The magnetic structures of GdCuSn, GdAgSn and GdAuSn.

We have determined the magnetic structures of GdCuSn, GdAgSn and GdAuSn using a combination of [Formula: see text]Gd Mössbauer spectroscopy and neutron powder diffraction. Each compound shows the same antiferromagnetic ordering of the Gd sublattice. The magnetic cell is doubled along the crystallographic a-axis (propagation vector [Formula: see text]) with the moments aligned along the hexagonal c-axis, forming alternating ferromagnetic sheets of up/down Gd moments along the a-axis.

Complex incommensurate helicoidal magnetic ordering of EuNiGe3.

(151)Eu Mössbauer spectroscopy and neutron powder diffraction are combined to show that the tetragonal (I4mm #107) compound EuNiGe3 orders magnetically below [Formula: see text] K and adopts a complex incommensurate helicoidal magnetic structure at 3.6 K, with a propagation vector [Formula: see text] and a Eu moment of 7.1(2) [Formula: see text]. On warming through 6 K an incommensurate sinusoidal modulation develops and dominates the magnetic order by 12 K.

Europium and manganese magnetic ordering in EuMn2Ge2.

The antiferromagnetic structures of both the manganese and europium sublattices in EuMn2Ge2 have been determined using thermal neutron diffraction. T(N)(Mn) = 714(5) K with the 3.35(5) µ(B) (at 285 K) Mn moments ordering according to the I4'/m'm'm space group. The Eu order is incommensurate with the 6.1(2) µ(B) (at 3.6 K) Eu moments oriented parallel to the c-axis with a propagation vector of k = [0.153(2) 0 0]. Both neutron diffraction and (151)Eu Mössbauer spectroscopy reveal evidence of magnetic short-range ordering of the Eu sublattice around and above T(N)(Eu) ∼ 10 K. The ordering temperature of the Eu sublattice is strongly affected by the sample's thermal history and rapid quenching from the melting point may lead to a complete suppression of that ordering.

Allelic Analysis of the Maize amylose-extender Locus Suggests That Independent Genes Encode Starch-Branching Enzymes IIa and IIb.

Starch branching enzymes (SBE) catalyze the formation of [alpha]-1,6-glucan linkages in the biosynthesis of starch. Three distinct SBE isoforms have been identified in maize (Zea mays L.) endosperm, SBEI, IIa, and IIb. Independent genes have been identified that encode maize SBEI and IIb; however, it has remained controversial as to whether SBEIIa and IIb result from posttranscriptional processes acting on the product of a single gene or whether they are encoded by separate genes. To investigate this question, we analyzed 16 isogenic lines carrying independent alleles of the maize amylose-extender (ae) locus, the structural gene for SBEIIb. We show that 22 d after pollination ae-B1 endosperm expressed little Sbe2b (ae)-hybridizing transcript, and as expected, ae-B1 endosperm also lacked detectable SBEIIb enzymatic activity. Significantly, we show that ae-B1 endosperm contained SBEIIa enzymatic activity, strongly supporting the hypothesis that endosperm SBEIIa and IIb are encoded by separate genes. Furthermore, we show that in addition to encoding the predominant Sbe2b-hybridizing message expressed in endosperm, the ae gene also encodes the major Sbe2b-like transcript expressed in developing embryos and tassels.

Immunological characterization of maize starch branching enzymes.

Highly purified fractions of three starch branching enzymes from developing maize (Zea mays L.) endosperm were used to prepare antisera in rabbits. In double diffusion experiments, no immunoprecipitate was observed when branching enzyme IIa or IIb was tested against branching enzyme I antiserum. No immunoprecipitate was formed when branching enzyme I was tested against branching enzyme IIa or IIb antiserum. Increasing amounts of antisera in the above combinations also failed to inhibit enzyme activity. Branching enzyme IIa antiserum cross-reacted and formed spurs with branching enzyme IIb when compared with branching enzyme IIa antigen. Comparison of branching enzyme IIb antiserum with branching enzyme IIa also resulted in an immunoprecipitate. Increasing levels of branching enzyme IIa antiserum inhibited branching enzyme IIb as did the reciprocal combination. The data indicated that branching enzymes IIa and IIb are immunologically similar while branching enzyme I is distinct. The data supports the classification of starch branching enzymes based on genetic, kinetic, and chromatographic properties.

Comparison of soluble starch synthases and branching enzymes from leaves and kernels of normal and amylose-extender maize.

