Progress 07/01/00 to 06/30/06
Outputs
While arginase in the animal urea cycle functions jettison excess N as urea, in germinating seeds arginase is involved in freeing N of arginine residues reserve proteins. Recently, plant arginases have been implicated in other roles, including defense. We showed that a second arginase (ARGAH2), closely linked to the first plant arginase gene (ARGAH1) identified on Arabidopsis chromosome 4, is functional in yeast. Both are localized to the mitochondrial matrix. ARGAH1 and ARGAH2 and are not expressed similarly in all organs and T-DNA insertion mutants demonstrate that the two genes are not functionally redundant. argah1 and argah2 mutant seeds had higher total nitrogen, but smaller amino acid pools and less free arginine than wild-type. Enzyme activities in the germinating mutants were differentially reduced; the resultant activities were additive to wild-type levels. Both arginases were induced following mechanical wounding, and disruption of either did not affect
wounding induction of the other. We suggest a possible link between the regulation of Arabidopsis mitochondrial arginases and mitochondrial nitric oxide synthase. Arabidopsis mitochondria have two basic amino acid transporters (BAC1 and BAC2) with partially overlapping specificities and differential expression in seedling development. Recombinant and purified BAC2, as previously reported for BAC1, transported various basic L-amino acids upon reconstitution in phospholipid vesicles. BAC1 and BAC2 displayed highest affinity for arginine with similar Km and Vmax. However, BAC2 transported citrulline for which BAC1 had little or no affinity. By semi-quantitative RT-PCR BAC1 transcript levels were higher than those of BAC2 in germinated seeds. However, BAC2 expression sharply and transiently increased two days after germination. Disruption of the Arabidopsis arginase structural genes (ARGAH1 or ARGAH2) accentuated the increases of transcript levels of BAC1 at germination and of BAC2 two
days after germination and from six days on. Early expression of BAC1 and BAC2 is consistent with the delivery of arginine, released from seed reserves, to mitochondrial arginase and the export of ornithine. Increase of BAC2 transcript levels later in seedling development is consistent with roles in NO, polyamine or proline metabolism - processes which involve involving arginine, citrulline and/or ornithine.
Impacts
Ability to manipulate arginine movement into various cell organelles-- mitochondria, chloroplasts, peroxisomes-- can control the production of important plant regulatory molecules, such as NO and polyamines. Our identification of a mitochondrial location for both arginase isozymes of Arabidopsis (as well as soybean arginase activity and mitochondrial signal peptide) and Arg/Citrulline transporters is basic to manipulation arginine pools and intracellular movement. We have already seen effects on NO-controlled processes in the arginase mutants.
Publications
- Goldraij A, Polacco JC (1999) Arginase is inoperative in developing soybean seeds. Plant Physiol 119: 297-304.
- Goldraij A, Polacco JC (2000) Arginine degradation by arginase in mitochondria of soybean seedling cotyledons. Planta 210: 652-658.
- Hoyos ME, Palmieri L, Wertin T, Arrigoni R, Polacco JC, Palmieri F (2003) Identification of a mitochondrial transporter for basic amino acids in Arabidopsis thaliana by functional reconstitution into liposomes and complementation in yeast. The Plant Journal 33: 1027-1033.
- Lombardo MC, Graziano M, Polacco JC, Lamattina L (2006) Nitric oxide functions as a positive regulator of root hair development. Plant Signaling & Behavior 1: 28-33.
- Palmieri L, Todd CD, Arrigoni R, Hoyos ME, Santoro A, Polacco JC (2006) Arabidopsis mitochondria have two basic amino acid transporters with partially overlapping specificities and differential expression in seedling development. BBA-Bionergetics. in press (available online)
|
Progress 01/01/04 to 12/31/04
Outputs
We have identified two Myb factors whose levels respond to P and N deprivation specifically. A disruption of one of them (the N-responsive one) leads to alteration in expression of N deficiency responsive genes in Arabidopsis (Todd et al. 2004). The promoters of many genes involved in N responsiveness undoubtedly contain common 'elements' or boxes. We have devised a screen of sequenced genomes for localization of these sequences in defined regions upstream of open reading frames (Reneker et al. 2004). In soybean, 'gene chips' or microarrays of 29000 genes (Vodkin et al. 2004) will allow breeders to distinguish varieties with respect to N partitioning, efficient N-fixation under drought stress, etc.
