The relationship between the rate of starch synthesis, the adenosine 5′-diphosphoglucose concentration and the amylose content of starch in developing pea embryos

Mutations that reduced the rate of starch synthesis in pea (Pisum sativum L.) embryos through effects on enzymes on the pathway from sucrose to adenosine 5′-diphosphoglucose (ADPglucose) also led to a reduction in the amylose content of the starch of developing embryos. Evidence is presented that this relationship between rate of synthesis and the composition of starch is due to the fact that amylopectin-synthesising isoforms of starch synthase have higher affinities for ADPglucose than the amylose-synthesising isoform. First, developing mutant embryos (rb, rug3 and rug4 mutants) displayed both reduced amylose contents in their starches and reduced ADPglucose contents relative to wild-type embryos. Second, incubation of detached, wild-type embryos for 6 h at high and low glucose concentrations resulted in differences in both ADPglucose content and the relative rates of amylose and amylopectin synthesis. At 0.25 M glucose both ADPglucose content and the proportion of synthesised starch that was amylose were about twice as great as at 25 μM glucose. Third, S _0.5 values for soluble (amylopectin-synthesising) starch synthases in developing embryos were several-fold lower than that for granule-bound (amylose synthesising) starch synthase. Estimates of the expected amylose contents of the starch of the mutant embryos, based on the reduction in their ADPglucose contents and on the S _0.5 values of the starch synthases, were very similar to the measured amylose contents. The implications of these results for the determination of starch composition are discussed. Received: 6 February 1999 / Accepted: 22 May 1999     

Sucrose Synthase in Legume Nodules Is Essential for Nitrogen Fixation

The role of sucrose synthase (SS) in the fixation of N was examined in the rug4 mutant of pea (Pisum sativum L.) plants in which SS activity was severely reduced. When dependent on nodules for their N supply, the mutant plants were not viable and appeared to be incapable of effective N fixation, although nodule formation was essentially normal. In fact, N and C resources invested in nodules were much greater in mutant plants than in the wild-type (WT) plants. Low SS activity in nodules (present at only 10% of WT levels) resulted in lower amounts of total soluble protein and leghemoglobin and lower activities of several enzymes compared with WT nodules. Alkaline invertase activity was not increased to compensate for reduced SS activity. Leghemoglobin was present at less than 20% of WT values, so O₂ flux may have been compromised. The two components of nitrogenase were present at normal levels in mutant nodules. However, only a trace of nitrogenase activity was detected in intact plants and none was found in isolated bacteroids. The results are discussed in relation to the role of SS in the provision of C substrates for N fixation and in the development of functional nodules.     

Symbiotic Nitrogen Fixation and Ammonium Nitrogen Assimilation in rrrbrb-, rrRbRb-, and RRrbrb-Pea Mutants

Wrinkled-seeded pea mutants (Pisum sativum L., genotypes rrrbrb-, rrRbRb-, and RRrbrb-) have seeds with reduced, but different, starch content and modified starch properties. Analysis of these mutants revealed an enhanced capacity of root nodules for symbiotic nitrogen fixation and of host plant organs for assimilation of ammonium nitrogen. This observation was confirmed by morphological data on organization of symbiotic system, by elevated nitrogenase activity, high protein accumulation in plants due to nitrogen fixation, and by enhanced activity of glutamine synthase in leaves and glutamate dehydrogenase in roots of mutants, as compared with the organs of wild-type pea. It is supposed that the aforementioned advantages of mutants are related to accumulation in seeds of elevated protein reserves that satisfy their demand for nitrogen during formation of symbiotic systems.     

Mutations in the gene encoding starch synthase II profoundly alter amylopectin structure in pea embryos.

Mutations at the rug5 (rugosus5) locus have been used to elucidate the role of the major soluble isoform of starch synthase II (SSII) in amylopectin synthesis in the developing pea embryo. The SSII gene maps to the rug5 locus, and the gene in one of three rug5 mutant lines has been shown to carry a base pair substitution that introduces a stop codon into the open reading frame. All three mutant alleles cause a dramatic reduction or loss of the SSII protein. The mutations have pleiotropic effects on the activities of other isoforms of starch synthase but apparently not on those of other enzymes of starch synthesis. These mutations result in abnormal starch granule morphology and amylopectin structure. Amylopectin contains fewer chains of intermediate length (B2 and B3 chains) and more very short and very long chains than does amylopectin from wild-type embryos. The results suggest that SSII may play a specific role in the synthesis of B2 and B3 chains of amylopectin. The extent to which these findings can be extrapolated to other species is discussed.     