Soluble starch synthases (SS) and branching enzymes (BE) from 20-day-old maize leaves and 22-day-old seeds of normal and amylose-extender (ae) were purified by DEAE-cellulose chromatography. Elution profiles of leaf extracts showed one major SS and two BE fractions from both genotypes. The SS fractions from normal and ae leaf extracts were capable of citrate-stimulated starch synthesis and had different reaction rates with various primers. The two BE fractions from normal leaf extracts differed significantly from each other but not when compared to the same BE from ae. Comparison of BE fractions from ae and normal leaves showed no differences based on chromatographic, kinetic, and immunological properties. Comparison of the leaf enzymes with endosperm enzymes showed major differences. Leaf extracts did not contain SSII or BEIIb observed in endosperm extracts. Developing ae endosperm lacks BEIIb activity and ae is the structural gene for BEIIb. The tissue specific expression of BEIIb in the endosperm provides the basis for explaining the tissue-specific expression of ae. We propose that as BEIIb is expressed in the endosperm, but not leaves, allelic substitution at the ae locus modifies only endosperm starch synthesis.

Soluble starch synthases and starch branching enzymes from cotyledons of smooth- and wrinkled-seeded lines of Pisum sativum L.

Soluble starch synthase and branching enzyme were purified from 18-day-old cotyledons of the smooth-seeded pea cultivar Alaska (RR) and wrinkled-seeded pea cultivar Progress #9 (rr) by DEAE-cellulose chromatography. Two coeluting peaks of primed and citrate-stimulated starch synthase activity and a major and minor peak of branching enzyme activity were observed in Alaska. However, in Progress #9, only one peak of synthase activity was found. When crude extracts of Progress #9 were centrifuged, over 70% of the starch synthase activity was recovered in the pelleted fraction, and additional washings of the pellet released no further activity. The addition of purified starch granules to Alaska crude extracts also resulted in the recovery of a greater proportion of synthase activity in pelleted fractions. The two peaks of branching enzyme activity in Alaska differed in their stimulation of phosphorylase, amylose branching activity, and activity in various buffers. The DEAE-cellulose profile of Progress #9 showed no distinct peak of branching enzyme and less than 10% of the total activity found in Alaska. The association of one form of soluble starch synthase with the pelleted fraction and the greatly reduced levels of branching enzyme provide a partial explanation for the appearance of high-amylose starch in Progress #9 cotyledons.

Gene dosage at the amylose-extender locus of maize: effects on the levels of starch branching enzymes.

Soluble starch branching enzymes and starch synthases from maize kernels of differing dosage of the ae locus were purified by DEAE-cellulose chromatography. A near-linear relationship between increasing dosage of the dominate amylose-extender allele (Ae) and branching enzyme IIb activity was found. In contrast, levels and properties of branching enzymes I and IIa, as well as the citrate-stimulated and primer-requiring starch synthases, remained unchanged. The near-linear increase in branching enzyme IIb activity with increasing doses of the Ae allele is consistent with the hypothesis that ae is the structural gene coding for branching enzyme IIb.

Properties of Citrate-stimulated Starch Synthesis Catalyzed by Starch Synthase I of Developing Maize Kernels.

Chromatography of extracts of maize on diethylaminoethyl-cellulose resolves starch synthase activity into two fractions (Ozbun, Hawker, Preiss 1971 Plant Physiol 48: 785-769). Only starch synthase I is capable of synthesis in the absence of added primer and the presence of 0.5 molar citrate. This enzyme fraction has been purified about 1,000-fold from maize kernels homozygous for the endosperm mutant amylose-extender (ae). Because ae endosperm lacks the starch-branching enzyme which normally purifies with starch synthase I, the final enzyme fraction was free of detectable branching enzyme activity. This allowed a detailed characterization of the citrate-stimulated reaction. The citrate-stimulated reaction was dependent upon citrate concentrations of greater than 0.1 molar. However, the reaction is not specific for citrate and malate also stimulated the reaction. Branching enzyme increased the velocity of the reaction about 4-fold but did not replace the requirement for citrate. Citrate reduced the K(m) for the primers amylopectin and glycogen from 122 and 595 micrograms per milliliter, respectively, to 6 and 50 micrograms per milliliter, respectively. The enzyme was found to contain 1.7 milligrams of anhydroglucose units per enzyme unit. Thus reaction mixtures contained 1 to 5 micrograms (5 to 25 micrograms per milliliter) of endogenous primer. The citrate-stimulated reaction could be explained by an increased affinity for this endogenous primer. The starch synthase reaction in the absence of primer is dependent upon several factors including endogenous primer concentration, citrate concentration as well as branching enzyme concentration.

