Molecular and serological comparisons of purified xanthine dehydrogenases from root nodules of three ureide-producing legume species

Xanthine dehydrogenase (XDH, EC 1.2.1.37) was immunopurified from root nodules of three legume species, soybean [ Glycine max (L.) Merr. cv. Pella], cowpea [ Vigna unguiculata (L.) Walp. cv. California Black Eye], and lima bean [ Phaseolus lunatus L. Henderson]. Polyclonal antibodies raised against each enzyme and monoclonal antibodies raised against soybean XDH were used to compare the three enzymes serologically. Double diffusion and enzyme-linked immunosorbent assays with polyclonal and monoclonal antibodies showed that the cowpea and lima bean enzymes are very similar immunologically but both differ measurably from soybean. Amino acid compositions of the legume nodule XDHs are presented as well. Although relatedness between these enzymes can be detected by immunological crossreactivity, each XDH has unique epitopes that can be used to distinguish the three proteins.     

Diffusion and reaction of oxygen in the central tissue of ureide-producing legume nodules

Previous simulation models for the diffusion and reaction of oxygen in legume nodules were based on infected cells and neglected adjacent uninfected cells. This study uses a three-dimensional model of the central zone of legume nodules made up of the two cell types represented by a geometrically defined, space-filling, binary combination of polyhedra, each with bevelled edges to allow for a network of intercellular gas spaces. The model predicted a distinctively compartmentalized distribution of [O₂] between uninfected and infected cells; with high O₂ concentrations for an uninfected cell being consistent with, and necessary for, efficient operation of uricase and ureide synthesis and low O₂ concentrations across most of the infected cell providing a suitable environment for N₂-fixation. Compartmentalization of O₂ also predicted significant O₂ fluxes between cell types, compromising maintenance of low [O₂] in infected cells, as well as high [O₂] in uninfected cells. The results predict that there might be significant resistance to O₂ diffusion across the cell : cell interface due to the plasmalemma and cell walls.     

Translocation from leaves to fruits of a legume,studied by a phloem bleeding technique: Diurnal changes and effects of continuous darkness

Summary Diurnal changes in the carbohydrates of leaf laminae and fruits and in the bleeding of sugar and amino acids from fruit phloem were followed by successive sampling from a population of Lupinus albus L. plants. Phloem sap was collected for a standard 5 min period from cut distal tips of attached fruits. Daily fluctuations in leaf dry matter resulted largely from changes in starch and sugar. Leaf sugar rose to a maximum in the afternoon, starch to a maximum at, or shortly after, dusk. Leaves lost sugar and starch from dusk to dawn. Phloem bleeding rate varied little over a daily cycle but sucrose levels fluctuated from a noon maximum of 12–13% (w/v) to a dawn minimum of 9–10%. The rhythm of phloem sugar levels matched closely those of fruit and leaf. Phloem amino acid levels fluctuated in phase with that of sucrose: the relative composition of the amino fraction did not vary significantly over the daily cycle. Pulse feeding of source leaves with ¹⁴CO₂ at different times in the photoperiod allowed study of the pattern of release of labelled photosynthate to the fruit phloem and the build up and depletion of ¹⁴C starch in leaves. Plants transferred to continuous darkness showed a rapid decline in output and concentration of phloem sap solutes, and translocated nitrogen to their fruits at only one quarter of the rate of control plants retained in natural daylight. The combined data from the experiments showed that the rate of output of sugar from cut phloem of a fruit was directly related to the current level of sugar in leaves. When leaf sugar levels were low (5–10 mg ml tissue water^-1) sugar in phloem was 10–11 times more concentrated than in source leaves, but at high leaf sugar levels (25–30 mg ml^-1) this concentration difference was only 3–4 fold.     