Nodulating ability of Rhizobium tropici is conditioned by a plasmid-encoded citrate synthase

Rhizobium species elicit the formation of nitrogen-fixing root nodules through a complex interaction between bacteria and plants. Various bacterial genes involved in the nodulation and nitrogen-fixation processes have been described and most have been localized on the symbiotic plasmids (pSym). We have found a gene encoding citrate synthase on the pSym plasmid of Rhizobium tropici, a species that forms nitrogen-fixing nodules on the roots of beans (PhasBoius vuigaris) and trees (Leucaena spp.). Citrate synthase is a key metabolic enzyme that incorporates carbon into the tricarboxylic acid cycle by catalysing the condensation of acetyl-CoA and oxalo-acetic acid to form citrate. R. tropici pcsA (the plasmid citrate synthase gene) is closely related to the corresponding genes of Proteobacteria. pcsA inactivation by a Tn5-mob insertion causes the bacteria to form fewer nodules (30–50% of the original strain) and to have a decreased citrate synthase activity in minimal medium with sucrose. A clone carrying the pcsA gene complemented ail the phenotypic alterations of the pcsA mutant, and conferred Rhizobium iegumino-sarum bv. phaseoli (which naturally lacks a plasmid citrate synthase gene) a higher nodulation and growth capacity in correlation with a higher citrate synthase activity. We have also found that pcsA gene expression is sensitive to iron availability, suggesting a possible role of pcsA in iron uptake.     

Nodule-enhanced expression of a sucrose phosphate synthase gene member (MsSPSA) has a role in carbon and nitrogen metabolism in the nodules of alfalfa (Medicago sativa L.)

Sucrose phosphate synthase (SPS) catalyzes the first step in the synthesis of sucrose in photosynthetic tissues. We characterized the expression of three different isoforms of SPS belonging to two different SPS gene families in alfalfa (Medicago sativa L.), a previously identified SPS (MsSPSA) and two novel isoforms belonging to class B (MsSPSB and MsSPSB3). While MsSPSA showed nodule-enhanced expression, both MsSPSB genes exhibited leaf-enhanced expression. Alfalfa leaf and nodule SPS enzymes showed differences in chromatographic and electrophoretic migration and differences in V _max and allosteric regulation. The root nodules in legume plants are a strong sink for photosynthates with its need for ATP, reducing power and carbon skeletons for dinitrogen fixation and ammonia assimilation. The expression of genes encoding SPS and other key enzymes in sucrose metabolism, sucrose phosphate phosphatase and sucrose synthase, was analyzed in the leaves and nodules of plants inoculated with Sinorhizobium meliloti. Based on the expression pattern of these genes, the properties of the SPS isoforms and the concentration of starch and soluble sugars in nodules induced by a wild type and a nitrogen fixation deficient strain, we propose that SPS has an important role in the control of carbon flux into different metabolic pathways in the symbiotic nodules.     

The importance of starch biosynthesis in the wrinkled seed shape character of peas studied by Mendel

The wrinkled-seed mutant (rr) of pea (Pisum sativum L.) arose through mutation of the gene encoding starch-branching enzyme isoform I (SBE1) by insertion of a transposon-like element into the coding sequence. Two isoforms of starch-branching enzyme have been documented in the developing pea embryo. The second isoform, SBEII, is expressed towards the later stages of embryo development while SBEI is expressed highly in the early stages. Due to mutation of SBEI the total amount of starch and the proportion of amylopectin, a branched starch polymer, are greatly reduced in the wrinkled (rr) line as compared to that in the wild-type, round (RR) line. Consequently, the level of sucrose in the rr line is nearly two fold that of the RR line. Increased sucrose concentration in the developing embryos of this mutant line causes increased uptake of water and thereby increases the cell size and fresh weight. During seed maturation in these mutant seeds a greater loss of water occurs. As a result, the wrinkled seed phenotype develops. Besides this morphological variation, the mutation also causes changes in the amount of lipid and of one storage protein, legumin. This review article discusses the role of the SBEI enzyme in causing such metabolic changes in the developing embryos with the implication that metabolism can play a central role in plant development.     