Impacts
The signals that plants respond to when N, and other nutrients, are limiting are not well understood. In addition, mineral and nutrient limitation is usually multivariate, so that the plant usually has to orchestrate multiple pathways for improving uptake and assimilation of more than one nutrient (eg P and N). Understanding the signalling will identify key genes, and these may provide QTL markers in breeding programs for cultivars which perform better under lowered nutrient availability (as may occur in marginal farmland, or in the third world where access to fertilizer is impossible).
Publications
- Todd, CD, Peiyu Z, Rodriguez Huete AM, Hoyos ME, Polacco JC (2004) Transcripts of MYB-like genes respond to phosphorus and nitrogen deprivation in Arabidopsis. Planta 219: 1003-1009.
- Reneker J, Shyu C-R, Zeng, P , Polacco JC, Gassmann W (2004) ACMES: Fast multiple-genome searches for short repeat sequences with concurrent cross-species information retrieval. Nuc Acid Res 32:W649-W653.
- Vodkin LO, Khanna A, Shealy R, Clough SJ, Delkin OG, Philip R, Zabala G, Thibaud-Nissen F, Sidarous M, Stromvik MV, Shoop E, Schmidt C, Retzel E, Erpelding J, Shoemaker RC, Rodriguez-Huete A, Polacco JC, Coryell V, Keim P, Gong G, Liu L, Pardinas J, Schweitzer P (2004) Microarrays for global expression using 27,500 sequenced cDNAs representing an array of developmental stages and physiological conditions of the soybean plant. Online, Bio Med Cent Genomics 5, 73 (http://www.biomedcentral.com/bmcgenomics/)
|
Progress 07/01/02 to 06/30/03
Outputs
OBJECTIVES: To isolate and characterize the genes involved in three aspects of arginine breakdown during soybean seed germination: (i) the entry of arginine into the mitochondrion, the site of arginase, (ii) the breakdown of arginine by arginase(s), and (iii) the Ni activation of urease, the enzyme which hydrolyzes urea, one of the arginase reaction products. i. We have cloned two arginine carriers of the inner mitochondrial membrane (BAC, or Basic Amino Acid MCP [mitochondrial carrier protein). Recombinant BAC-MCP's were integrated into membranes of artificial vesicles, as well as into transgenic yeast mutants lacking an ornitine/arginine MCP (arg11)d other hosts. ii. We have characterized two arginases from Arabidopsis. Both are active in yeast and both have mitochondrial transit N-terminal peptides. iii. In characterizing a putative mutant (AJ6) in Ni insertion we determined that it contained a missense alteration in the ubiquitous urease structural gene and that
this variant and a second allelic alteration complemented each other. This INTERALLELIC COMPLEMENTATION provided us insight into plant urease structure. and showed it to be similar to that of bacterial urease, though the latter is a nonamer and the plant enzyme a trimer.
Impacts
Nitrogen (N) is the most limiting mineral nutrient in crop productivity. Urea is one of the major forms of N fertilizer worldwide. It is important to understand the structure and function of plant ureases which break down much of this urea. We focus on the placement of nickel atoms into urease-- without nickel urease is inactive. We also focus on urease structure because this will help us design plant ureases which are resistant to urease innhibitors. Since much of plant N is stored and/or moved as arginine it is important to understand how arginine is broken down. One of the products of arginase-catalyzed breakdown of arginine is urea, and urease is required for complete mobilization, or recycling, of arginine N.
Publications
- Todd CD, Polacco JC (2004) Soybean cultivars Williams 82 and Maple Arrow produce both urea and ammonia during ureide degradation. J Exp Bot 55: 867-877.