Transport of organic solutes in Phloem and xylem of a nodulated legume

Collections of xylem exudate of root stumps or detached nodules, and of phloem bleeding sap from stems, petioles, and fruits were made from variously aged plants of Lupinus albus L. relying on nodules for their N supply. Sucrose was the major organic solute of phloem, asparagine, glutamine, serine, aspartic acid, valine, lysine, isoleucine, and leucine, the principal N solutes of both xylem and phloem. Xylem sap exhibited higher relative proportions of asparagine, glutamine and aspartic acid than phloem sap, but lower proportions of other amino acids. Phloem sap of petioles was less concentrated in asparagine and glutamine but richer in sucrose than was phloem sap of stem and fruit, suggesting that sucrose was unloaded from phloem and amides added to phloem as translocate passed through stems to sinks of the plant. Evidence was obtained of loading of histidine, lysine, threonine, serine, leucine and valine onto phloem of stems but the amounts involved were small compared with amides. Analyses of petiole phloem sap from different age groups of leaves indicated ontogenetic changes and effects of position on a shoot on relative rates of export of sucrose and N solutes. Diurnal fluctuations were demonstrated in relative rates of loading of sucrose and N solutes onto phloem of leaves. Daily variations in the ability of stem tissue to load N onto phloem streams were of lesser amplitude than, or out of phase with fluctuations in translocation of N from leaves. Data were related to recent information on C and N transport in the species.     

Xylem and Phloem transport and the functional economy of carbon and nitrogen of a legume leaf

Exchanges of CO₂ and changes in content of C and N were studied over the life of a leaf of Lupinus albus L. These data were combined with measurements of C:N weight ratios of xylem (upper stem tracheal) and phloem (petiole) sap to determine net fluxes of C and N between leaf and plant. Phase 1 of leaf development (first 11 days, leaf to one-third area) showed increasing net import of C and N, with phloem contributing 61% of the imported C and 18% of the N. ¹⁴C feeding studies suggested the potential for simultaneous import and export through phloem over the period 9 to 12 days. Phase 2 (11-20 days, leaf attaining maximum area and net photosynthesis rate) exhibited net import through xylem and increasing export through phloem. Eighty-two% of xylem-delivered N was consumed in leaf growth, the remainder exported in phloem. Phase 3 (20-38 days) showed high but declining rates of photosynthesis, translocation, and net export of N. Phase 4 (38-66 days) exhibited substantial losses of N and declining photosynthesis and translocation of C. C:N ratio of xylem sap remained constant (2.3-2.6) during leaf life; petiole phloem sap C:N ratio varied from 25 to 135 over leaf development. The relationships between net photosynthesis and N import in xylem were: phase 1, 4.8 milligrams C per milligram N; phase 2, 24.7 milligrams C per milligram N; phase 3, 91.9 milligrams C per milligram N; and phase 4, 47.7 milligrams C per milligram N.     

Phloem in developing cotton fruits: 6(5)carboxyfluorescein as a tracer for functional phloem

Carbohydrate and water transport into developing cotton (Gossypiumhirsutum L.) fruits is essential for normal fruit development,and phloem has been suggested to play a major role in this transport.To study transport into cotton fruits of different developmentalstages, a phloem tracer [6(5)carboxyfluorescein] was appliedto sympodial leaves. Tracer distribution into the subtendedfruit was determined using epifluorescence microscopy. Phloemthroughout the entire capsule wall and central column was functionalfrom early stages of fruit development. Vasculature of the capsulewall consisted of a complex three-dimensional network. Vascularbundles ended in radially-oriented club-shaped structures, whichconsisted mainly of fibres, with phloem on the outside of thesestructures. The vascular system of the capsule wall was connectedto the central column by amphicribral bundles in the partitions.The central column had two adaxial (ventral) vascular bundlesper carpel. These vascular bundles branched into the placentalregion of the central column, and connected to the seeds viathe funiculi. Amphicribral vascular bundles diverged from theseadaxial bundles into the partition. The results of this studyconfirmed previous observations on the vascular anatomy of thecapsule wall and describe the vascular anatomy of the centralcolumn of cotton fruits Key words: Cotton, Gossypium hirsutum L., phloem, fruit, carboxyfluorescein, vasculature     