Cadmium in white lupin nodules: Impact on nitrogen and carbon metabolism

The aims of this work were to investigate the microlocalisation of cadmium (Cd) in Lupinus albus L. cv. Multolupa nodules, and to determine its effects on carbon and nitrogen metabolism. Nodulated white lupin plants were grown in a growth chamber with or without Cd (150 μM). Energy-dispersive X-ray microanalysis showed the walls of the outer nodule cortex cells to be the main area of Cd retention, helping to reduce the harmful effect Cd might have on the amount of N₂ fixed by the bacteroids. Sucrose synthase activity declined by 33% in the nodules of the Cd-treated plants, and smaller reductions were recorded in glutamine synthetase, aspartate aminotransferase, alkaline invertase and NADP-dependent isocitrate dehydrogenase activities. The Cd treatment also sharply reduced nodule concentrations of malate, succinate and citrate, while that of starch doubled, but that of sucrose experienced no significant change. In summary, the present results show that white lupins accumulate significant amounts of Cd in their root nodules. However, the activity of some enzymes involved in ammonium assimilation did decline, promoting a reduction in the plant N content. The downregulation of sucrose synthase limits the availability of carbon to the bacteroids, which might interfere with their respiration. Carbon metabolism therefore plays a primary role in the impaired function of the white lupin root nodule caused by Cd, while N metabolism appears to have a more secondary involvement.     

Correlation between NaCl Sensitivity of Rhizobium Bacteria and Ineffective Nodulation of Leguminous Plants

A sodium chloride (NaCl)-sensitive mutant of Rhizobium fredii USDA191, which contained a single copy of Tn5-Mob transposed into chromosomal DNA, was obtained by Tn5-Mob random insertion. The growth rate of this mutant was lower than that of the wild type in the presence of 0.2 M NaCl and it seemed to lack the inductive ATP production in response to the addition of NaCl. This mutant induced the formation of small and whitish nodules on lateral roots of soybeans, which were negative for acetylene reduction activity, indicating that the nodules were ineffective for nitrogen fixation. The mutant also reduced the weight of above-ground portions and roots to 64 and 55%, respectively, compared with the weight of the plants inoculated with the wild-type cells. These results suggest that NaCl sensitivity of Rhizobium bacteria is one of the important factors for nodule formation and nitrogen fixation.     

Carbohydrates and carbohydrate enzymes in developing cotton ovules

Patterns of carbohydrates and carbohydrate enzymes were investigated in developing cotton ovules to establish which of these might be related to sink strength in developing bolls. Enzymatic analysis of extracted tissue indicated that beginning 1 week following anthesis, immature cotton seeds (Gossypium hirsutum L. cv. Coker 100A glandless) accumulated starch in the tissues which surround the embryo. Starting at 15 days post anthesis (DPA), this starch was depleted and starch simultaneously appeared in the embryo. Sucrose entering the tissues surrounding the embryo was rapidly degraded, apparently by sucrose synthase; the free hexose content of these tissues reached a peak at about 20 DPA. During the first few weeks of development these tissues contained substantial amounts of hexose but little sucrose; the reverse was true for cotton embryos. Embryo sucrose content rose sharply from the end of the first week until about 20 DPA; it then remained roughly constant during seed maturation. Galactinol synthase (EC 2.4.1.x) appeared in the embryos approximately 25 days after flowering. Subsequently, starch disappeared and the galactosides raffinose and stachyose appeared in the embryo. Except near maturity, sucrose synthase (EC 2.4.1.13) activity in the embryos predominated over that of both sucrose phosphate synthase (EC 2.4.1.14) and acid invertase (EC 3.2.1.26). Activities of the latter enzymes increased during the final stages of embryo maturation. The ratio of sucrose synthase to sucrose phosphate synthase was found to be high in young cotton embryos but the ratio reversed about 45 DPA, when developing ovules cease being assimilate sinks. Insoluble acid invertase was present in developing cotton embryos, but at very low activities; soluble acid invertase was present at significant activities only in nearly mature embryos. From these data it appears that sucrose synthase plays an important role in young cotton ovule carbohydrate partitioning and that sucrose phosphate synthase and the galactoside synthesizing enzymes assume the dominant roles in carbohydrate partitioning in nearly mature cotton seeds. Starch was found to be an important carbohydrate intermediate during the middle stages of cotton ovule development and raffinose and stachyose were found to be important carbohydrate pools in mature cotton seeds.     

