Ureide Metabolism in Non-nodulated Phaseolus vulgaris L.

The distribution of ureide-N was studied throughout vegetativeand reproductive growth of non-nodulated Phaseolus vulgarisL. (bushbean) grown in nitrate nutrient solution. Largest increasesin ureide-N per plant were correlated with flowering and earlypod formation and with seed filling. Highest amounts of ureidesper organ were measured in stems and axillary trifoliates. Highestconcentrations (µmol ureide-N g^–1 fr. wt.) weremeasured in young developing organs and stems. Seeds did notaccumulate ureides until the ureide content of pods had reacheda maximum. Results obtained using the inhibitor of xanthine oxidase, allopurinol,are consistent with the origin of ureides via purine degradationbut alternative pathways cannot be discounted. Leaves and stems were shown to have the ability to degrade allantoatevia an enzymic process.     

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)     

Effects of Different Inorganic Nitrogen Sources on Photosynthetic Carbon Metabolism in Primary Leaves of Non-nodulated Phaseolus vulgaris L

Young bean plants (Phaseolus vulgaris L. var Saxa) were fed with three different types of inorganic nitrogen, after being grown on nitrogen-free nutrient solution for 8 days. The pattern of ¹⁴CO₂ fixation was investigated in photosynthesizing primary leaf discs of 11-day-old plants (3 days with nitrogen source) and in a pulse-chase experiment in 13-day-old plants (5 days with nitrogen source).

Ammonium caused, in contrast to nitrate nutrition, a higher level of ¹⁴C incorporation into sugar phosphates but a lower incorporation of label into malate, glycolate, glycerate, aspartate, and alanine. The labeling kinetics of glycine and serine were little changed by the nitrogen source. Ammonium feeding also produced an increase in the ratio of extractable activities of ribulose-1,5-bisphosphate carboxylase to phosphoenolpyruvate carboxylase and an increase in dark respiration and the CO₂ compensation concentration. Net photosynthesis was higher in plants assimilating nitrate.

The results point to stimulated turnover of the photosynthetic carbon reduction cycle metabolites, reduced phosphoenolpyruvate carboxylation, and altered turnover rates within the photosynthetic carbon oxidation cycle in ammonium-fed plants. Mechanisms of the regulation of primary carbon metabolism are proposed and discussed.

     

Temperature Effects on Mitochondrial Respiration in Phaseolus acutifolius A. Gray and Phaseolus vulgaris L

Electron transport, using succinate as a substrate, was measured polarographically in mitochondria isolated from Phaseolus vulgaris and P. acutifolius plants at 25°C and 32°C. Mitochondria isolated from P. vulgaris plants grown at 32°C had reduced electron transport and were substantially uncoupled. Growth at 32°C had no effect on electron transport or oxidative phosphorylation in P. acutifolius compared to 25°C grown plants. Mitochondria isolated from 25°C grown P. vulgaris plants measured at 42°C were completely uncoupled. Similarly treated P. acutifolius mitochondria remained coupled. The uncoupling of P. vulgaris was due to increased proton permeability of inner mitochondrial membrane. The alternative pathway was more sensitive to heat than the regular cytochrome pathway. At 42°C, no alternative pathway activity was detected. The substantially greater heat tolerance of P. acutifollus compared to P. vulgaris mitochondrial electron transport suggests that mitochondrial sensitivity to elevated temperatures is a major limitation to growth of P. vulgaris at high temperatures and is an important characteristic conveying tolerance in P. acutifolius.     

Nitrogen fixation in breeding lines of Phaseolus vulgaris L.

Nitrogen fixation in nine common bean (Phaseolus vulgaris L.) lines was estimated using the ¹⁵N isotope dilution method at two locations in two seasons. In the first season at one location no N₂ fixation was detected while in the second season up to 51 kg N ha^–1 were estimated. There were significant differences between lines and correlations between trials were significant for the amounts of N₂ fixed, but not for total shoot nitrogen. The plants that fixed the most nitrogen nodulated rapidly after germination. Differences in maximum nodule mass, but not specific nodule activity, were detected also.     

Zinc Nutrition and Starch Metabolism in Phaseolus vulgaris L

The effect of Zn nutrition on leaf starch metabolism was studied in two navy bean (Phaseolus vulgaris L.) varieties, Sanilac and Saginaw. Sanilac is much more susceptible to Zn deficiency than is Saginaw. The variables examined in these two strains were starch content, the activity of soluble starch synthetase (ADP-glucose: starch α-4-glucosyltransferase, EC 2.4.1.b), and the size and number of starch grains. All of these variables decreased during Zn deficiency. The reductions were much greater in Sanilac than in Saginaw. Thus, positive correlations exist between the relative changes in these variables in Sanilac and Saginaw under low Zn and their genotypic difference in growth response to low Zn. These results are taken to purport that the above observations most likely represent characteristic responses to Zn deficiency. We therefore suggest that, as a possible role in plant metabolism, Zn is involved in starch formation.     

