Dale G. Blevins

Characterization of FRO1, a Pea Ferric-Chelate Reductase Involved in Root Iron Acquisition

To acquire iron, many plant species reduce soil Fe(III) to Fe(II) by Fe(III)-chelate reductases embedded in the plasma membrane of root epidermal cells. The reduced product is then taken up by Fe(II) transporter proteins. These activities are induced under Fe deficiency. We describe here the FRO1 gene from pea (Pisum sativum), which encodes an Fe(III)-chelate reductase. Consistent with this proposed role, FRO1 shows similarity to other oxidoreductase proteins, and expression of FRO1 in yeast conferred increased Fe(III)-chelate reductase activity. Furthermore,FRO1 mRNA levels in plants correlated with Fe(III)-chelate reductase activity. Sites of FRO1expression in roots, leaves, and nodules were determined.FRO1 mRNA was detected throughout the root, but was most abundant in the outer epidermal cells. Expression was detected in mesophyll cells in leaves. In root nodules, mRNA was detected in the infection zone and nitrogen-fixing region. These results indicate that FRO1 acts in root Fe uptake and they suggest a role in Fe distribution throughout the plant. Characterization of FRO1 has also provided new insights into the regulation of Fe uptake. FRO1expression and reductase activity was detected only in Fe-deficient roots of Sparkle, whereas both were constitutive in brzand dgl, two mutants with incorrectly regulated Fe accumulation. In contrast, FRO1 expression was responsive to Fe status in shoots of all three plant lines. These results indicate differential regulation of FRO1 in roots and shoots, and improper FRO1 regulation in response to a shoot-derived signal of iron status in the roots of thebrz and dgl mutants.

The Arabidopsis AtOPT3 Protein Functions in Metal Homeostasis and Movement of Iron to Developing Seeds

The Arabidopsis thaliana AtOPT3 belongs to the oligopeptide transporter (OPT) family, a relatively poorly characterized family of peptide/modified peptide transporters found in archebacteria, bacteria, fungi, and plants. A null mutation in AtOPT3 resulted in embryo lethality, indicating an essential role for AtOPT3 in embryo development. In this article, we report on the isolation and phenotypic characterization of a second AtOPT3 mutant line, opt3-2, harboring a T-DNA insertion in the 5′ untranslated region of AtOPT3. The T-DNA insertion in the AtOPT3 promoter resulted in reduced but sufficient AtOPT3 expression to allow embryo formation in opt3-2 homozygous seeds. Phenotypic analyses of opt3-2 plants revealed three interesting loss-of-function phenotypes associated with iron metabolism. First, reduced AtOPT3 expression in opt3-2 plants resulted in the constitutive expression of root iron deficiency responses regardless of exogenous iron supply. Second, deregulation of root iron uptake processes in opt3-2 roots resulted in the accumulation of very high levels of iron in opt3-2 tissues. Hyperaccumulation of iron in opt3-2 resulted in the formation of brown necrotic areas in opt3-2 leaves and was more pronounced during the seed-filling stage. Third, reduced AtOPT3 expression resulted in decreased accumulation of iron in opt3-2 seeds. The reduced accumulation of iron in opt3-2 seeds is especially noteworthy considering the excessively high levels of accumulated iron in other opt3-2 tissues. AtOPT3, therefore, plays a critical role in two important aspects of iron metabolism, namely, maintenance of whole-plant iron homeostasis and iron nutrition of developing seeds.

Root Growth Inhibition in Boron-Deficient or Aluminum-Stressed Squash May Be a Result of Impaired Ascorbate Metabolism

Although cessation of growth is the most apparent symptom of boron deficiency, the biochemical function of boron in growth processes is not well understood. We propose that the action of boron in root meristems is associated with ascorbate metabolism. Total inhibition of root growth in squash (Cucurbita pepo L.) plants transferred to boron-free medium coincided with a major decrease (up to 98%) in the ascorbate concentration of root apices. Under low-boron conditions, in which root growth was partially inhibited, ascorbate concentration declined in proportion to growth rate. The decline in ascorbate concentration in boron-deficient root tips was not related to ascorbate oxidation. Ascorbate added to the medium improved root growth in plants supplied with insufficient boron. Increasing concentrations of aluminum in the nutrient medium caused progressive inhibition of root growth and a parallel reduction in ascorbate concentration of root apices. Elevated boron levels improved root growth under toxic aluminum conditions and produced root apices with higher ascorbate concentrations. To our knowledge, this is the first report of a correlation between boron nutrition, ascorbate concentration in root apices, and growth. These findings show that root growth inhibition resulting from either boron deficiency or aluminum toxicity may be a consequence of disrupted ascorbate metabolism.

Subcellular organization of ureide biogenesis from glycolytic intermediates and ammonium in nitrogen-fixing soybean nodules.

Subcellular organelle fractionation of nitrogen-fixing nodules of soybean (Glycine max (L.) Merr.) indicates that a number of enzymes involved in the assimilation of ammonia into amino acids and purines are located in the proplastids. These include asparagine synthetase (EC 6.3.1.1), phosphoribosyl amidotransferase (EC 2.4.2.14), phosphoglycerate dehydrogenase (EC 1.1.1.95), serine hydroxymethylase (EC 2.1.2.1), and methylene-tetrahydrofolate dehydrogenase (EC 1.5.1.5). Of the two isoenzymes of asparate aminotransferase (EC 2.6.1.1) in the nodule, only one was located in the proplastid fraction. Both glutamate synthase (EC 1.4.1.14) and triosephosphate isomerase (EC 5.3.1.1) were associated at least in part with the proplastids. Glutamine synthetase (EC 6.3.1.2) and xanthine dehydrogenase (EC 1.2.1.37) were found in significant quantities only in the soluble fraction. Phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) was found mostly in the soluble fraction, although small amounts of it were detected in other organelle fractions. These results together with recent organelle fractionation and electron microscopic studies form the basis for a model of the subcellular distribution of ammonium assimilation, amide synthesis and uredie biogenesis in the nodule.