Evidence for independent genetic control of the multiple forms of maize endosperm branching enzymes and starch synthases.

Soluble starch synthase and starch-branching enzymes in extracts from kernels of four maize genotypes were compared. Extracts from normal (nonmutant) maize were found to contain two starch synthases and three branching enzyme fractions. The different fractions could be distinguished by chromatographic properties and kinetic properties under various assay conditions. Kernels homozygous for the recessive amylose-extender (ae) allele were missing branching enzyme IIb. In addition, the citrate-stimulated activity of starch synthase I was reduced. This activity could be regenerated by the addition of branching enzyme to this fraction. No other starch synthase fractions were different from normal enzymes. Extracts from kernels homozygous for the recessive dull (du) allele were found to contain lower branching enzyme IIa and starch synthase II activities. Other fractions were not different from the normal enzymes. Analysis of extracts from kernels of the double mutant ae du indicated that the two mutants act independently. Branching enzyme IIb was absent and the citrate-stimulated reaction of starch synthase I was reduced but could be regenerated by the addition of branching enzyme (ae properties) and both branching enzyme IIa and starch synthase II were greatly reduced (du properties). Starch from ae and du endosperms contains higher amylose (66 and 42%, respectively) than normal endosperm (26%). In addition, the amylopectin fraction of ae starch is less highly branched than amylopectin from normal or du starch. The above observations suggest that the alterations of the starch may be accounted for by changes in the soluble synthase and branching enzyme fractions.

Starch characteristics in cultures of normal and mutant maize endosperm.

Vigorously growing suspension cultures of 'normal', amylose-extender (ae) and waxy (wx) maize endosperm were established from near isogenic lines of maize inbred A636. The recovery of the ability to produce vigorous cultures of ae and wx endosperm by backcrossing demonstrate the genetic control of endosperm growth in vitro. Phenotypic expression of the endosperm mutants in culture was studied by examining the properties of starch accumulated in endosperm cultures and starch from developing and mature kernels of the same genotype. After 9 months in culture, the amylose contents of the starch in normal callus tissue and normal endosperm tissue were not significantly different, 28.2% and 31.7%, respectively. Starch granules from normal cultures and endosperm stained blue-black with iodine and were round to polygonal in shape. The starches of wx endosperm and callus cultures contained no amylose, and wx starch granules stained brown-orange with iodine. Although, wx starch granules were primarily round, a few granules with "jagged edges" were observed in starch samples isolated from cultures and kernels. The percent amylose in starch from ae callus was significantly lower than the amylose content of starch from ae endosperm tissue, 39.9% and 67.7%, respectively. Starch granules from ae endosperm and cultures were smaller than normal and wx starch granules. Irregular starch granules which are typical of ae endosperm were present in ae callus tissue, but were less frequently observed. We conclude that specific endosperm mutant phenotypes are expressed in vitro.

Genetic control of plastid carotenoids and transformation in the skin of Cucurbita pepo L. fruit.

The influence of allelic state of gene B on skin pigmentation in two cultivars of Cucurbita pepo L. has been studied. Total carotenoids were lower at early stages of fruit development in cultivar (cv.) 'Early Prolific' (EP) BB YY fruit skin, than in EP B (+) B (+) YY fruit skin, but no differences were observed in total skin carotenoids twenty days after anthesis. Total carotenoids were lower in cv. 'Fordhook Zucchini' (FZ) BB yy fruit skin, than in FZ B (+) B (+) yy fruit skin at all developmental stages from anthesis to maturity. Both green and yellow tissues contained typical foliar carotenoids. The carotenoids from yellow fruit skin of both EP genotypes and of FZ BB were characterized by a low carotene: xanthophyll ratio, with a high proportion of the xanthophylls esterified to fatty acids. The xanthophylls of the yellow tissues were esterified with 12∶0, 14∶0, 16∶0 fatty acids. The carotenoids from the green fruit skin of FZ B (+) B (+) had a higher percentage of carotenes (primarily ß-carotene) and a lower percentage of esterified xanthophylls. Spectral shapes of carotenoid fractions from all yellow tissues were similar and distinguishable from those of green FZ B (+) B (+) tissue. The results of these studies are discussed in terms of the genetic control of plastid transformation in Cucurbita pepo L.

Enzymes Catalyzing the Reversible Conversion of Fructose-6-Phosphate and Fructose-1,6-Bisphosphate in Maize (Zea mays L.) Kernels.