Cytokinin Effects on Leaf Architecture in Phaseolus vulgaris L.

Treatment of expanding primary leaves of bean plants (Phaseolnsvulgaris L. cv. Limburgse vroege) with benzyladenine (BA) orkinetin at 0.5 mM for five consecutive days resulted in thickerleaves showing a significant decrease in intercellular air spacevolume. Compared with control plants, exposed mesophyll cellsurface area was lower per unit tissue volume, but unchangedwhen expressed per unit leaf surface area. Stomata of treatedplants were not fully closed in the dark and they did not openas wide as controls in the middle of the light period, suggestingthat the treatment resulted in impaired stomatal action. Allthe effects mentioned were more pronounced after treatment withBA, compared to kinetin. In spite of their magnitude, the observedchanges in leaf structure and function did not seem to havean important effect on total leaf diffusion resistance to carbondioxide during the course of the light period. Key words: Cytokinins, Leaf architecture     

Nitrogen assimilation and partitioning in two nitrogen-fixing cultivars of Phaseolus vulgaris L.

Two cultivars of Phaseolus vulgaris L., one responsive (Mexico 309) and one less-responsive (Rio Tibagi) to nodulation with Rhizobium were grown in Leonard jars in a greenhouse. Bean plants were either inoculated with a strain of Rhizobium leguminosarum bv. phaseoli (UMR-1899), a vesicular-arbuscular mycorrhizal (VAM) fungus (Glomus etunicatum) or were left non-inoculated (controls). At two harvests (21 and 28 days post-emergence), extracts containing soluble proteins and free amino acids were prepared from leaves, roots and nodules of field beans. Nodulated plants contained a significantly higher concentration of protein and amino acids in all plant parts. Nitrogen-fixing beans invested a significantly greater proportion of total N as protein-N and amino acid-N as compared to VAM or control beans. Abundant nodule-specific proteins (nodulins) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), identified and quantified using scanning densitometry. Rio Tibagi nodules contained a significantly lower concentration of glutamine synthetase (GS) subunits than did Mexico 309 nodules. Glutamate synthase (GOGAT) and GS activities were low relative to other legumes. The transferase/synthetase ratio for GS was also low indicating that the synthetase activity was caturated and was operating at maximal level in these young N₂-fixing associations. Specific nodule activity (SNA) and the level of GS were correlated (r=0.90, p<0.05) for both cultivars at both harvests. GS activity was only 8 or 24% higher than SNA in nodules of Mexico 309 or Rio Tibagi cultivars, respectively, under conditions where substrate was not limiting. This suggests that early in the functioning of this symbiosis N assimilation by GS is the rate-limiting step in N₂ fixation by these two bean cultivars, each with a different symbiotic efficiency. Phaseolus breeding programs that attempt to improve N₂ fixation in beans should identify germplasm that expresses elevated levels of nodule-specific GS or GOGAT, and this material should be used along with effective R. leguminosarum bv. phaseoli strains that have already been selected, to determine superior host-microsymciont associations.     

Growth and Nitrogen Fixation of Phaseolus vulgaris L. at Two Irradiances II. Nitrogen Fixation

Phaseolus vulgaris cv. Glamis plants grown at 7 and 28 W m^–228 W m^–2 in controlled environment cabinets showed copiousnodulation and high levels of acetylene reducing activity. Earlydifferences in nodulation were apparent before differences inphotosynthesis and were attributed to an effect of far-red lighton nodule development. Total plant nitrogen content was greater at 28 W m^–2 thanat 7 W ^–2 but nitrogen content as a percentage of d. wtwas greater at the lower irradiance level. Total acetylene reducing activity (nmol. min^–1 root^–1)was greater at 28 W m^–2 than at 7 W ^–2, but therewas no irradiance effect on specific activity (nmol. min^–1g d. wt of pink nodules^–1 or nmol. min^–1 pink nodule^–1). Transfer of 40-day-old plants from 7 W m^–2 to 28 W m^–2resulted in increased nodulesize(due toincreased size of infectedcells), accompanied by increased total, but not specific, acetylenereducing activity. Transfer of plants from 28 W m^–2 to 7 W m^–2 resultedin a fall of total acetylene reducing activity within 24 h,and senescence of large nodules. Specific acetylene reducingactivity was unaffected The results are interpreted as an effect of light on the productionof nitrogen fixing tissue, rather than on nitrogenase activity.     

