Craig A. Atkins

Purine Biosynthesis. Big in Cell Division, Even Bigger in Nitrogen Assimilation

Synthesis of the purine ring is a central metabolic function of all cells. The products, AMP and GMP, provide purine bases for DNA and RNA, as well as for a number of essential coenzymes (NAD, NADP, FAD, and coenzyme A) and signaling molecules (e.g. cAMP; Fig. [1][1]). ATP serves as the energy

Optimising biological N2 fixation by legumes in farming systems

Whether grown as pulses for grain, as green manure, as pastures or as the tree components of agro-forestry systems, the value of leguminous crops lies in their ability to fix atmospheric N2, so reducing the use of expensive fertiliser-N and enhancing soil fertility. N2 fixing legumes provide the basis for developing sustainable farming systems that incorporate integrated nutrient management. By exploiting the stable nitrogen isotope 15N, it has been possible to reliably measure rates of N2 fixation in a wide range of agro-ecological field situations involving many leguminous species. The accumulated data demonstrate that there is a wealth of genetic diversity among legumes and their Rhizobium symbionts which can be used to enhance N2 fixation. Practical agronomic and microbiological means to maximise N inputs by legumes have also been identified.

Ion Circulation via Phloem and Xylem Between Root and Shoot of Nodulated White Lupin

The exchange rates of mineral cations in the xylem and phloem between root and shoot of white lupin (Lupinus albus L., cv. Ultra) were measured using nodulated plants grown in a defined liquid culture medium low in Na and lacking nitrogen. Harvests were taken at 39 and 49 days after sowing and plant parts analysed for C, N, and the mineral cations K(+), Mg(++), Ca(++), and Na(+). Respiration losses of carbon by nodulated roots were assessed using Pettenkofer assemblies, and concentrations of C, N, and cations in xylem and phloem assayed by collecting root bleeding xylem sap and phloem sap of stem base during the day and night at several times throughout the study period. Flow rates of C and N between root and shoot were determined as in earlier modeling studies (Pate et al., 1979 b), using data on consumption of C by roots, increments of C and N in shoot and root dry matter and, C : N ratios in xylem and phloem sap. Using ratios of cation : C in xylem and phloem, net flow of the various ions between shoot and root were computed. The data showed substantial return of K(+) and Mg(++) from shoot to root with phloem translocate. This return flow provided roots with more K(+) or Mg(++) than was required for growth. It was estimated that 76 % or 87 % of the phloem-borne K(+) and Mg(++) respectively reentered the xylem and was thus circulated within the plant. Rates of return flow to roots and circulation within the plant were very small for Ca(++) and for Na(+) under the conditions of the experiment.

Cellular and Subcellular Organization of Pathways of Ammonia Assimilation and Ureide Synthesis in Nodules of Cowpea (Vigna unguiculata L. Walp.)

Fractionation of cell organelles of nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp) by discontinuous and continuous sucrose density centrifugation indicated that starch-containing plastids possessed the complete pathway for purine nucleotide synthesis together with significant activities of some other enzymes associated with the provision of substrates in purine synthesis; triosephosphate isomerase (EC 5.3.1.1), NADH-glutamate synthase (EC 2.6.1.53), aspartate aminotransferase (EC 2.6.1.1), phosphoglycerate oxidoreductase (EC 1.1.1.95), and methylene tetrahydrofolate oxidoreductase (EC 1.5.1.5). Enzymes of purine oxidation, xanthine oxidoreductase (EC 1.2.3.2), and urate oxidase (EC 1.7.3.3) were recovered in the soluble fraction; glutamine synthetase (EC 6.3.1.2) occurred in bacteroids and in the cytosol. Intact, infected (bacteroid-containing) and uninfected cells were prepared by enzymatic maceration of the central zone of the nodule and partially separated by centrifugation on discontinuous sucrose gradients. Glutamine synthetase was largely restricted to infected cells whereas plastid enzymes, de novo purine synthesis, and urate oxidase were present in both cell types. Although the levels of all enzymes assayed were higher in infected cells, both cell types possessed the necessary enzyme complement for ureide formation. A model for the cellular and subcellular organization of nitrogen metabolism and the transport of nitrogenous solutes in cowpea nodules is proposed.

Evidence for a Purine Pathway of Ureide Synthesis in N2-fixing Nodules of cowpea [Vigna unguiculata (L.) WALP.]