The significance of the glycolytic and gluconeogenic conversion of fructose-6-phosphate and fructose-1,6-bisphosphate on sugar metabolism was investigated in maize (Zea mays L.) kernels. Maximum extractable activities of the pyrophosphate (PPi) dependent phosphofructokinase, fructose-1,6-bisphosphatase, and the ATP-dependent phosphofructokinase were measured in normal and four maize genotypes, which accumulate relatively more sugars and less starch, to determine how these enzymes are affected by the genetic lesions. Normal endosperm accumulated more dry matter than the high sugar/low starch genotypes, but protein contents did not differ greatly among the genotypes. Mutation of several starch biosynthetic enzymes had little impact on the activities of PPi-dependent phosphofructokinase, fructose-1,6-bisphosphatase, and ATP-dependent phosphofructokinase, despite the altered capacity of the cell to synthesize starch. The PPi-dependent phosphofructokinase appeared to be more active toward glycolysis in all genotypes studied. Activity of the PPi-dependent phosphofructokinase in shrunken (low sucrose synthase genotype) did not differ from the activity in other genotypes, suggesting that the gluconeogenic production of PPi may not be the primary role of the enzyme. As expected, shrunken kernels contained more sugars and less starch than normal kernels throughout kernel development except at the very early stages. Developmental profiles of normal kernels also showed marked changes in the PPi-dependent phosphofructokinase activity, whereas the level of ATP-dependent phosphofructokinase activity remained relatively steady during kernel development. In addition, the ATP-dependent phosphofructokinase, and not the PPi-dependent phosphofructokinase, appeared to correlate more closely with respiration rate. These findings suggest that glycolysis catalyzed by the ATP-dependent phosphofructokinase may serve primarily to support energy production, and glycolysis catalyzed by the PPi-dependent phosphofructokinase may contribute mainly to generation of biosynthetic intermediates.

Alterations in Carbohydrate Intermediates in the Endosperm of Starch-Deficient Maize (Zea mays L.) Genotypes.

Metabolite levels in kernels of selected starch-deficient mutants of maize (Zea mays L.) were investigated to gain insight into partitioning of carbohydrate metabolism during kernel development. Several free sugars, hexose phosphates, triose phosphates, fructose-2,6-bisphosphate, and pyrophosphate were measured in normal, shrunken, shrunken-2, amylose extender dull waxy, and brittle genotypes, which were in a near-isogenic W64A background. These mutants were selected to include at least one lesion in both the cytosolic (shrunken) and amyloplastic (shrunken-2) compartments. All the starch-deficient genotypes contained elevated levels of fructose-2,6-bisphosphate and triose phosphates but reduced levels of pyrophosphate, indicating an enhanced glycolytic utilization of carbohydrates in response to the reduced utilization of sugars for starch synthesis. The shrunken kernels (sucrose synthase deficient) contained reduced levels of glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate, and this reduction paralleled the reduction in starch accumulation, but levels of triose phosphates were elevated. In shrunken-2 kernels, glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate, dihydroxyacetone phosphate, and glyceraldehyde-3-phosphate were increased, but fructose-1,6-bisphosphate was lower. These findings support the view that hexose phosphate transport across the amyloplast envelope is more important for starch biosynthesis than transport of triose phosphates. The amylose extender dull waxy mutation showed less dramatic effects on hexose phosphates, but the triose phosphates were greatly increased. The brittle mutation, which has an unknown lesion, showed distinctly similar changes in metabolite levels with shrunken-2, suggesting that the lesion may be associated with the amyloplast.

Expression of the large plastid gene, ORF2280, in tomato fruits and flowers.

The expression of ORF2280, a large plastid gene of unknown function, was examined in tomato leaves, in a developmental series of tomato fruits, and in tomato flowers. Western blots indicated that much more ORF2280 protein is present in fruits and flowers than in leaves. The most abundant proteins detected, 68 and 59 kDa, are present in about equal amounts in fruits of all stages; they are even more abundant in flowers. A 170-kDa ORF2280 protein is also present in fruit of all stages; it is most abundant in small green fruit. The presence of higher levels of ORF2280 proteins in tomato fruits and flowers indicates that it may have a specialized function in these nonphotosynthetic tissues.

Nitrogenase Activity Associated with Roots and Stems of Field-Grown Corn (Zea mays L.) Plants.