Metabolism and translocation of allantoin in ureide-producing grain legumes

Transfer of the nitrogen and carbon of allantoin to amino acids and protein of leaflets, stems and petioles, apices, peduncles, pods, and seeds of detached shoots of nodulated cowpea (Vigna unguiculata L. Walp. cv. Caloona) plants was demonstrated following supply of [2-¹⁴C], [1,3-¹⁵N]allantoin in the transpiration stream. Throughout vegetative and reproductive growth all plant organs showed significant ureolytic activity and readily metabolized [2-¹⁴C]allantoin to ¹⁴CO₂. A metabolic pathway for ureide nitrogen utilization via allantoic acid, urea, and ammonia was indicated. Levels of ureolytic activity in extracts from leaves and roots of nodulated cowpea were consistently maintained at higher levels than in non-nodulated, NO₃⁻ grown plants.

[¹⁴C]Ureides were recovered in extracts of aphids (Aphis craccivora and Macrosiphum euphorbieae) feeding at different sites on cowpea plants supplied with [2-¹⁴C]allantoin through the transpiration stream or to the upper surface of single leaflets. The data indicated that the ureides were effectively transferred from xylem or leaf mesophyll to phloem, and then translocated in phloem to fruits, apices, and roots.

     

Onset of Phloem Export from Senescent Petals of Daylily

During senescence, petals of attached daylily (Hemerocallis hybrid cv Cradle Song) flowers lost 95% sugar and 65% dry weight over the first 24 h, with 30% of dry weight loss coming from nonsugar components. Detaching flowers did not delay senescence, but halted loss of carbohydrate and amino acid, suggesting that loss in the intact state was due to phloem export. Petal autolysis occurred mainly in the interveinal parenchyma, causing vascular strands to begin separating from the petal mass. Such vascular strands still stained with tetrazolium and accumulated sucrose, indicating a retained viability. Their sucrose accumulation rates were high in comparison with those of other plant tissues, and the accumulated product was mainly sucrose. Sucrose synthesis took place in the senescent petal, and sucrose was the principal sugar in phloem exudate, whereas hydroxyproline and glutamine were the main transport amino acids. [14C]Sucrose applied to attached senescent flowers was rapidly translocated to other parts of the plant, particularly developing flower buds. Thus, onset of phloem export allowed most of the soluble carbohydrate and amino acid in the senescing flower to be retrieved by the plant. Additional salvaged material came from proteins and possibly from structural carbohydrate. Over a 12-h period, the flower switched from acting as a strong carbohydrate sink during expansion to become a strong source during senescence. This rapid reversal offers potential for phloem transport studies.     

Subcellular distribution of superoxide dismutase in leaves of ureide-producing leguminous plants

The subcellular localization of superoxide dismutase (SOD; EC. 1.15.1.1) was studied in leaves of two ureide-producing leguminous plants ( Phaseolus vulgaris L. cv. Contender and Vigna unguiculata [L.] Walp). In leaves of Vigna and Phaseolus , three superoxide dismutases were found, an Mn-SOD and two Cu, Zn-containing SODs (I and II). Chloroplasts, mitochondria, and peroxisomes were purified by differential and density-gradient centrifugation using either Percoll or sucrose gradients. The yields obtained in intact chloroplasts and peroxisomes from Vigna were considerably higher than those achieved for Phaseolus . Purified chloroplasts only contained the Cu, Zn-SOD II isozyme, but in mitochondria both Mn-SOD and Cu, Zn-SOD I isozymes were present. In purified peroxisomes no SOD activity was detected. The absence of SOD activity in leaf peroxisomes from Vigna contrasts with results reported for the amide-metabolizing legume Pisum sativum L. where the occurrence of Mn-SOD was demonstrated in leaf peroxisomes (del Río et al. 1983. Planta 158: 216–224; Sandalio et al. 1987. Plant Sci. 51: 1–8). This suggests that in leaf peroxisomes from Vigna plants the generation of O₂^- radicals under normal conditions probably does not take place.     