Purification and properties of soybean nodule xanthine dehydrogenase

Abstract Xanthine dehydrogenase (EC 1.2.1.37), an essential enzyme for ureide metabolism was purified from the cytosol fraction of soybean nodules. The purified xanthine dehydrogenase was shown to be homogeneous by electrophoresis and a pI of 4.7 was determined by isoelectric focusing. The enzyme had a molecular weight of 285,000 and two subunits of molecular weight 141,000 each. The holoenzyme contained 1.7 (±0.7) mol Mo and 8.1 (±2.0) mol Fe/mol enzyme and the enzyme also contained FMN and is thus a molybdoironflavoprotein. Soybean xanthine dehydrogenase is the second enzyme in plants demonstrated to contain Mo and the first xanthine-oxidizing enzyme reported to contain FMN, rather than FAD as the flavin cofactor.

Ethylene production, cluster root formation, and localization of iron(III) reducing capacity in Fe deficient squash roots

Dicots and non-graminaceous monocots have the ability to increase root iron(III) reducing capacity in response to iron (Fe) deficiency stress. In squash (Cucurbita pepo L.) seedlings, Fe(III) reducing capacity was quantified during early vegetative growth. When plants were grown in Fe-free solution, the Fe(III) reducing capacity was greatly elevated, reached peak activity on day 4, then declined through day 6. Root ethylene production exhibited a temporal pattern that closely matched that of Fe(III) reducing capacity through day 6. On the 7th day of Fe deficiency, cluster root morphology developed, which coincided with a sharp increase in the root Fe(III) reducing capacity, although ethylene production decreased. Localization of Fe(III) reducing capacity activity was observed during the onset of Fe deficiency and through the development of the root clusters. It was noted that localization shifted from an initial pattern which occurred along the main and primary lateral root axes, excluding the apex, to a final localization pattern in which the reductase appeared only on secondary laterals and cluster rootlets.

Ureide biogenesis in leguminous plants

Abstract Allantion and allantoic acid are the predominant forms of organic nitrogen produced by N-fixing nodules of some important legume species. Only recently has the significance and biosynthetic origin of these substances in leguminous plants been realized.

Phosphorus nutritional effects on root hydraulic conductance, xylem water flow and flux of magnesium and calcium in squash plants

Previous studies have found that P nutrition of plants is an important factor in the uptake and translocation of Mg and Ca, and increasing root osmotic hydraulic conductance (Lo) and osmotically driven xylem exudate flow (Jv). Experiments were designed to determine if the observed changes in Mg and Ca uptake and translocation, Jv, and Lo from altered P nutrition are related or are separate functions. When six-week old squash (Cucurbita pepo L.) plants grown in perlite were treated with P levels ranging from 50 to 400 μM P for seven days, Jv and Lo increased as P treatment level increased. Xylem exudate concentrations of Mg and Ca were maintained as Jv increased, resulting in an increase in total flux of these mineral elements. The increase in Mg and Ca flux in the xylem exudate correlated with increased shoot Mg and Ca levels as P nutritional level was raised. Further studies with greenhouse grown plants indicated that the increases in Jv, Lo, and Mg and Ca flux were more responsive to changes in P nutritional level than to similar changes in levels of other anions. In hydroponically grown squash plants, xylem exudate was collected for a 20 min period after 0, 2 and 4 h in treatments of 50 and 500 μM P or after P treatment was increased from 50 to 500 μM. Immediately after nutrient solution P was increased (time 0), there was a 33% increase in Jv and a 22% increase in Lo when compared to the 50 μM P treatment. The Jv and Lo of the 50–500 μM P treatment did not equal levels of the continuous 500 μM control at time 0, but were similar after 2 and 4 h. Flux of Mg and Ca did not increase as rapidly as Jv in the 50–500 treatment indicating that regulation of Mg and Ca uptake and xylem loading by P may lag behind that of water movement.

Response of soybean to foliar‐applied boron and magnesium and soil‐applied boron 1

Abstract Annual plants may partition carbon (C) preferentially to reproductive structures slowing root elongation and subsequent nutrient uptake. Although foliar applications of nitrogen (N), phosphorus (P), potassium (K), and sulfur (S) supplement uptake by roots, soybean [Glycine max (L.) Merr.] yield increases have not been found in most studies. Experiments were designed to determine if foliar applications of boron (B), magnesium (Mg), or B+Mg would increase soybean yield and if soybean would respond to B applied to the soil several weeks prior to planting. Foliar B or Mg applied separately four times during reproductive growth did not affect soybean yield. However, four foliar applications of B+Mg increased soybean yield 12% at Mt. Vernon and 4% at Columbia over a three‐year period. Two foliar applications of B+Mg during the late reproductive stages increased soybean yield 8% over a two‐year period. The yield increase from foliar B+Mg treatment resulted from an increased number of pods on the main st...

Ureide catabolism in nitrogen-fixing legumes.

Abstract The ureides, allantoin and allantoate, are the major nitrogen transport compounds in certain N 2 -fixing legumes. A novel pathway of allantoate catabolism that releases 4NH 3 , 2CO 2 and glyoxylate from allantoate, independent of urease action, has been proposed for soybeans.