Summary The principal nitrogenous solutes produced from N 2 -fixation and exported from the nodules of cowpea [ Vigna unguiculata (L.) WALP.] were allantoin and allantoic acid. The following evidence suggests that these ureides were derived from purines. 1. Nodules were particularly active in enzymes of purine oxidation (xanthine dehydrogenase, uricase, allantoinase). 2. These enzymes were found largely restricted to the cytosol of the bacteroid containing cells (ie in close proximity to the sites of N 2 fixation) an-d increased in activity with nodule development. 3. Allopurinol (4-hydroxypyrazolo [3,4-d] pyrimidine) was an effective inhibitor of the xanthine dehydrogenase extracted from cowpea nodules and low concentrations (0.08-0.31 mM) applied to the root systems of intact plants in liquid culture rapidly (within 1 hr) inhibited ureide export, reduced the nodule pools of ureides and caused a concomitant accumulation of xanthine in nodules. 4. Glycine-2, 14 C supplied to slices of nodule tissue was more readily incorporated into allantoin and allantoic acid than glucose-U, 14 C or acetate-1,2, 14 C.

Macromolecules in phloem exudates - a review

Proteomic and transcriptomic analyses using the growing resources of genomic information have been applied to identification of macromolecules in exudates collected from phloem. Most of the analyses rely on collection of exudate following incisions made to the vasculature, but some limited data are available for exudates collected from excised aphid stylets. Species examined, to date, include a number of cereals (rice, barley, and wheat), a number of cucurbits, castor bean, members of the genus Lupinus, brassicas, and Arabidopsis. As many as 1,100 proteins, some hundreds of transcripts, and a growing number of small ribonucleic acids (RNAs), including micro-RNAs, have been identified across the species with a high degree of commonality. Questions relating to the nature and extent of contamination of sieve element contents with those of surrounding companion cells and nonvascular cells are addressed together with likely functions of identified macromolecules. The review considers likely translocation and systemic signaling functions among the macromolecular inventory of phloem exudates.

Transformation of a grain legume (Lupinus angustifolius L.) via Agrobacterium tumefaciens-mediated gene transfer to shoot apices

Transgenic plants of Lupinus angustifolius L. (cvs. Unicrop and Merrit) were routinely generated using Agrobacterium-mediated gene transfer to shoot apices. The bar gene for resistance to phosphinothricin (PPT, the active ingredient of the herbicide Basta) was used as the selectable marker. After co-cultivation, the shoot apex explants were transferred onto a PPT-free regeneration medium and their tops were thoroughly wetted with PPT solution (2 mg/ml). The multiple axillary shoots developing from the shoot apices were excised onto a medium containing 20 mg/l PPT. The surviving shoots were transferred every second week onto fresh medium containing 20 mg/l PPT. At each transfer, the number of surviving shoots decreased, until it stabilized. Indeed, some of these chimeric shoots surviving the PPT selection, eventually produced new green healthier axillary shoots which could be transferred to soil. This whole process took from 5 to 9 months after co-cultivation. Average transformation frequencies of 2.8% for cv. Unicrop and of 0.4% for the commercial cultivar Merrit were achieved. Molecular analysis of T0, T1, and T2 generations demonstrated stable integration of the foreign gene into the plant genome and expression of the integrated gene. Transformed plants of the T1 and T2 generations were resistant in glasshouse trials where the herbicide Basta (0.1 mg/ml) was sprayed onto whole plants. These results demonstrate that Agrobacterium-mediated gene transfer to preorganised meristematic tissue combined with axillary regeneration can form the basis of a routine transformation system for legume crop species which are difficult to regenerate from other explants.

The extrafloral nectaries of cowpea (Vigna unguiculata (L.) Walp.) II. Nectar composition, origin of nectar solutes, and nectary functioning

Nectar was collected from the extrafloral nectaries of leaf stipels and inflorescence stalks, and phloem sap from cryopunctured fruits of cowpea plants. Daily sugar losses as nectar were equivalent to only 0.1–2% of the plants current net photosynthate, and were maximal in the fourth week after anthesis. Sucrose:glucose:fructose weight ratios of nectar varied from 1.5:1:1 to 0.5:1:1, whereas over 95% of phloem-sap sugar was sucrose. [14C]Sucrose fed to leaves was translocated as such to nectaries, where it was partly inverted to [14C]glucose and [14C]fructose prior to or during nectar secretion. Invertase (EC 3.2.1.26) activity was demonstrated for inflorescence-stalk nectar but not stipel nectar. The nectar invertase was largely associated with secretory cells that are extruded into the nectar during nectary functioning, and was active only after osmotic disruption of these cells upon dilution of the nectar. The nectar invertase functioned optimally (phloem-sap sucrose as substrate) at pH 5.5, with a starting sucrose concentration of 15% (w/v). Stipel nectar was much lower in amino compounds relative to sugars (0.08–0.17 mg g-1 total sugar) than inflorescence nectar (22–30 mg g-1) or phloem sap (81–162 mg g-1). The two classes of nectar and phloem sap also differed noticeably in their complements of organic acids. Xylem feeding to leaves of a range of 14C-labelled nitrogenous solutes resulted in these substrates and their metabolic products appearing in fruit-phloem sap and adjacent inflorescence-stalk nectar. 14C-labelled asparagine, valine and histidine transferred freely into phloem and appeared still largely as such in nectar. 14C-labelled glycine, serine, arginine and aspartic acid showed limited direct access to phloem and nectar, although labelled metabolic products were transferred and secreted. The ureide allantoin was present in phloem, but absent from both types of nectar. Models of nectary functioning are proposed.