Bauhinia larsenii, a fossil legume from Guangxi, China

Fossil leaves and a branch with a leaf and a pod attached have been discovered from Ningming County, Guangxi Autonomous Region, China, and are described as Bauhinia larsenii sp. nov. (Leguminosae: Cercideae). The strata from which the Bauhinia fossils were collected, namely the Ningming Formation, were dated as Late Eocene–Oligocene based on a combination of pollen, fauna and flora. The affinity of the new species to Bauhinia section Micralvesia subsection Viridescentes species is also discussed.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 147 , 437–440.     

Optimization of DNA isolation from legume nodules

AIMS: The aim of this study was to optimize DNA extraction from legume nodules to obtain large amounts of high-quality genomic DNA. METHODS AND RESULTS: Nodules of different legume species were used. Varied concentrations of guanidine thiocyanate (from 6 mol l(-1) to 0.05 mmol l(-1)), a component of DNAzol, were tested. The quality of DNA extract was determined by PCR-RFLP. The best results were obtained with 0.5 mmol l(-1) guanidine thiocyanate, which resulted in greater DNA yield than with higher and lower concentrations or with DNAzol. CONCLUSION: The procedure using 0.5 mmol l(-1) guanidine thiocyanate yields the highest DNA amount when compared with previously described protocols and offers a reliable method to isolate DNA from nodules of different origins. SIGNIFICANCE AND IMPACT OF THE STUDY: Irrespective of nodule origin, DNA yield was increased significantly, by two (e.g., Vigna nodules) to seven (Acacia auricoliformis nodules) times. In addition, the proposed procedure's costs are lower than those using the DNAzol.     

Phloem mobility of magnesium

Magnesium-28 was applied to specific leaves of bean (Phaseolus vulgaris) and barley (Hordeum vulgare) plants. After 24 hours, as much as 7% of the absorbed Mg was exported from the treated bean leaves and 11% was transported basipetally from the treated zone of the barley leaves. Transport of Mg did not occur past a heat-killed section of the treated leaf, thereby indicating that translocation took place via the phloem. Mg movement in the phloem was also evident in autoradiograms of bean stem segments in which the xylem was separated from the phloem by a thin sheet of plastic.     

Nitrogen yield advantage from grass–legume mixtures is robust over a wide range of legume proportions and environmental conditions

Current challenges to global food security require sustainable intensification of agriculture through initiatives that include more efficient use of nitrogen (N), increased protein self‐sufficiency through homegrown crops, and reduced N losses to the environment. Such challenges were addressed in a continental‐scale field experiment conducted over 3 years, in which the amount of total nitrogen yield (N_tot) and the gain of N yield in mixtures as compared to grass monocultures (N_gainmix) was quantified from four‐species grass–legume stands with greatly varying legume proportions. Stands consisted of monocultures and mixtures of two N₂‐fixing legumes and two nonfixing grasses. The amount of N_tot of mixtures was significantly greater (P ≤ 0.05) than that of grass monocultures at the majority of evaluated sites in all 3 years. N_tot and thus N_gainmix increased with increasing legume proportion up to one‐third of legumes. With higher legume percentages, N_tot and N_gainmix did not continue to increase. Thus, across sites and years, mixtures with one‐third proportion of legumes attained ~95% of the maximum N_tot acquired by any stand and had 57% higher N_tot than grass monocultures. Realized legume proportion in stands and the relative N gain in mixture (N_gainmix/N_tot in mixture) were most severely impaired by minimum site temperature (R = 0.70, P = 0.003 for legume proportion; R = 0.64, P = 0.010 for N_gainmix/N_tot in mixture). Nevertheless, the relative N gain in mixture was not correlated to site productivity (P = 0.500), suggesting that, within climatic restrictions, balanced grass–legume mixtures can benefit from comparable relative gains in N yield across largely differing productivity levels. We conclude that the use of grass–legume mixtures can substantially contribute to resource‐efficient agricultural grassland systems over a wide range of productivity levels, implying important savings in N fertilizers and thus greenhouse gas emissions and a considerable potential for climate change mitigation.     

