Cellular and subcellular organization of pathways of ammonia assimilation and ureide synthesis in nodules of cowpea (Vigna unguiculata L. Walp)

Shelp, Barry J.; Atkins, Craig A.; Storer, Paul; Canvin, David T.

doi:10.1016/0003-9861(83)90229-1

Cited by 87 publications

(54 citation statements)

References 26 publications

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“…The formation of ureides in root nodules involves linked metabolic pathways of de novo purine synthesis and catabolism (17). Some of the enzymes of purine biosynthesis are found in the proplastids of infected cells (18). On the other hand, uricase II and other enzymes of ureide metabolism have been detected in uninfected cells of soybean nodules (4).…”

mentioning

confidence: 99%

Purification, cDNA Cloning, and Developmental Expression of the Nodule-Specific Uricase from Phaseolus vulgaris L.

Sánchez

Campos²,

Padilla³

et al. 1987

Plant Physiol.

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Nodule-specific uoricase (uricase I) from Phasolus vulgas L. was purified to homogeneity by chromatographic methods. Purification data indicated that uricas II is approximately 2% of the total soluble protein from mature nodules. Specific antiserum was raised and used to determine the developmental expression and for immunoselection of polysomes. Uricase II was antigenically detected early in nodule development, 2 to 3 days before nitrogen fixation. Uricase-encoding cDNA clones were isolated by hybridizing a nodule-specific pUC9 cDNA library with labeled mRNA from immunoselected polysomes and a 35,000 molecular weight uricase II-encoding cDNA from soybean. An homologous clone (pNF-UR07) was used to assess the expression pattern of the specific transcript during development. Northern-blot analysis indicated that uricase II mRNA is exclusively expressed in nodule tissue.

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mentioning

confidence: 99%

Purification, cDNA Cloning, and Developmental Expression of the Nodule-Specific Uricase from Phaseolus vulgaris L.

Sánchez

Campos²,

Padilla³

et al. 1987

Plant Physiol.

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“…However, if the bacteroid/plastid fraction was snap frozen in liquid N 2 and thawed, only the plastids were lysed so that their contents could be collected as a soluble extract. Assays of this soluble extract for a bacteroid-marker enzyme, ␤-hydroxybutyrate dehydrogenase (␤-HBD; Shelp et al, 1983), yielded a low level of activity (9 nmol min Ϫ1 mg Ϫ1 protein), indicating some small degree of lysis of bacteroids after this freeze-thaw treatment. Lysing the bacteroid pellet by Triton X-100 treatment after removing the soluble plastid extract released the ␤-HBD activity (91 nmol min Ϫ1 mg Ϫ1 protein).…”

Section: Purification and Partial Characterization Of Mitochondrial Amentioning

confidence: 99%

“…No ␤-HBD activity could be recovered in the washed mitochondrial suspension whether or not detergent was added. Furthermore, the soluble plastid extract was essentially free of mitochondrial contamination as indicated by a very low level of Glu dehydrogenase activity (marker for plant mitochondria; Shelp et al, 1983). The plastid fraction typically showed 1 to 4 nmol Glu-dependent NAD-reduction min Ϫ1 mg Ϫ1 protein compared with 130 nmol Glu-dependent NAD-reduction min Ϫ1 mg Ϫ1 protein for the mitochondria fraction.…”

Section: Purification and Partial Characterization Of Mitochondrial Amentioning

confidence: 99%

“…AIRS was assayed spectrophotometrically as described by Schrimsher et al (1986), and ␤-HBD and Glu dehydrogenase as described by Shelp et al (1983) and Atkins et al (1975), respectively. Intactness of membranes in bacteroid preparations was assessed by measuring the activity of ␤-HBD before and after treatment of bacteroids with Triton X-100.…”

Section: Enzyme Assaysmentioning

confidence: 99%

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Dual Intracellular Localization and Targeting of Aminoimidazole Ribonucleotide Synthetase in Cowpea

Goggin¹,

Lipscombe²,

Fedorova³

et al. 2003

Plant Physiology

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De novo purine biosynthesis is localized to both mitochondria and plastids isolated from Bradyrhizobium sp.-infected cells of cowpea (Vigna unguiculata L. Walp) nodules, but several of the pathway enzymes, including aminoimidazole ribonucleotide synthetase (AIRS [EC 6.3.3.1], encoded by Vupur5), are encoded by single genes. Immunolocalization confirmed the presence of AIRS protein in both organelles. Enzymatically active AIRS was purified separately from nodule mitochondria and plastids. N-terminal sequencing showed that these two isoforms matched the Vupur5 cDNA sequence but were processed at different sites following import; the mitochondrial isoform was five amino acids longer than the plastid isoform. Electrospray tandem mass spectrometry of a trypsin digest of mitochondrial AIRS identified two internal peptides identical with the amino acid sequence deduced from Vupur5 cDNA. Western blots of proteins from mitochondria and plastids isolated from root tips showed a single AIRS protein present at low levels in both organelles. 35 S-AIRS protein translated from a Vupur5 cDNA was imported into isolated pea (Pisum sativum) leaf chloroplasts in vitro by an ATPdependent process but not into import-competent mitochondria from several plant and non-plant sources. Components of the mature protein are likely to be important for import because the N-terminal targeting sequence was unable to target green fluorescent protein to either chloroplasts or mitochondria in Arabidopsis leaves. The data confirm localization of the protein translated from the AIRS gene in cowpea to both plastids and mitochondria and that it is cotargeted to both organelles, but the mechanism underlying import into mitochondria has features that are yet to be identified.In root nodules of legumes of the tribe Phaseoleae, symbiotically fixed N 2 is assimilated by de novo purine biosynthesis, after which the purines are oxidized to allantoin and allantoic acid (Atkins and Smith, 2000). It is in this form that fixed N is exported in xylem from the nodule to the shoot. The 10 enzymes of the purine biosynthetic pathway have been well studied in relation to their role in cancer metabolism, and the genes encoding them cloned from a wide variety of organisms. However, in plants, far less is known about the enzymes of the pathway, although genes encoding a number have now been isolated (Atkins and Smith, 2000).The cDNA sequences of all the plant purine biosynthetic (pur) genes studied have 5Ј extensions relative to the coding regions of the corresponding Escherichia coli genes (Senecoff and Meagher, 1993;Chapman et al., 1994;Schnorr et al., 1994Schnorr et al., , 1996Kim et al., 1995;Senecoff et al., 1996;Smith et al., 1998). These extensions potentially encode N-terminal, organelle-targeting sequences and indicate that in contrast to other eukaryotes (Gooljarsingh et al., 2001), the pathway is likely to be organelle-localized in plants. This was confirmed in a recent study that showed that in cowpea (Vigna unguiculata L. Walp) nodules, both the mitochondria ...

