Nitrogen Fixation and Allantoin Formation in Soybean Plants

The activity of nitrogenase and the concentration of ammonia and allantoin (+ allantoic acid) in root nodules were measured throughout the growth period of soybean plants. Nitrogenase activity measured by acetylene reduction increased with plant growth and reached a maximum level at the flowering period. The level of ammonia and allantoin concentration in nodules was parallel with increased nitrogenase activity. At the late reproductive stage (pod-forming period), nitrogenase activity showed a marked decrease, but the ammonia and allantoin in the nodules remained at a constant level. Detached nodules from 56 day-old soybean plants were exposed to ¹⁵N₂ gas, and the distribution of ¹⁵N among nitrogen compounds was investigated. Enrichment of ¹⁵N in allantoin and allantoic acid reached a fairly high level after 90 min of nitrogen fixation; ca. 22% of ¹⁵N in acid-soluble nitrogen compounds was incorporated into allantoin + allantoic acid. In contrast, enrichment of ¹⁵N in amide nitrogen was relatively low. No significant ¹⁵N was detected in the RNA fraction. The data suggested that ureide formation in nitrogen-fixing root nodules did not take place through the breakdown of nucleic acids, but directly associated with the assimilating system of biologically fixed nitrogen.     

Leaf Senescence and Abscisic Acid in Leaves of Field-grown Soybean

Leaf senescence in field-grown soybean (Merrill) as defined by the period after full expansion, was studied by measuring abscisic acid (ABA), total soluble protein, and chlorophyll in leaves through the later part of the growing season. ABA concentrations increased significantly at the end of the season when leaves had started to turn yellow, well after total soluble protein and chlorophyll had started to decline. The results indicate that events occurring before leaf yellowing are more significant in evaluating leaf senescence since the yellowing condition and rise in ABA are effects of changes in physiological activity beginning when leaves are still green.     

Allantoic Acid Synthesis in Soybean Root Nodule Cytosol via Xanthine Dehydrogenase

Allantoin and allantoic acid are the major forms of nitrogen transported from soybean nodules to other parts of the plant. Neither the pathway or the site of ureide synthesis has been demonstrated in root nodules.     

Allometric Relationships in Field-grown Soybean

Allometric relationships in plants uncover size-correlated variationsin form and development and characterize the relative growthof a part of a plant in comparison with a whole. Stable allometricrelationships in ontogeny can be used as components of cropmodels and to estimate plant parameters that are difficult tomeasure. Our objective was to discover whether stable allometricrelations exist in ontogeny of field-grown soybeans (GlycinemaxL.). We used field data on vegetative stages, plant height,stem weight, and leaf weight of 16 soybean cultivars measuredon farms and on the experiment station of Mississippi StateUniversity during 1993–1995 growing seasons. The numberof observed crops for each cultivar ranged from one to 14. Stemheights displayed linear log-log dependencies on the vegetativestage, before and after the breakpoint stage, which typicallywas between the ‘fourth node’ and ‘sixth node’vegetative stages. Slopes of the log-log dependencies afterthe breakpoint stage were similar in all cultivars. Stem masshad log-log linear dependencies on stem height. Slopes of thesedependencies differed among cultivars grown under the same conditions,and among crops of the same cultivar grown under different conditions.Water stress could be a modifier of these relationships. Theproportion of leaf weights in the total weight of leaves andstems decreased linearly as the vegetative development progressed.Since allometric relations are stable for a specific crop, theycan be used to forecast vegetative development as soon as theyare established.Copyright 1998 Annals of Botany Soybean,Glycine maxL., allometry, vegetative development.     

Allantoin and Allantoic Acid in the Nitrogen Economy of the Cowpea (Vigna unguiculata [L.] Walp.)

The ureides, allantoin and allantoic acid, represented major fractions of the soluble nitrogen pool of nodulated plants of cowpea (Vigna unguiculata [L.] Walp. cv. Caloona) throughout vegetative and reproductive growth. Stem and petioles were the principal sites of ureide accumulation, especially in early fruiting.

Labeling studies using ¹⁴CO₂ and ¹⁵N₂ and incubation periods of 25 to 245 minutes indicated that synthesis of allantoin and allantoic acid in root nodules involved currently delivered photosynthate and recently fixed N, and that the ureides were exported from nodule to shoot via the xylem. From 60 to 80% of xylem-borne N consisted of ureides; the remainder was glutamine, asparagine, and amino acids. Allantoin predominated in the soluble N fraction of nodules and fruits, allantoin and allantoic acid were present in approximately equal proportions in xylem exudate, stems, and petioles.