Corn (Zea mays L.) plants were assayed for nitrogenase activity (C(2)H(2) reduction) during early ear development. Hybrid corn and inbred lines were grown separately at two experimental fields in New Jersey. Acetylene-dependent ethylene production was observed a few hours after harvest, from the field, on intact plants, root-soil cores, lower stem segments, and excised roots, all assayed under air and not preincubated previously. Incubation of excised roots at 1% O(2) resulted in lower rates of C(2)H(2) reduction. The time course of C(2)H(2) reduction by excised roots, assayed in air, was similar for all genotypes studied (two hybrids, eight inbreds, and a cross of corn x teosinte) and indicated that a long preincubation at reduced O(2) is not absolutely required for early detection of nitrogenase activity. Isolation of N(2)-fixing bacteria from within the roots and stems, together with the diurnal fluctuation of nitrogenase activity in response to day/night cycles, were indicative of a close association with plant function. Collectively, the results provided strong evidence for the occurrence of nitrogenase activity associated with corn plants growing in a temperate climate and dependent upon indigenous N(2)-fixing bacteria.

Restriction enzyme analysis of tomato chloroplast and chromoplast DNA.

Plastid DNA was isolated from the chloroplasts of tomato (Lycopersicon esculentum var Traveler 76) leaves and the chromoplasts of ripe tomato fruit. Comparisons of the two DNAs were made by restriction endonuclease analysis using PvuII, HpaI, and Bg1I. No differences in the electrophoretic banding patterns of the restricted plastid DNAs were detected, indicating that no major rearrangements, losses, or gains of plastid DNA accompany the transition from chloroplast to chromoplast.

Enzyme activities associated with maize kernel amyloplasts.

Activities of the enzymes of gluconeogenesis and of starch metabolism were measured in extracts of amyloplasts isolated from protoplasts derived from 14-day-old maize (Zea mays L., cv Pioneer 3780) endosperm. The enzymes triosephosphate isomerase, fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, phosphohexose isomerase, phosphoglucomutase, ADPG pyrophosphorylase, UDPG pyrophosphorylase, soluble and bound starch synthases, and branching enzyme were found to be present in the amyloplasts. Of the above enzymes, ADPG pyrophosphorylase had the lowest activity per amyloplast. Invertase, sucrose synthase and hexokinase were not detected in similar amyloplast preparations. Only a trace of the cytoplasmic marker enzyme alcohol dehydrogenase could be detected in purified amyloplast fractions. In separate experiments, purified amyloplasts were lysed and then supplied with radioactively labeled glucose-6-phosphate, glucose-1-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, glucose, fructose, sucrose, and 3-0-methylglucose in the presence of adenosine triphosphate or uridine triphosphate. Of the above, only the phosphorylated substrates were incorporated into starch. Incorporation into starch was higher with added uridine triphosphate than with adenosine triphosphate. Dihydroxyacetone phosphate was the preferred substrate for uptake by intact amyloplasts and incorporation into starch. In preliminary experiments, it appeared that glucose-6-P and fructose-1,6-bisphosphate may also be taken up by intact amyloplasts. However, the rate of uptake and incorporation into starch was relatively low and variable. Additional study is needed to determine conclusively whether hexose phosphates will cross intact amyloplast membranes. From these data, we conclude that: (a) Triose phosphate is the preferred substrate for uptake by intact amyloplasts. (b) Amyloplasts contain all enzymes necessary to convert triose phosphates into starch. (c) Sucrose breakdown must occur in the cytosol prior to carbohydrate transfer into the amyloplasts. (d) Under the conditions of assay, amyloplasts are unable to convert glucose or fructose to starch. (e) Uridine triphosphate may be the preferred nucleotide for conversion of hexose phosphates to starch at this stage of kernel development.

Survey of plastid RNA abundance during tomato fruit ripening: the amounts of RNA from the ORF 2280 region increase in chromoplasts.

A comprehensive survey of the levels of plastid RNAs at progressive stages of tomato fruit ripening was conducted by hybridizing total RNA with labeled Pst I fragments that cover almost the entire tomato plastid genome and with gene-specific probes. Two different cultivars of tomato (Lycopersicon esculentum Mill.) were examined, Traveler 76 and Count II. One of the tomato probes, P7, revealed a pronounced increase in the amount of an 8.3 kb RNA in ripe fruit. The homologous region of the tobacco plastid genome contains several genes for ribosomal proteins and a large unidentified open reading frame (2280 codons). Little change was observed in the levels of many transcripts during ripening. However, in some cases (e.g. psbA and psbC/D) the amount of RNA decreased during ripening of Count II but showed little or no change in Traveler 76. The contrast between Traveler 76 and Count II tomatoes shows that the level of plastid transcripts can vary substantially during fruit ripening with no obvious effect on the chloroplast to chromoplast transition. The large RNA from the P7 region may encode a protein that functions predominantly in chromoplasts.