- Todd, CD, Peiyu Z, Rodriguez Huete AM, Hoyos ME, Polacco JC (2004) Transcripts of MYB-like genes respond to phosphorus and nitrogen deprivation in Arabidopsis. Planta accepted.
- Reneker RJ, Shyu C-R, Zeng, P , Polacco JC, Gassmann W (2004) ACMES: Fast multiple-genome searches for short repeat sequences with concurrent cross-species information retrieval. Nuc Acid Res accepted.
- Hoyos ME, Palmieri L, Wertin T, Arrigoni R, Polacco JC, Palmieri F (2003) Identification of a mitochondrial transporter for basic amino acids in Arabidopsis thaliana by functional reconstitution into liposomes and complementation in yeast. Plant J 33: 1027-1033.
- Goldraij A, Beamer LJ, Polacco JC (2003) Interallelic complementation at the ubiquitous urease coding locus of soybean. Plant Physiol 132: 1801-1810.
- Zeng P, Vadnais DA, Zhang Z, Polacco JC (2003) Refined glufosinate selection in Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill] Plant Cell Reports 22: 478 - 482
|
Progress 01/01/02 to 12/31/02
Outputs
OBJECTIVES: To characterize genes involved in Arg (Arg) breakdown during soybean germination: (i) the entry of Arg into the mitochondrion, the site of arginase, and (ii) the Ni activation of urease, the enzyme which hydrolyzes urea, one of the arginase reaction products. 1. Clone the Arg carrier(s) of the inner mitochondrial membrane (Arg MCP). 2. Functionally integrate recombinant Arg MCP into membranes of artificial vesicles. 3. Clone urease accessory proteins. beyond UreG. 4. Characterize a putative new accessory protein mutant, AJ6. APPROACH: 1. To clone the Arg MCP(s) we will take advantage of the similarity among MCP's to identify candidates and confirm them by complementation cloning in a yeast arg11 knockout which lacks the major Arg MCP. 2. Recombinant Arg MCP will be integrated into membranes of artificial vesicles, as well as in transgenic yeast, bacteria and other hosts. 3. To clone more urease accessory proteins we will pursue recently isolated soybean
homologs to bacterial ureD and ureF. Functionality will be determined in S. pombe urease mutants. 4. To test the hypothesis that Aj6 has a lesion in a unique plant urease accessory gene we will carefully repeat allelism tests, determine Ni movement and urease isozyme levels in mutant aj6/aj6. PROGRESS: NICKEL- Ni is an active site component of both plant and bacterial ureases. We now know that the soybean Eu3 urease accessory gene encodes a homolog of bacterial ureG, quite similar to that of bacteria except for a His-rich N-terminal extension, making it also similar to hypB, a bacterial ureG homolog which activates the Ni metalloenzyme, hydrogenase, not found in plants. A relationship between urease and hydrogenase with respect to Ni availability was evidenced by our finding that soybean eu3 mutants harbor PPFM bacteria (pink-pigmented facultative methylotrophs, Methylobacterium spp.) which are urease and hydrogenase-negative in planta and which behave as if they are Ni-deprived in
free-living culture. This year we have identified urease accessory genes ureD, ureF and ureG of tomato, Arabidopsis and soybean as well as thos of the fungus Schizosaccharomyces pombe. Plant UreF is functional in that it corrected the pombe ureF mutant. NITROGEN- Urease is re-incorporates the urea produced from arginase-catalyzed catabolism of seedling Arg. Arg constitutes 18% of total soybean seed N. We had shown that arginase is transcriptionally regulated, that it is mitochondrial and that Arg enters the mitochondria by facilitated diffusion. This year we have cloned and expressed two distinct Arabidopsis putative Arg mitochondrial inner membrane carrier proteins (Arg mcp's). Expressed in yeast they complemented the arg11 mutant defective in mitochondrial ornithine-Arg. E. coli-expressed proteins were incorporated into in vitro proteoliposomes. One Arg mcp favored external Arg (over lysine and histidine) for facilitating efflux of radiolabeled internal Arg. However, though this MCP
(Atm-BAC1) favors Arg as a basic amino acid substrate, it does not appear to be exclusively expressed in the germinating seedling.