Lectin genes from the legume Medicago truncatula

We report the cloning and characterization of two lectin genes from Medicago truncatula, designated Mtlec1 and Mtlec2. The two genes show a high degree of homology and apparently belong to a small multigene family. Mtlec1 appears to encode a functional lectin with 277 amino acids, whereas Mtlec2 is probably non-functional, since a frameshift mutation (insertion of two nucleotides) leads to premature translation termination after only 98 amino acids. The deduced amino acid sequence of the polypeptide MtLEC1 suggests that this lectin is a metalloprotein with Glc/Man specificity.     

Phloem translocation of a reduced oligogalacturonide inRicinus communis L

We have re-examined the evidence against the phloem mobility of oligogalacturonide elicitors using a reduced oligogalacturonide in the phloem translocation system ofRicinus communis var. Gibsonii. A tritium-labelled end-reduced oligogalacturonide of degree of polymerisation 6 was injected into the hollow centre of the petiole of four- to five-week-old plants. Two experimental procedures were followed. In the first, the whole plant was harvested and dissected after 5 h incubation. In the second, phloem sap was collected from an incision in the main stem below the injected petiole; collection started 2 h after incubation and continued for a further 3 h. Determination of the total radiolabel present in the dissected plant showed that at least 8% of the applied activity was exported from the injected leaf, most of this being recovered from the main stem below the injected petiole and the roots. The activity in the phloem exudate showed that the rate of export of radiolabel was already at its maximum by the end of the 2-h incubation period. Radiolabelled material recovered from the main stem was found to be highly comparable to starting material when subjected to thin-layer chromatography. These results demonstrate the phloem mobility of reduced oligogalacturonides of low degree of polymerisation and therefore re-establish the potential for oligogalacturonides to act as systemic signals.     

Nodulation of legume trees from South East Brazil

Summary An extended survey of nodulation of legume trees from South-East Brazilian forests was conducted. Six new species from the Caesalpinioideae, 23 from the Mimosoideae and 27 from the Papilionoideae are reported to have nodules. Nitrogenase activity (acetylene reduction) was tested for all nodules and rhizobia were isolated from the most active.     

Three-phase partitioning of trypsin inhibitor from legume seeds

Three-phase partitioning (TPP) was used to isolate trypsin inhibitors from navy bean (NB), red kidney bean (RK) and adzuki bean (AZ) from the Royal Project Foundation in Thailand. The method was to mix the crude extract with solid ammonium sulfate (30% saturation, w/v) and tert-butanol (t-butanol) in order to obtain the three phases. The trypsin inhibitor was purified to 5-, 14- and 7-fold with 315%, 441% and 228% recovery for NB, RK and AZ, respectively. The SDS-PAGE showed the major inhibitor band with the molecular weights (MWs) of 132, 118 and 13 kDa for NB, RK and AZ, respectively. The fractions from NB and AZ showed higher pH stability compared to that of the RK, and they had the optimum pH ranges of 7–9. The highest relative inhibitory activity of the fractions of NB and RK were found at 50 °C, and all fractions were quite stable at 90 °C for 60 min of incubation. Increasing the concentration of salt (up to 3%, w/v) did not significantly decrease the inhibitory activity of all fractions (p > 0.05). The trypsin inhibitors from the three legumes were unable to inhibit the autolysis of Pacific whiting and arrowtooth flounder muscles.     

Three new legume species from South America

Three new South American legumes are described:Acosmium cardenasii (Leguminosae: Sophoreae) known only from the type locality, San Ignacio de Velasco, Bolivia;Moldenhauera nitida (Leguminosae: Caesalpinioideae) from the southeastern flanks of the Serra do EspinhaÇo in the State of Minas Gerais, Brazil; andSclerolobium prancei (Leguminosae: Caesalpinioideae) known only from the Rio Madeira headwaters, Territory of RondÔnia, Brazil. A key to the Brazilian species ofMoldenhauera is included.