flux density, continuous fluorescent light). During the first 24 h, stomata remained open, and plants rapidly wilted as leaf transpiration exceeded root water absorption. During this phase, ABA did not accumulate in leaves or in roots. After 24 h, ABA content increased in both tissues, leaf diffusion resistance increased, and plants rehydrated and regained turgor. No osmotic adjustment was associated with turgor recovery. Following turgor recovery, stomata remained closed, and ABA levels in both roots and leaves were elevated compared with controls. The application of ABA (0.1 mm) to the root system of the plants throughout exposure to 3°C prevented the chilling-induced water stress. Excised leaves fed 0.1 mm ABA via the transpiration stream had greater leaf diffusion resistance at 20 and 3°C compared with non-ABA fed controls, but the amount of ABA needed to elicit a given degree of stomatal closure was higher at 3°C compared with 20°C. These findings suggest that endogenous ABA may play a role in ameliorating plant water status during chilling.     

Abscisic Aldehyde Is an Intermediate in the Enzymatic Conversion of Xanthoxin to Abscisic Acid in Phaseolus vulgaris L. Leaves

The enzymatic conversion of xanthoxin to abscisic acid by cell-free extracts of Phaseolus vulgaris L. leaves has been found to be a two-step reaction catalyzed by two different enzymes. Xanthoxin was first converted to abscisic aldehyde followed by conversion of the latter to abscisic acid. The enzyme activity catalyzing the synthesis of abscisic aldehyde from xanthoxin (xanthoxin oxidase) was present in cell-free leaf extracts from both wild type and the abscisic acid-deficient molybdopterin cofactor mutant, Az34 (nar2a) of Hordeum vulgare L. However, the enzyme activity catalyzing the synthesis of abscisic acid from abscisic aldehyde (abscisic aldehyde oxidase) was present only in extracts of the wild type and no activity could be detected in either turgid or water stressed leaf extracts of the Az34 mutant. Furthermore, the wilty tomato mutants, sitiens and flacca, which do not accumulate abscisic acid in response to water stress, have been shown to lack abscisic aldehyde oxidase activity. When this enzyme fraction was isolated from leaf extracts of P. vulgaris L. and added to extracts prepared from sitiens and flacca, xanthoxin was converted to abscisic acid. Abscisic aldehyde oxidase has been purified about 145-fold from P. vulgaris L. leaves. It exhibited optimum catalytic activity at pH 7.25 in potassium phosphate buffer.     

Metabolism of Abscisic Acid in Developing Seeds of Phaseolus vulgaris L. and its Correlation to Germination and {alpha}-Amylase Activity

Bean seeds were unable to germinate during their developmentwhich was characterized either by an increasing ABA content(expressed as ng ABA/seed) or by a fairly high and constantABA concentration (expressed as ng ABA g^–1 fr. wt). During seed maturation the mother plant induces a dormant stateby depressing the ABA catabolism and keeping the endogenousABA at a high level to prevent both the premature hydrolysisof starch (-amylase activity) and the germination of the morphogeneticallyadult yet immature seeds. The depth of this induced dormancyis positively correlated with endogenous ABA concentration. Application of exogenous ABA to fully mature seeds, which containno endogenous ABA and show a very active ABA catabolism, onlyprolonged the lag phase but had no influence on the furtherdevelopment of the -amylase activity.     

Changes after Decapitation in Concentrations of Indole-3-Acetic Acid and Abscisic Acid in the Larger Axillary Bud of Phaseolus vulgaris L. cv Tender Green

Early changes in the concentrations of indole-3-acetic acid (IAA) and abscisic acid (ABA) were investigated in the larger axillary bud of 2-week-old Phaseolus vulgaris L. cv Tender Green seedlings after removal of the dominant apical bud. Concentrations of these two hormones were measured at 4, 6, 8, 12 and 24 hours following decapitation of the apical bud and its subtending shoot. Quantitations were accomplished using either gas chromatography-mass spectrometry-selected ion monitoring (GS-MS-SIM) with [¹³C₆]-IAA or [²H₆]-ABA as quantitative internal standards, or by an indirect enzyme-linked immunosorbent assay, validated by GC-MS-SIM. Within 4 hours after decapitation the IAA concentration in the axillary bud had increased fivefold, remaining relatively constant thereafter. The concentration of ABA in axillary buds of decapitated plants was 30 to 70% lower than for buds of intact plants from 4 to 24 hours following decapitation. Fresh weight of buds on decapitated plants had increased by 8 hours after decapitation and this increase was even more prominent by 24 hours. Anatomical assessment of the larger axillary buds at 0, 8, and 24 hours following decapitation showed that most of the growth was due to cell expansion, especially in the intermodal region. Thus, IAA concentration in the axillary bud increases appreciably within a very few hours of decapitation. Coincidental with the rise in IAA concentration is a modest, but significant reduction in ABA concentration in these axillary buds after decapitation.     