The rb Mutation of Peas Causes Structural and Regulatory Changes in ADP Glucose Pyrophosphorylase from Developing Embryos

A mutation at the rb locus of pea (Pisum sativum L.) alters the shape, reduces the starch content, and increases the lipid and sucrose contents of the seed. These effects are probably all consequences of a reduction of up to 40-fold in the maximum catalytic activity of ADP glucose pyrophosphorylase in the developing embryo of the mutant relative to the wild type. We have investigated how the mutation brings about this reduction in activity. The purified enzyme from mutant embryos has a specific activity about 10-fold lower than that from wild-type embryos, and it is much more sensitive to the effectors inorganic phosphate and 3-phosphoglycerate than the wild-type enzyme. Both wild-type and mutant enzymes consist of polypeptides of around 50 kilodaltons. One of the polypeptides of the purified wild-type enzyme is missing from the mutant enzyme. We deduce that in the wild-type embryo this protein may interact with other subunits to confer a high specific activity and a low susceptibility to effectors on the enzyme.     

Evidence for Mitochondrial Regulation of Photosynthesis by a Starchless Mutant of Nicotiana sylvestris

Mutant NS458 of Nicotiana sylvestris (Speg. et Comes) contains a defective plastid phosphoglucomutase and accumulates only trace amounts of starch. Determinations of carbon partitioning using tracer d-[3-¹⁴C]glyceric acid showed that the maximal CO₂ assimilation by mature leaves of the mutant at saturating [CO₂] and light and low [O₂] was close to the flux for sucrose formation in the wild type. The mutant is characterized by exceptionally slow oscillations in maximal CO₂ assimilation. The postulate that these slow oscillations follow changes in the cytosolic rate of sucrose phosphate synthesis has been investigated. Studies with wild-type and mutant leaf discs subjected to various treatments failed to indicate that any significant activation-inactivation cycle in sucrose-P synthase activity can occur. The rate of sucrose phosphate synthesis, however, might be altered by variations in the supply of uridine UDP-glucose which is controlled by the rate of ATP regeneration (via UTP regeneration). Treating mutant leaf protoplasts and young leaves with oligomycin, an inhibitor of mitochondrial ATP regeneration, reduced photosynthesis by as much as 25 and 40%, respectively. The wild type failed to show inhibition by oligomycin, i.e. its effect is masked when starch and sucrose synthesis can interact. It is concluded that maximal CO₂ assimilation in the mutant is fine tuned by mitochondrial metabolism such that interactions between sucrose synthesis and mitochondrial processes may generate the observed oscillations.     

Evidence that the rb Locus Alters the Starch Content of Developing Pea Embryos through an Effect on ADP Glucose Pyrophosphorylase

The aim of this work was to discover whether the rb locus of peas (Pisum sativum L.) affects seed starch content through action on an enzyme of starch synthesis in the developing embryo. The phenotypic effects of this locus are like those of the better characterised, unlinked r locus, which affects seed starch content through action on starch-branching enzyme. Embryos recessive at one or both of these loci (RRrbrb, rrRbRb, rrrbrb) have lower starch contents from an early stage of development than embryos dominant at these loci (RRRbRb). Maximum catalytic activities of enzymes of the pathway from sucrose to starch (sucrose synthase EC 2.4.1.13, UDP glucose pyrophosphorylase EC 2.7.7.9, ADP glucose pyrophosphorylase EC 2.7.7.27, ADP glucose-starch synthase EC 2.4.1.21, starch-branching enzyme EC 2.4.1.18) were compared in developing embryos of three lines of rbrb peas and four lines of RbRb peas. The only consistent difference between the two sorts of embryo was in the activity of ADP glucose pyrophosphorylase, which was at least tenfold lower in rbrb than in RbRb embryos. The activity in rbrb embryos was in most cases less than the estimated rate of starch synthesis of RRRbRb embryos. We conclude that the effect of the rb locus on the starch content of pea seeds is mediated through an alteration in the activity of ADP glucose pyrophosphorylase in the developing embryo.     

Physiological comparisons of root and stem nodules of Aeschynomene scabra and Sesbania rostrata

A few legume species possess the ability to form N₂-fixing nodules on stems as well as on roots. Little is known of the functional characteristics of stem nodules, or to what extent they differ from root nodules. Stem and root nodules of greenhouse-grown plants of Aeschynomene scabra (inoculated with the photosynthetic rhizobial strain BTAi 1) and Sesbania rostrata (inoculated with Azorhizobium caulinodans strain BTSr 3) were examined for assimilation of ¹⁴CO₂ in the light and dark, soluble carbohydrate and starch contents, acetylene reduction activity, relative efficiency of nitrogenase in terms of uptake-hydrogenase activity, glutamine synthetase and glutamate synthase, and reduced N and ureide contents. In general, stem nodules possessed higher enzyme activities and metabolite contents than did root nodules, suggesting that they fix N₂ with greater energy efficiency. This greater efficiency correlated with photosynthesis in the cortex of stem nodules. Differences in enzyme activities and metabolite contents between the stem nodules on A. scabra and those on S. rostrata probably result either from legume-species characteristics or from the photosynthetic capability of strain BTAi 1.     