Extracts of the plant tissue fraction of nitrogen-fixing cowpea nodules contained glutamate synthase (EC 2.6.1.53) and glutamine synthetase (EC 6.3.1.2), but little activity of glutamate dehydrogenase (EC 1.4.1.3). High levels of uricase (EC 1.7.3.3) and allantoinase (EC 3.5.2.5) were also detected. Allantoinase but little uricase was found in extracts of leaflets, pods, and seeds.

Balance sheets were constructed for production, storage, and utilization of ureide N during growth. Virtually all (average 92%) of the ureides exported from roots was metabolized on entering the shoot, the compounds being presumably used as N sources for protein synthesis.

     

Isolating Stem Cells from Soft Musculoskeletal Tissues

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1^st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures. Download video file.^{(88M, mp4)}     

The Biosynthesis of delta-Aminolevulinic Acid in Higher Plants: II. Formation of C-delta-Aminolevulinic Acid from Labeled Precursors in Greening Plant Tissues

δ-Aminolevulinic acid was accumulated by greening cucumber (Cucumis sativus L. var. Alpha green) cotyledons, barley (Hordeum sativum var. Numar) leaves, and bean (Phaseolus vulgaris L. var. Red Kidney) leaves in the presence of various ¹⁴C-labeled precursors and levulinic acid, a competitive inhibitor of δ-aminolevulinic acid dehydrase. The radioactivity in the accumulated δ-aminolevulinic acid was measured.     

Effects of Allopurinol [4-Hydroxypyrazolo(3,4-d)Pyrimidine] on the Metabolism of Allantoin in Soybean Plants

Some studies on the effects of xanthine oxidase inhibitor allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine] on allantoin metabolism of soybean plants (Glycine max cv. Tamanishiki) are reported. Soybean seedlings, aseptically germinated for 96 hours on agar containing 1 millimolar allopurinol, contained only slight amounts of allantoin, allantoic acid, and urea as compared with controls. Analysis of purines and pyrimidines of the allopurinol-treated seedlings showed marked accumulation of xanthine both in the cotyledons and seedling axes. No hypoxanthine accumulation was found. Xanthine accumulation due to allopurinol treatment was relatively low after the cotyledons had fallen. For nodulated plants, allopurinol caused a significant drop in allantoin (+allantoic acid) in the stems and nodules, accompanied by a striking accumulation of xanthine in the nodules. The xanthine concentration in the nodules far exceeded that in the germinated seedlings. Allopurinol at a concentration of 50 micromolar strongly inhibited xanthine oxidase prepared from soybean nodules.

The results suggested that the main pathway of allantoin formation in soybean plants was through purine decomposition, via xanthine-uric acid. It was specially noted that a very active purine-decomposing system existed in soybean nodules.

     

Stem Cells and Stem Cell-derived Tissues and Their Use in Safety Assessment

Toxicology has long relied on animal models in a tedious approach to understanding risk of exposure to an uncharacterized molecule. Stem cell-derived tissues can be made in high purity, quality, and quantity to enable a new approach to this problem. Currently, stem cell-derived tissues are primarily “generic” genetic backgrounds; the future will see the integration of various genetic backgrounds and complex three-dimensional models to create truly unique in vitro organoids. This minireview focuses on the state of the art of a number of stem cell-derived tissues and details their application in toxicology.     

The Biosynthesis of delta-Aminolevulinic Acid in Higher Plants: I. Accumulation of delta-Aminolevulinic Acid in Greening Plant Tissues

δ-Aminolevulinic acid dehydrase activity in cucumber (Cucumis sativus L. var. Alpha green) cotyledons did not change as the tissue was allowed to green for 24 hours. δ-Aminolevulinic acid accumulated in greening cucumber cotyledons, and barley (Hordeum sativum L. var. Numar) and bean (Phaseolus vulgaris L. var. Red Kidney) leaves incubated in the presence of levulinic acid, a specific competitive inhibitor of δ-aminolevulinic acid dehydrase. The rate of δ-aminolevulinic acid accumulation in levulinic acid-treated cucumber cotyledons paralleled the rate of chlorophyll accumulation in the controls, and the quantity of δ-aminolevulinic acid accumulated compensated for the decrease in chlorophyll accumulation. When levulinic acid-treated cucumber cotyledons were returned to darkness, δ-aminolevulinic acid accumulation ceased.     