Abscisic Acid Accumulation and Water Relations of Four Cultivars of Phaseolus vulgaris L. under Drought

Larqué-Saavedra, A., Rodriguez, M. T., Trejo, C. andNava, T. 1985. Abscisic acid accumulation and water relationsof four cultivars of Phaseolus vulgaris L. under drought.—J.exp. Bot 36: 1787–1792. Plants of four cultivars of Phaseolus vulgaris L. differingin drought resistance were grown in pots under greenhouse conditionsand prior to flowering water was withheld from the pots untilthe mid-day transpiration rate reached values below 1.0 µgH₂O cm^–2 s^–1 (designated the ‘drought’stage). At this point leaves were harvested on 3 or 4 occasionsover 24 h to determine the abscisic acid (ABA) concentration,total water potential (), solute potential (₁) and turgor potential(_p). Results showed that values of , ₁, and _p differed between cultivarswhen they reached the ‘drought’ stage. The stomatalsensitivity to changes in and _p, was as follows: Michoacán12A3 > Negro 150 Cacahuate 72 > Flor de Mayo. These datacorrelated well with the pattern of drought resistance reportedfor the cultivars. ABA accumulation at the ‘drought’ stage differedbetween cultivars at each sampling time, but overall differencesin ABA level between cultivars were not significant. ABA levelsdid not, therefore, correlate with the drought resistance propertiesreported for the cultivars. Results are discussed in relationto and hour of the day when bean samples were taken for ABAanalysis. Key words: Phaseolus vulgaris L., drought resistance, abscisic acid     

Effect of Fruits on Dormancy and Abscisic Acid Concentration in the Axillary Buds of Phaseolus vulgaris L

The mechanism regulating the growth of adult plants in two determinate bean (Phaseolus vulgaris L.) cultivars was investigated. “Redkloud” plants flowered, formed fruits, and ceased shoot growth earlier than “Redkote” plants. Redkloud attained a smaller plant size, compared to Redkote, by imposing dormancy on axillary buds at an earlier age. In both cultivars, cessation of bud growth coincided with maximum combined fruit length per plant. Removal of fruits caused resumption of axillary bud growth within 4 to 5 days. The amount of new growth induced by fruit removal depended on the cultivar and plant age. In fully developed Redkloud plants, where shoot growth had already ceased, total leaf and shoot number per plant nearly doubled within 2 weeks following fruit removal. A much smaller response was observed in the still growing Redkote plants. Fruits, therefore, are assumed to play a major role in the regulation of shoot growth and total plant size through the control of axillary bud dormancy. It seems that smaller plant size, earlier maturity, and earlier senescence of Redkloud, compared to Redkote, were the result of earlier flowering, and accomplished in part through the growth-inhibiting action of fruits.     

Effect of Older Fruits on Abortion and Abscisic Acid Concentration of Younger Fruits in Phaseolus vulgaris L

In two cultivars of bean (Phaseolus vulgaris L., Redkloud and Redkote) the older fruits growing at the base of racemes aborted less frequently than the younger ones above them. When older fruits at the base of racemes were removed, the abortion rate of the younger ones was reduced and their abscisic acid (ABA) concentration was lowered. Thirteen days after fruit removal, 36 to 45% of the younger fruits remained viable on treated plants while less than 12% of the younger fruits were viable on control plants. On these intact controls the ABA concentration of young fruits was at least twice that of defruited plants. A similar difference was found when the ABA content was expressed on a per fruit basis, suggesting a direct regulatory influence of older fruits over the ABA content of younger fruits.     

Alleviation of Salt Stress in Common Bean (Phaseolus vulgaris) by Exogenous Abscisic Acid Supply

In this work the effect of abscisic acid (ABA) and 100 mM NaCl on common bean (Phaseolus vulgaris var. Coco) growth, nitrogenase activity, and nodule metabolism was studied. Experiments were carried out in a controlled environmental chamber and plants, at the vegetative growth stage (16 days old), were treated with ABA (1 μM and 10 μM) and 48 h later were exposed to saline treatment. Results revealed that plant dry weight, nodule dry weight, nitrogen fixation (acetylene reduction activity and ureides content), and most enzymes of ammonium and ureides metabolism were affected by both ABA and NaCl. The addition of 1 μM ABA to the nutrient solution before the exposure to salt stress reduced the negative effect of NaCl. Based on our results, we suggest that ABA application improves the response of Phaseolus vulgaris symbiosis under saline stress conditions, including the nitrogen fixation process and enzymes of ammonium assimilation and purine catabolism.     

Cytokinins in Germinating Seeds of Phaseolus vulgaris L. I. Changes in Endogenous Levels Within the Cotyledons

Ethanolic extracts from the cotyledons of mature dry Phaseolusvulgaris L. seed yielded cytokinin-like activity which co-chromatographedwith zeatin and ribosylzeatin. Under conditions which stimulatedgermination and cotyledon expansion, the level of these cytokininsdecreased rapidly in both intact embryos and excised cotyledons.In the excised cotyledons the decrease was continuous, resultingin very low levels of cytokinin being detected after 4 daysof incubation. With the embryonic axis present, however, theinitial decrease was arrested and reversed after 3 days. Thissuggests that the cotyledons do not synthesize cytokinins butthat these hormones are imported from the embryonic axis, particularlyonce radicle growth is well under way. Phaseolus vulgaris, bean, cotyledons, cytokinins, germination