Phenolic and Oxidative Metabolism as Bioindicators of Nitrogen Deficiency in French Bean Plants (Phaseolus vulgaris L. cv. Strike)

Abstract: The aim of the present work was to determine the effect of abiotic stress, such as nitrogen (N) deficiency, on phenol and oxidative metabolism. In addition, we analyzed whether the response of the two metabolic processes is a good bioindicator of N deficiency in French bean plants. The N was applied to the nutrient solution in the form of NH₄NO₃ at 1.35 mM (N1), 2.7 mM (N2) and 5.4 mM (N3), this latter dosage being considered optimal. The results indicated that application of 1.35 and 2.70 mM of N can be defined as suboptimal or deficient, as it depressed foliar biomass of the French bean plants in our experiment. In addition, abiotic stress from the application of these N dosages stimulated the enzymes PPO, POD and CAT, and inhibited PAL and SOD activities, resulting in the lowest foliar accumulation of phenolic compounds and H₂O₂.     

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.     

Invertase Activity, Carbohydrate Metabolism and Cell Expansion in the Stem of Phaseolus vulgaris L.

In the stem of Phaseolus vulgaris L. the specific activity ofacid invertase was highest in the most rapidly elongating internode.Activity of the enzyme was very low in internodes which hadcompleted their elongation, in young internodes before the onsetof rapid elongation, and in the apical bud. From shortly afterits emergence from the apical bud the elongation of internode3 was attributable mainly to cell expansion. Total and specificactivities of acid invertase in this internode rose to a maximumat the time of most rapid elongation and then declined. Transferof plants to complete darkness, or treatment of plants withgibberellic acid (GA₃), increased the rate of internode elongationand final internode length by stimulating cell expansion. Bothtreatments rapidly increased the total and specific activitiesof acid invertase in the responding internodes; peak activitiesof the enzyme occurred at the time of most rapid cell expansion. In light-grown plants, including those treated with GA₃, rapidcell and internode elongation and high specific activities ofacid invertase were associated with high concentrations of hexosesugar and low concentrations of sucrose. As cell growth ratesand invertase activities declined, the concentration of hexosefell and that of sucrose rose. In plants transferred to darkness,stimulated cell elongation was accompanied by a rapid decreasein hexose concentration and the disappearance of sucrose, indicatingrapid utilization of hexose. No sucrose was detected in theapical tissues of light-grown plants. The results are discussed in relation to the role of acid invertasein the provision of carbon substrates for cell growth. Key words: Cell expansion, Acid invertase, Hexose, Sucrose, Phaseolus     

Photosynthesis, Carbohydrate Metabolism, and Export in Beta vulgaris L. and Phaseolus vulgaris L. during Square and Sinusoidal Light Regimes

Rates of photosynthesis, sucrose synthesis, starch accumulation and degradation were measured in sugar beet (Beta vulgaris L.) and bean (Phaseolus vulgaris L.) plants under a square-wave light regime and under a sinusoidal regime that simulated the natural daylight period. Photosynthesis rate increased in a measured manner in direct proportion to the increasing light level. In contrast to this close correspondence between photosynthesis and light, a lag in photosynthesis rate was seen during the initial hour under square-wave illumination. The leaf appeared to be responding to limits set by carbon metabolism rather than by gas exchange or light reactions. Under the sinusoidal regime starch degradation occurred during the first and last 2 hours of the photoperiod, likely in response to photosynthesis rate rather than directly to light level. Starch broke down when photosynthesis was below a threshold rate and accumulated above this rate. Under square-wave illumination, accumulation of starch did not begin until irradiance was at full level for an hour or more and photosynthesis was at or near its maximum. Under a sinusoidal light regime, sucrose synthesis rate comprised carbon that was newly fixed throughout the day plus that from starch degradation at the beginning and end of the day. Synthesis of sucrose from recently fixed carbon increased with increasing net carbon fixation rate while its formation from degradation of starch decreased correspondingly. The complementary sources of carbon maintained a relatively steady rate of sucrose synthesis under the changing daytime irradiance.     

Physiological and Anatomical Effects of Growth Temperature on Phaseolus vulgaris L. Cultivars

Two Phaseolus vulgaris L. cultivars were grown at 20/15, 25/20,and 30/25 °C day/night temperatures in growth chambers witha 16 h thermoperiod corresponding to the photoperiod. When thefirst trifoliolate leaf was fully expanded rates of CO₂ exchange(CER) were measured at 27 °C and saturating light usinginfrared gas analysis. Stomatal (r_s) and mesophyll resistances,CO₂ compensation points, activities of the enzymes ribulosebisphosphate carboxylase (RuBPCase), glycolate oxidase (GAO),malate dehydrogenase (MDH), and fructose-1, 6 diphosphate (FDP),chlorophyll content, Hill activities, and leaf anatomy at boththe light and electron microscope level were also investigatedin these leaves. Rates of CO₂ exchange in the light, transpiration rate, andchlorophyll content increased with increasing growth temperaturewhile leaf thickness, specific leaf weight, RuBPCase activity,compensation point, and stomatal resistance decreased. Mesophyllresistance also decreased when calculated assuming zero chloroplastCO₂ concentration (rm, o), but not when calculated assuminga chloroplast CO₂ concentration equal to the CO₂ compensationconcentration (rm, g). Average leaf size was maximal in 25/20°C plants while dark respiration, MDH activity, stomataldensity, and starch were minimal. The activities of GAO andFDP and Hill activity were not affected by temperature pretreatment.     