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“…NADlinked xanthine dehydrogenase, also in infected cells, oxidizes the bases to uric acid but the next reaction of the pathway (oxidation of uric acid to allantoin by uricase) occurs in uninfected cells and requires O 2 . Assays of uricase activity following enzymatic separation of the two cell types and their organelles (Shelp et al 1983;Atkins, Smith & Storer 1997), together with immunogold-labelling using polyclonal antibodies to the purified enzyme (Webb & Newcomb 1987), indicate that uricase is almost exclusively localized in microbodies of uninfected cells.…”

Section: Introductionmentioning

confidence: 99%

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

Thumfort

Layzell

Atkins

1999

Plant Cell & Environment

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It is not surprising then that the uricase reaction is believed to be severely limited by O 2 supply in vivo (Rainbird & Atkins 1981;Atkins 1991). If the pathway of purine synthesis was not closely regulated, accumulation of relatively insoluble uric acid could possibly limit the flux of fixed N from the nodule. However, cowpea nodules do not show measurable accumulation of uric acid (Atkins et al. 1988) and, even under conditions of reduced external pO 2 , ureide synthesis is maintained at a high rate (Atkins, Dakora & Storer 1990). Thus, either the affinity of uricase for O 2 is much higher in vivo than measured for the purified enzyme in vitro or the [O 2 ] in uninfected cells is considerably higher than that in adjacent infected cells. High intracellular [O 2 ] (Oi) in uninfected cells requires high pO 2 in the intercellular gas spaces, yet such concentrations are incompatible with a view of nodule functioning that does not allow for a resistance to O 2 entry into the infected cell .Most previous mathematical models for the diffusion and reaction of O 2 in nodules assumed [O 2 ] within the cytosol of the infected cell would be close to being equilibrated with pO 2 in the intercellular gas spaces of the tissue (Bergersen 1982;Sheehy et al. 1987;Gaito, Hunt & Layzell 1989;Bergersen 1994). Thus pO 2 in the intercellular gas spaces needed to satisfy uricase would be expected to raise Oi in the infected cells above the range in which nitrogenase activity is maintained. However, more recent simulations of diffusion into infected cells (Thumfort, Atkins & Layzell 1994, Thumfort, Layzell & Atkins 1999 predicted that saturation of leghaemoglobin (Lb) in the cyosol close to the cell : gas space interface would limit facilitated diffusion and provide an intrinsic mechanism for a variable diffusive resistance to O 2 entry. Bergersen (1996) also predicted significant gradients of [O 2 ] in the cytosol of infected cells, but attributed this to disequilibrium between O 2 and Lb due to loading of deoxygenated Lb near the cell surface. This region of the cell also shows a concentration of organelles (mitochondria and plastids) so that the volume of cytosol and amount of Lb is significantly reduced compared to the rest of the infected cell (Millar, Day & Bergersen et al. 1995). One consequence to resistance at this point in the diffusive pathway is that pO 2 in the gas spaces surrounding both infected and uninfected cells could be as high as 1-2% (12-25 mM;Thumfort et al. 1994Thumfort et al. , 1999 but would still maintain average Oi levels in infected cells as low as 10-60 nM. Such high pO 2 in the gas spaces could satisfy the low affinity of uricase for O 2 .All earlier mathematical models of nodules considered a central zone tissue comprising only infected cells and intercellular gas spaces. The present study models the diffusion and metabolism of O 2 more realistically. A geometrically detailed computer simulation was developed to incorporate the three-dimensional shape and packing of both cell types, at the f...

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Cellular and subcellular organization of pathways of ammonia assimilation and ureide synthesis in nodules of cowpea (Vigna unguiculata L. Walp)

Cited by 87 publications

References 26 publications

Purification, cDNA Cloning, and Developmental Expression of the Nodule-Specific Uricase from Phaseolus vulgaris L.

Purification, cDNA Cloning, and Developmental Expression of the Nodule-Specific Uricase from Phaseolus vulgaris L.

Dual Intracellular Localization and Targeting of Aminoimidazole Ribonucleotide Synthetase in Cowpea

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

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