Gibberellins and the Legume-Rhizobium Symbiosis : I. Endogenous Gibberellins of Lima Bean (Phaseolus lunatus L.) Stems and Nodules

The content of gibberellin-like substances in nodules formed by Bradyrhizobium species strain 127E14 on roots of lima bean (Phaseolus lunatus L.) has been previously found to be relatively high. The objectives of the present study were to purify and identify the endogenous gibberellins from the stems and nodules of lima bean. By sequential silica gel partition column chromatography, C₁₈ reverse-phase high performance liquid chromatography, and combined gas chromatography-mass spectrometry, the gibberellins A₁, A₃, A₁₉, A₂₀, A₂₉, and A₄₄ were identified from root nodules. Gibberellins A₁, A₃, A₁₉, A₂₀, and A₄₄ were also identified from lima bean stem tissue. These data provide the first mass spectral-based evidence that gibberellins are present in leguminous root nodules. The presence of the gibberellins identified indicates that the early 13-hydroxylation gibberellin biosynthetic pathway predominates in stem and nodule tissue. However, it is not known if the gibberellins within the nodules are produced in situ, or if they are imported from some remote host plant tissue.     

Possible Involvement of Hydrogen Peroxide and Salicylic Acid in the Legume-Rhizobium Symbiosis

H₂O₂ content was studied in the roots and epicotyls of pea (Pisum sativum L.) with normal (cultivar Marat) and disturbed (non-nodulating mutant K14a and hypernodulating mutant Nod3) regulation of root nodulation after inoculation with active industrial strain of Rhizobium leguminosarum bv. viceae 250a/CIAM 1026. Pea biotypes differed by H₂O₂ content in roots and epicotyls. Exogenous salicylic acid (SA) (0.2 mM) affected H₂O₂ and SA levels in roots in an inoculation-dependent manner. The involvement of hydrogen peroxide and SA as signaling molecules as well as of antibacterial agents in the pea-rhizobium interaction at the initial stages of symbiosis is proposed.__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 3, 2005, pp. 300–305.Original Russian Text Copyright © 2005 by Glyan’ko, Makarova, Vasil’eva, Mironova.     

Metabolism under Microaerobic Conditions of Mitochondria from Cowpea Nodules

A method is described for isolating mitochondria from nodules of cowpea (Vigna unguiculata [L.] Walp.) under completely anaerobic conditions. The mitochondria were immediately active when incubated aerobically with substrates, and their respiration rates were higher than mitochondria prepared in air. The mitochondria lacked fumarate reductase and were not inhibited by 5% CO₂. When incubated under microaerobic conditions, their respiration could be measured by leghemoglobin spectroscopy. Microaerobic respiration was inhibited approximately 50% by 1 millimolar malonate, and was completely inhibited by cyanide. O₂ uptake and the ATP/O ratio declined under microaerobic conditions, and therefore ATP production may be low in the environment of infected nodule cells.     

The Effect of Ageing on the Leghemoglobin of Cowpea Nodules

Experiments were conducted to study the effect of ageing of the host and senescence of the nodules on the content and composition of the proteins of leghemoglobin in nodules of cowpea (Vigna sinensis L.). Total and heme protein contents of leghemoglobin were the highest in the red nodules and dwindled with the onset of nodule senescence. In the fully senescent green nodules the total proteins decreased to a very low level, while the heme proteins were almost absent. Disc electrophoretograms of leghemoglobin obtained from red and brown nodules showed nine bands out of which only four gave positive test for heme. Greening of the nodules resulted in a considerable reduction in the intensity of the bands. Senescence of the host plant resulted in a reduction in the number of leghemo-globin proteins in all types of nodules and brought about a drastic change in their electrophoretic mobility.     

Lectin Content of the Lima Bean During Development of the Seed and Seedling

The seed of lima beans contains a haemagglutinating substance(lectin) of value in immunochemical research and in typing humanbloods. During the early growth of the seed, this substanceincreases rapidly in comparison with dry weight of the seed,but subsequently increases in proportion to dry weight. Duringgermination of the seed and growth of the seedling, lectin decreasesrapidly but small quantities may be detected in various plantparts. Although the activity of the substance may be inhibitedby N-acetyl-D-galactosamine, a reaction reversible by dialysis,the titre was not increased nor was the specificity changedby dialysis of the fresh extract.     