Partitioning of K+, Na+, Mg++ , and Ca++ through Xylem and Phloem to Component Organs of Nodulated White Lupin under Mild Salinity

Summary Rates of cation (K + , Na + , Mg ++ , Ca ++ ) transport via xylem and phloem and of cation exchange among component organs of effectively nodulated white lupin ( Lupinus albus L. cv. Ultra) were determined during an 8-day period at flowering under mild salinity (10 mol m −3 NaCl). Carbon, nitrogen and mineral cations were analyzed in roots, leaflets, stem, and petiole tissue of each of four strata of leaves of the shoot, in root bleeding (xylem) sap and in phloem saps obtained from stems and petioles of the corresponding strata. Respiration of roots and respiration and photosynthesis of stem segments and leaves of each stratum of the shoot were measured. Carbon and nitrogen flows within the shoot and between root and shoot were then estimated as in earlier studies (Pate et al., 1979 b). Using these flow profiles, ratios of each cation to carbon in transport fluids and increments of ions in plant parts during the study period and patterns of partitioning of individual cations were estimated. The data revealed high rates of transport in phloem and xylem within the shoot for K + , and to a lesser extent also for Mg ++ and Na + . Substantial rates of cycling of K + and Mg ++ between shoot and root were observed. K + was translocated preferentially towards young, growing organs, but Na + mainly deposited in stems (partly in exchange for K + ) and transported preferentially to the root. Acropetal translocation of K + in phloem occurred preferentially towards the inflorescence, in xylem principally towards lateral branches. The flow model for K + suggested progressive enrichment of the upward moving xylem stream with K + through mobilization of K + from mature stem tissues, combined with selective phloem to xylem transfer of K + in the stem vasculature. Translocation of Na + to the root was partly due to preferential xylem to phloem transfer within stem tissue. Basipetal transport of Na + was particularly evident when the external supply of NaCl was removed. Phloem mobility of Na + is discussed in relation to the relatively limited salt tolerance of white lupin.

Effect of salinity on growth of four strains of Rhizobium and their infectivity and effectiveness on two species of Acacia

Two Rhizobium strains (WU1001 and WU1008) were isolated from nodules of Acacia redolens growing in saline areas of south-west Australia, and two strains selected from the University of Western Australias culture collection (WU429 isolated from A. saligna and WU433 from A. cyclops). The growth of each in buffered, yeast extract mannitol broth culture was largely unaffected by salt up to 300 mM NaCl. A slight increase in lag time occurred at concentrations of 120 mM NaCl and above, but cell number at the static phase was not affected. Each of the four Rhizobium strains tested accumulated Na+ but showed decreasing levels of sugar with increasing salt in the external medium. Amino acid levels also increased, in some cases by more than tenfold. However, the relative proportion of each remained fairly constant in the bacteria, irrespective of salt treatment. Only trace quantities of proline were detected and there was no increase in this amino acid with salt. Acidic amino acids (glutamate and aspartate) remained as a constant proportion.Rhizobium strains WU429, WU1001 and WU1008 produced effective nodules on both A. cyclops and A. redolens grown in sand with up to 80 mM NaCl (added in nutrient solutions free of nitrogen). Strain WU433 was highly infective on both Acacia species tested at low salt concentrations (2–40 mM NaCl), but infection was sensitive to salt levels at 120 mM NaCl and above. Nodules formed with strain WU433 were, however, ineffective on both A. redolens and on A. cyclops and showed nil or negligible rates of acetylene reduction at all salt concentrations. Strains WU429, WU1001 and WU1008 in combination with a highly salt-tolerant provenance of A. redolens formed symbioses which did not vary significantly in nodule number and mass, specific nodule activity or total N content irrespective of salt level up to 160 mM NaCl. On a more salt sensitive provenance of A. redolens and on A. cyclops the infectivity and effectivity of the Rhizobium strains tested usually decreased as the external salt concentration increased. These data are interpreted to indicate that tolerance of the legume host was the most important factor determining the success of compatible Rhizobium strains in forming effective symbioses under conditions of high soil salinity.