The assimilation and allocation of nutrients by symbiotic and aposymbiotic pea aphids, Acyrthosiphon pisum

The failure of a nutritionally balanced diet to ameliorate the impact of symbiont disruption in the pea aphid Acyrthosiphon pisum (Harris) was investigated using two approaches. The assimilation of dietary nutrients by aphids was investigated using chemically-defined diets containing ³ H-labelled inulin and ¹⁴C-labelled sucrose or amino acids. Symbiotic aphids (i.e., aphids containing their bacteria) had a high sucrose demand and assimilated 72% of sucrose ingested in the diet, whereas the assimilation of sucrose by aposymbiotic aphids (in which the bacteria had been disrupted), was significantly reduced to 47%. The assimilation of individual dietary amino acids by symbiotic aphids varied between 61 and 92%, and there was no impact on the feeding or assimilation rate when the aphids were fed a phloem sap-like diet containing a reduced amount of essential amino acids. Consequently, the absolute amount of each essential amino acid assimilated by symbiotic aphids feeding on a phloem sap-like diet was reduced by 36–59%. Aposymbiotic aphids consistently assimilated a lower proportion of ingested amino acids, and lysine in particular was poorly assimilated from the diet. In a second experiment, the allocation of free amino acids in the haemocoel to aphid embryos was investigated following microinjection of ¹⁴C-labelled amino acids. After 2 h, radiolabel could be detected at varying levels from the embryo complement of both symbiotic and aposymbiotic aphids, indicating rapid but selective uptake by the embryos. The essential amino acids phenylalanine and lysine were incorporated into the protein fraction of embryo tissues, but the rate of incorporation per unit biomass was approximately 4-fold higher in the embryos of aposymbiotic aphids, possibly reflecting increased demand due to the lack of amino acid provisioning from the symbiotic bacteria.     

Multiple, distinct isoforms of sucrose synthase in pea

Genes encoding three isoforms of sucrose synthase (Sus1, Sus2, and Sus3) have been cloned from pea (Pisum sativum). The genes have distinct patterns of expression in different organs of the plant, and during organ development. Studies of the isoforms expressed as recombinant proteins in Escherichia coli show that they differ in kinetic properties. Although not of great magnitude, the differences in properties are consistent with some differentiation of physiological function between the isoforms. Evidence for differentiation of function in vivo comes from the phenotypes of rug4 mutants of pea, which carry mutations in the gene encoding Sus1. One mutant line (rug4-c) lacks detectable Sus1 protein in both the soluble and membrane-associated fractions of the embryo, and Sus activity in the embryo is reduced by 95%. The starch content of the embryo is reduced by 30%, but the cellulose content is unaffected. The results imply that different isoforms of Sus may channel carbon from sucrose towards different metabolic fates within the cell.     

A Cascade of Sequentially Expressed Sucrose Transporters in the Seed Coat and Endosperm Provides Nutrition for the Arabidopsis Embryo

Developing plant embryos depend on nutrition from maternal tissues via the seed coat and endosperm, but the mechanisms that supply nutrients to plant embryos have remained elusive. Sucrose, the major transport form of carbohydrate in plants, is delivered via the phloem to the maternal seed coat and then secreted from the seed coat to feed the embryo. Here, we show that seed filling in Arabidopsis thaliana requires the three sucrose transporters SWEET11, 12, and 15. SWEET11, 12, and 15 exhibit specific spatiotemporal expression patterns in developing seeds, but only a sweet11;12;15 triple mutant showed severe seed defects, which include retarded embryo development, reduced seed weight, and reduced starch and lipid content, causing a “wrinkled” seed phenotype. In sweet11;12;15 triple mutants, starch accumulated in the seed coat but not the embryo, implicating SWEET-mediated sucrose efflux in the transfer of sugars from seed coat to embryo. This cascade of sequentially expressed SWEETs provides the feeding pathway for the plant embryo, an important feature for yield potential.