Formation of Nitrosylleghemoglobin in Nodules of Nitrate-Treated Cowpea and Pea Plants

The formation of nitrosylleghemoglobin (LbNO) was examined incowpea and pea nodules in relation to the inhibition of nitrogenfixation by nitrate. Leghemoglobin was of the ferrous type andwas mainly converted to LbNO in cowpea nodules when the acetylene-reducingactivity decreased to 45% of control values as a result of thesupply of nitrate. In nodules of nitrate-treated pea plants,leghemoglobin was also of the ferrous type and LbNO was a minorcomponent of leghemoglobin. The levels of LbNO isolated fromnodules corresponded to the levels of LbNO calculated from equilibriumconstants for LbNO and the concentration of nitrite in nodules.The dissociation rate constants for LbNO from both cowpea andpea were much smaller than those for LbO₂ or LbCO, as is alsothe case in soybean. These results indicate that the inhibition of the functionsof leghemoglobin, due to the accumulation of LbNO, induces adecrease in nitrogen fixation in cowpea nodules, and that theinhibition of nitrogen fixation in pea nodules is not relatedto the formation of LbNO. (Received July 2, 1990; Accepted October 9, 1990)     

Faba Bean Necrotic Yellows Virus Naturally Infects Phaseolus Bean and Cowpea in the Coastal Area of Syria

In an attempt to identify possible summer hosts of the faba bean necrotic yellows virus (FBNYV), a field survey was conducted in the coastal area of Syria. Using a monoclonal antibody to FBNYV in indirect ELISA, FBNYV was detected in a large number of samples from Phaseolus vulgaris L. and in a few samples from Vigna unguiculata (L.) Walp. in which it caused severe symptoms. This is the first report of natural infection of P. vulgaris and V. unguiculata with FBNYV.     

Growth Activity of the Gibberellins of Dwarf French Bean, Potato, and Lettuce

Zones with similar Rf to gibberellic acid (GA) on chromatogramsof ethylacetate extracts of dwarf French bean and potato leavesand stems and lettuce heads all promoted growth of disks ofetiolated dwarf French bean leaves in darkness. The presence of a gibberellin was confirmed by tests with stemsof dwarf pea, but that the substance was not GA was shown bythe lack of effect in the tests with lettuce hypocotyls. Thetests with dwarf maize, effects on d-3 and d-5, but not d-1,identified it as gibberelliin A₅ (bean factor II). Materialthat promoted the growth of lettuce hypocotyls occurred in zonesclose to the starting-line of chromatograms of extracts of beanand lettuce leaves. Calibration of the growth tests with GA gave an estimate ofthe gibberellin content of the tissues extracted. In the dwarf-peatest the apparent gibberellin contents were similar to the valuesin the leaf-disk test, but very low values occurred in the maizetests.     

Seasonal Distribution of Carbohydrates in Nodules and Stem Exudate from Field-grown Soya Bean Plants

The concentration of carbohydrates in tap root nodules fromfield-grown soya bean [Glycine max (L.) Merr.] plants was verysimilar to the concentration of compounds previously reportedin greenhouse-grown nodules during vegetative growth of seedlings.The concentration of D-pinitol, sucrose and starch in nodulesdeclined during rapid fruit growth, but the concentration ofother compounds did not decline. The availability of carbohydratein nodules during fruit growth did not seem likely to be thecause of the decline in nitrogen-fixing activity of noduleswhich has been reported by others. All compounds except glucoseand , -trehalose declined to concentrations near zero duringa 10-day period of nodule decay. However, the decline in carbohydratedid not appear to cause nodule senescence because it did notprecede the period of decay and because decayed nodules containedsubstantial quantities of glucose and , -trehalose. Seasonalmean concentrations (72 samples from 24 dates) of compounds,in mg carbohydrate per g f. wt of nodule, were: sucrose, 2.84;D-pinitol, 1.14; D-chiro-inositol, 1.27; glucose, 1.40; , -trehalose,1.34; myo-inositol, 0.65; maltose, 0.31; and fructose, 0.21. Quantities of sugars and cyclitols in stem exudate collectedin the field on 13 dates were small (< 10 percent) relativeto the quantity of nitrogenous compounds transported from rootsto shoots. The seasonal pattern of pinitol transport in thexylem was very similar to the seasonal pinitol concentrationin nodules. A large increase in sugar concentration in stemexudate subsequent to 80 days after planting supports the viewthat lack of carbohydrate was not a cause of nodule senescence. Glycine max (L.) Merr, soya bean, cyclitols, , -trehalose, starch, D-pinitol, carbohydrates, root nodules, senescence     

Reexamination of the Intracellular Localization of de Novo Purine Synthesis in Cowpea Nodules

Sucrose and Percoll density gradient centrifugation were used to separate organelles from the central zone tissue of cowpea (Vigna unguiculata L. Walp. cv Vita 3: Bradyrhizobium strain CB 756) nodules. Enzyme activity analysis has shown that both plastids and mitochondria have a full complement of enzymes for de novo purine synthesis. In vitro activities of individual component enzymes (glycinamide ribonucleotide synthetase, EC 6.3.4.13; glycinamide ribonucleotide transformylase, EC 2.1.2.2; aminoimidazole ribonucleotide synthetase, EC 6.3.3.1; aminoimidazole carboxamide ribonucleotide transformylase, EC 6.3.2.6; and adenylosuccinate-AMP lyase, EC 4.3.2.2) as well as of the whole purine pathway (from ribose-5-phosphate to inosine monophosphate) were similar in the two organelles. No significant cytosolic or bacteroidal activity of any of the purine pathway enzymes was detected on assay. These findings are contrary to earlier studies (M.J. Boland, K.R. Schubert [1983] Arch Biochem Biophys 220: 179-187; B.J. Shelp C.A. Atkins, P.J. Storer, D.T. Canvin [1983] Arch Biochem Biophys 224: 429-441) that concluded that enhanced expression of purine synthesis in nodules of ureide-forming species is localized to plastids. Significantly increased recovery of activity of key pathway enzymes (particularly of labile aminoimidazole ribonucleotide synthetase) coupled with improved assay methods and the use of Percoll in addition to sucrose for gradient centrifugation have together contributed to much higher reaction rates and more definitive analyses of particulate fractions.     