Growth and Nitrogen Fixation of Phaseolus vulgaris L. at Two Irradiances 1. Growth

Plants of Phaseolus vulgaris grown at 7 and 28 W m^–2 showedno differences in rate of development of leaves or flowers.At 7 W m-Z plants had longer internodes, more succulent stemsand leaves, higher ratios of shoot:root and greater leaf areasthat those at 28 W m^–2. These differences were establishedprior to detectable differences in photosynthesis and couldpartly be attributed to an increased proportion of far-red light. Although the final d. wt, carbon content, and fruit yield werehigher at 28 W m^–2, plants at 7 W m^–2 apparentlyhad similar relative growth rates and greater photosyntheticefficiency. Dry weight differences are most easily interpretedas resulting from the establishment of an earlier net carbongain at 28 W m^–2 than at 7 W m^–2.     

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.     

The influence of aluminium availability on phosphate uptake in Phaseolus vulgaris L. and Phaseolus lunatus L.

Aluminium toxicity is one of the major limiting factors of crop productivity on acid soils. High levels of available aluminium in soil may induce phosphorus deficiency in plants. This study investigates the influence of Aluminium (Al) on the phosphate (P_i) uptake of two Phaseolus species, Phaseolus vulgaris L. var. Red Kidney and Phaseolus lunatus L. The two bean species were treated first with solutions of Al at different concentrations (0, 25, 50 and 100 μM, pH 4.50) and second with solutions of P_i (150 μM) at pH 4.50. The higher the Al concentration the higher the Al concentration sorbed but P. vulgaris L var. Red Kidney adsorbed significantly more Al than P. lunatus L. Both species released organic acids: P. vulgaris L var. Red Kidney released fumaric acid and P. lunatus L. fumaric and oxalic acids which could have hindered further Al uptake.The two bean species showed a sigmoid P_i uptake trend but with two different mechanisms. P. vulgaris L var. Red Kidney showed a starting point of 3 h whereas P. lunatus L. adsorbed P_i immediately within the first minutes. In addition, P. vulgaris L var. Red Kidney presented significantly higher P_i uptake (higher uptake rate ‘k’ and higher maximum adsorption ‘a’ of the kinetic uptake model). The Al treatments did not significantly influence P_i uptake. Results suggest that P. lunatus L. might adopt an external Al detoxification mechanism by the release of oxalic acid. P. vulgaris L var. Red Kidney on the other hand seemed to adopt an internal detoxification mechanism even if the Al sorbed is poorly translocated into the shoots. More detailed studies will be necessary to better define Al tolerance and/or resistance of Phaseolus spp.     

Metabolism of gibberellins in a cell-free system from immature seeds of Phaseolus vulgaris L.

The biosynthetic steps from gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) to C₁₉-GAs were studied by means of a cell-free system from the embryos of immature Phaseolus vulgaris seeds. Stable-isotope-labeled GAs were used as substrates and the products were identified by gas chromatography-mass spectrometry. Gibberellin A₁₂-aldehyde was converted to GA₄ via non-hydroxylated intermediates and to GA₁ via 13-hydroxylated intermediates. 13-Hydroxylation took place at the beginning of the pathway by the conversion of GA₁₂-aldehyde to GA₅₃-aldehyde. The conversion of GA₂₀ to GA₅ and GA₆ was also shown but no 2-hydroxylating activity was found. Endogenous GAs from embryos and testas of 17-dold seeds were re-examined by gas chromatography-selected ion monitoring using stable-isotopelabeled GAs as internal standards. Gibberellins A₉, A₁₂, A₁₅, A₁₉, A₂₃, A₂₄, and A₅₃ were identified for the first time in P. vulgaris, in addition to GA₁, GA₄, GA₅, GA₆, GA₈, GA₁₇, GA₂₀, GA₂₉, GA₃₇, GA₃₈ and GA₄₄, which were previously known to occur in this species. The levels of all GAs, except the 2-hydroxylated ones, were greater in the embryos than in the testas. Conversely, the contents of GA₈ and GA₂₉, both 2-hydroxylated, were much higher in the testas than in the embryos.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - GC-SIM gas chromatography-selected ion monitoring - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - m/z ion of mass