Impacts
Control of Arg movement into intracellular compartments of soybean will allow us to alter levels of Arg in foods as well as levels of Arg derivatives such as polyamines and nitric oxide (NO), compounds can have important effects on plant development and disease resistance. To exploit the plant-PPFM relationship, we are identifying the bacterial product that stimulates germination of aged soybean seeds.
Publications
- Bacanamwo M, Witte CP, Lubbers MW, Polacco JC (2002) Activation of the urease of Schizosaccharomyces pombe by the UreF accessory protein from soybean. Molec Genet Genom MGG 268:525-34.
|
Progress 01/01/01 to 12/31/01
Outputs
NICKEL: Ni is an active site component of both plant and bacterial ureases. In previous years we showed that the soybean Eu3 urease accessory gene encoded a homolog of bacterial ureG, quite similar to that of bacteria accept for a His-rich N-terminal extension, making it also similar to hypB, a bacterial ureG homolog which activates the Ni metalloenzyme, hydrogenase, not found in plants. A relationship between urease and hydrogenase with respect to Ni availability was evidenced by our finding that soybean eu3 mutants harbor PPFM bacteria (pink-pigmented facultative methylotrophs, Methylobacterium spp.) which are urease and hydrogenase-negative in planta and which behave as if they are Ni-deprived in free-living culture. This year we have identified urease accessory genes ureD, ureF and ureG of the fungus Schizosaccharomyces pombe as well as mutants lacking these proteins. The ureF mutant was corrected with plant UreF. The plant UreD transcript appears to be subjected
to alternative splicing, and soybean mutant AJ6, deficient in ubiquitous, but not embryo-specific urease, appears to accumulate a new, mutant UreD variant. NITROGEN: In soybean, a ureide source of urea was discounted by us in the variety Williams 82. However, there is a report that ureides are degraded to urea in varieties with drought-tolerant N-fixation. Urease is important for re-incorporating the urea produced from catabolism of Arg which conbstitutes 18% of total soybean seed N. Upon germination much Arg is broken down by arginase. To examine arginase regulation at the cell biological level we determined that it was mitochondrial and that Arg entered the mitochondria by facilitated diffusion. This year we have cloned and expressed two distinct Arabidopsis putative Arg mitochondrial inner membrane carrier proteins (Arg mcp's). Expressed in yeast they complemented the arg11 mutant defective in mitochondrial ornithine-Arg. E. coli-expressed proteins were incorporated into in vitro
proteoliposomes. One Arg mcp favored external Arg (over lysine and histidine) for facilitating efflux of radiolabeled internal Arg. A goal is to ablate this transporter in source leaves leading to great delivery of Arg to the developing embryo.
Impacts
Control of Arg movement in to intracellular compartments of soybean and other plants will allow us to alter levels of Arg in foods as well as levels of Arg derivatives such as polyamines and nitric oxide (NO). The latter two compounds can have important effects on plant development and disease resistance. We are learning more of the PPFM-plant association to manipulate it for improvement of plant performance.
Publications
- Coello P.; Maughan J.P.; Mendoza A.; Philip R.; Bollinger D.W.; Veum T.L.; Vodkin L.O.; Polacco J.C. (2001) Generation of low phytic acid Arabidopsis seeds expressing an E. coli phytase during embryo development. Seed Sci Res 11, 285-292.