The Effect of the Roots on Calcium Ascent in Bean Stems

Bean roots enhance calcium translocation in the stems by supplyingto the ascending sap other previously accumulated cations. Theseions apparently compete with calcium for binding sites in thestem.     

Cadmium-Induced Structural and Ultrastructural Changes in the Vascular System of Bush Bean Stems

The effects of a phytotoxic cadmium concentration (4.45 . 10^?5 M) on the structure and ultrastructure of bean plant stems were analysed by light (LM), transmission electron (TEM) and scanning electron microscopy (SEM). Cadmium significantly reduced both the number and the size of tracheary elements. Cadmium-induced electron dense depositions, which seemed to obstruct partially some xylem vessels, were found only in the later maturing tracheary elements (helical, scalariform or reticulate structure), but not in the annular structured early differentiated ones. Plants exposed to Cd showed less fiber development than the control plants. In the plants treated with Cd abnormally high amounts of calcium oxalate crystals were found in the paratracheary parenchyma cells of the bottom of the stems. The levels of soluble Ca²⁺ in the expressed stem sap of Cd-treated plants was significantly decreased, while substantial amounts of soluble Cd were detected. The probable mechanisms of the structural alterations observed are discussed.     

Gibberellins in Leaves of Alstroemeria hybrida: Identification and Quantification in Relation to Leaf Age

In alstroemeria (Alstroemeria hybrida), leaf senescence is retarded effectively by the application of gibberellins (GAs). To study the role of endogenous GAs in leaf senescence, the GA content was analyzed by combined gas chromatography and mass spectrometry. Five 13-hydroxy GAs (GA₁₉, GA₂₀, GA₁, GA₈, and GA₂₉) and three non-13-hydroxy GAs (GA₉ and GA₄) were identified in leaf extracts by comparing Kováts retention indices (KRIs) and full scan mass sprectra with those of reference GAs. In addition, GA₁₅, GA₄₄, GA₂₄, and GA₃₄ were tentatively identified by comparing selected ion monitoring results and KRIs with those of reference GAs. A number of GAs were detected in conjugated form as well. Concentrations of GAs in alstroemeria changed with the development of leaves. The proportion of biologically active GA₁ and GA₄ decreased with progressive senescence and the fraction of conjugated GAs increased. Received May 26, 1997; accepted August 12, 1997     

Semi-Automatic Quantification of Subsolid Pulmonary Nodules: Comparison with Manual Measurements

Rationale

Accurate measurement of subsolid pulmonary nodules (SSN) is becoming increasingly important in the management of these nodules. SSNs were previously quantified with time-consuming manual measurements. The aim of the present study is to test the feasibility of semi-automatic SSNs measurements and to compare the results to the manual measurements.

Methods

In 33 lung cancer screening participants with 33 SSNs, the nodules were previously quantified by two observers manually. In the present study two observers quantified these nodules by using semi-automated nodule volumetry software. Nodules were quantified for effective diameter, volume and mass. The manual and semi-automatic measurements were compared using Bland-Altman plots and paired T tests. Observer agreement was calculated as an intraclass correlation coefficient. Data are presented as mean (SD).

Results

Semi-automated measurements were feasible in all 33 nodules. Nodule diameter, volume and mass were 11.2 (3.3) mm, 935 (691) ml and 379 (311) milligrams for observer 1 and 11.1 (3.7) mm, 986 (797) ml and 399 (344) milligrams for observer 2, respectively. Agreement between observers and within observer 1 for the semi-automatic measurements was good with an intraclass correlation coefficient >0.89. For observer 1 and observer 2, measured diameter was 8.8% and 10.3% larger (p<0.001), measured volume was 24.3% and 26.5% larger (p<0.001) and measured mass was 10.6% and 12.0% larger (p<0.001) with the semi-automatic program compared to the manual measurements.

Conclusion

Semi-automated measurement of the diameter, volume and mass of SSNs is feasible with good observer agreement. Semi-automated measurement makes quantification of mass and volume feasible in daily practice.     

Antidiabetic Potential of Protein Hydrolysates and Peptide Fractions from Lima Bean (Phaseolus lunatus L): An In Vitro Study

International Journal of Peptide Research and Therapeutics - Characterized by uncontrolled, long-term high blood sugar levels, diabetes mellitus affects ever increasing numbers of people worldwide....