Ent-kaurenic Acid and Its Related Compounds from Glandular Trichome Exudate and Leaf Extracts of Polymnia sonchifolia

Yacon (Polymnia sonchifolia) leaves possess glandular trichomes on the surface. The exudate from the glandular trichome and the leaf are itself rich in ent-kaurenic acid (ent-kaur-16-en-19-oic acid). A kaurene derivative, 15-α-angeloyloxy-ent-kauren-19-oic acid 16-epoxide, was isolated from the leaves, together with two known angeloyloxykaurenic acids. The high content of ent-kaurenic acid in the leaf suggests that these diterpenes play a certain physiological role, since the glandular trichome exudates of other species function in their defensive mechanism.     

Ascorbic Acid in Neural Tissues

Abstract: Large amounts of ascorbic acid were readily removed from neural tissue by washing with warmed saline solutions. In areas where the original level was highest, such as cortex and cerebellum, a higher percentage was removed than from areas of lower concentration, such as pons-medulla. The residual level in both types of tissue was similar. During scurvy, the ascorbic acid retained in the guinea pig brain is more readily removed by washing than is that of the normal brain.     

Exogenous Jasmonic and Abscisic Acids Act Differentially in Elongating Tissues from Oat Stem Segments

Segments can be cut from the peducular-1 internode of oat (Avena sativa L.) shoots so as to contain the graviresponsive, auxin-sensitive leaf sheath pulvinus, and the gibberellin-sensitive internodal tissue. These two growth-capable tissues were used to study the effects and interactions of jasmonic acid (JA) and abscisic acid (ABA) in regulating cell elongation. When supplied alone at physiologic concentrations (10⁻⁵, 10⁻⁴ m), JA promoted growth and cell wall synthesis in the internodal tissue, whereas by itself, ABA inhibited internodal elongation and even inhibited JA-promoted growth. When gibberellic acid (GA₃) was used to stimulate internodal elongation, JA and ABA caused similar levels of inhibition and, at certain concentrations, were synergistic. Inhibition by ABA was initiated several hours earlier than inhibition by JA, and only the ABA effect could be partially overcome by 10⁻³ m aminoethoxyvinylglycine. Both JA and ABA inhibited elongation of pulvinar tissue that was induced to grow by gravistimulus or auxin, although here JA was more potent than ABA at equimolar concentrations. When 10⁻⁵ m fusicoccin was used as a general nonphysiologic growth stimulus, JA had no effect on the internode but inhibited the pulvinus, whereas ABA had no effect on the pulvinus but inhibited the internode. These results provide strong physiologic evidence that JA and ABA act by different mechanisms in the regulation of elongation, at least in this representative grass. Received May 28, 1996; accepted November 7, 1996     

Sucrose-Starch Conversion in Heterotrophic Tissues of Plants

The historical progress in recent years pertaining to the sucrose-starch conversion in heterotrophic tissues of plants has been described. Special attention has been focused on the enzymatic breakdown of sucrose to produce hexose units that are transported to the amyloplast compartment by means of specific translocator molecules and act as glucose donors for starch biosynthesis. Although the current prevailing view is that variable mechanisms operate in different plant tissues and organs, it is often argued that the following enzymic steps are essential in the overall step of sucrose to starch conversion: sucrose + UDP -> UDPGlc + Fru(sucrose synthase-SS) (UDPGlc UDPGlc + PPi -» G1P + UTPpyrophosphorylase-UGPase) (ADPGlc GlP + ATP -» ADPGlc + PPipyrophosphorylase-AGPase) (starch ADPGlc -> starchsynthase) The presence of an ADPGlc-specific translocator in the amyloplast envelope has been demonstrated in a number of plant sources, which indicates the potential role of ADPGlc-synthesizing machineries located in the cytosol of starch-storing cells. Although it was initially believed that AGPase is present exclusively in the amyloplast compartment, the presence of a cytosolic enzyme has been shown in some cereals. The SS has a potential to produce ADPGlc, but the general belief is that this is not a dominant reaction in the mechanism of starch biosynthesis. Numerous experimental trials have been reported by many scientists employing transgenic plants transformed with cDNAs either in antisense- or sense- orientation encoding enzymes which are presumably involved in the process of sucrose-starch conversion. Although great caution is needed to interpret the data obtained, the general picture is contradictory to the mechanism presented above. It now appears that serious reconsideration is needed for the possible mechanism of SS-catalyzed ADPGlc formation and its subsequent link to starch formation. In the newly proposed mechanistic scheme, which appears to be consistent with the results by other scientists as well, hexokinase, phosphoglucomutase (PGM), and ADPGlc formation by AGPase are components in the cyclic turnover of starch molecules in the amyloplast compartment.