- Koenig RL, Morris RO, Polacco JC (2002) tRNA Is the Source of Low-Level trans-Zeatin Production in Methylobacterium spp. J Bacteriol 184, 1832-1842.
|
Progress 01/02/00 to 12/31/00
Outputs
NICKEL: We are interested in two biological roles for Ni, as a component of the active sites of hydrogenase and urease. The former is a bacterial enzyme and is reported to be important for the recapture of energy lost when nitrogenase reduces protons instead of N2. The latter is both a plant and bacterial enzyme. In soybean, urease is important for re-incorporating the urea produced from catabolism of arginine (a ureide urea source is still controversial). Both enzymes require accessory genes for proper insertion of Ni. We have shown that the Eu3 urease accessory gene of soybean encodes a ureG homolog, quite similar to that of bacteria accept for a His-rich N-terminal extension. We are examining the relationship between hydrogenase and urease activation by Ni, in Rhizobium leguminosarum. We are attempting to correct a HypB mutant, deficient in hydrogenase activation, with the ureG homolog of plants. A relationship between urease and hydrogenase with respect to Ni
availability is evidenced by our earlier finding that soybean mutants unable to incorporate Ni into the plant urease active site harbor pink-pigmented facultative methylotrophs (PPFM's = Methylobacterium spp.) which are urease and hydrogenase-negative in planta and which behave as if they are Ni-deprived in free-living culture. Recently, mutations in the hydrogenase activation genes, HypA and HypB of Helicobacter, were found to increase dramatically the Ni requirement for urease production (lab of R Meier). NITROGEN: Arginine conbstitutes 18% of total soybean seed N. Upon germination much of it is broken down by arginase. We have shown that arginase is mitochondrial, that it is induced at the mRNA, protein and activity levels upon germination and that it is primarily, if not solely, responsible for the generation of seedling urea. During seed development, however, though arginase is potentially active it does not operate to hydrolyze Arg, thus avoiding a futile urea cycle. To
determine whether the mitochondrial membrane is a barrier to Arg entry during seed development we compared Arg uptake by mitochondria isolated from pre and post-germination cotyledons. There was no difference in rate or in substrate concentration dependence. The uptake was specific and the apparently coupled mitochondria did not need to generate a membrane potential for uptake. These properties suggest the action of an inner mitochondrial membrane carrier protein (MCP) which catalyzes an Arg antiport with a positively charged counter substrate (such as ornithine). We cloned two potential MCP's from Arabidopsis and they complemented a yeast arg11 mutant defective in ornithine efflux from the mitochondrion. We are currently examining the subcellular location of these transporters as well as attempting to incorporate them into in vitro liposomes.
Impacts
Control of Arg movement in to intracellular compartments of soybean and other plants will allow us to alter levels of Arg in foods as well as levels of Arg derivatives such as polyamines and nitric oxide (NO). The latter two compounds can have important effects on plant development and disease resistance.
Publications
- Freyermuth SK, Bacanamwo M, Polacco JC. (2000) The soybean Eu3 gene encodes a Ni-binding protein necessary for urease activity. The Plant Journal 21, 53-60.
- Goldraij A, Polacco JC. (2000) Arginine degradation by arginase in mitochondria of soybean seedling cotyledons. Planta 210, 652-658.
|
Progress 01/01/99 to 12/31/99
Outputs
Urease is produced by most microorganisms and all plants and plays an important role in recycling massive amounts of urea from industrial and biological sources, worldwide. The widespread presence of plant urease speaks to its evolutionary importance, especially considering the conservation of both structural and accessory genes, the latter for emplacement of Ni into the urease active site. Our goals were (1) to elucidate the mechanism of Ni incorporation into the soybean urease active site, and (2) to determine whether urease and arginase play a role in mobilization of N during soybean seed development. Under 1 we established that the Eu2 and Eu3 genes encode urease accessory factors for placing Ni on the apoureases encoded by Eu1 (embryo urease) and Eu4 (ubiquitous urease). The Eu3 gene product (Eu3) is similar to bacterial UreG by the criteria of antigenicity and P-loop (nucleotide-binding site). Eu3 also binds Ni, a trait expected from the deduced its deduced
HIS-rich N-terminus. Eu3 is absent in eu3-e1/eu3-e1 while Eu3-e3/Eu3-e3 mutants specify an Eu3 transcript with valine in place of alanine at residue 142 (A142V). Eu3 (A142V) retained Ni-binding ability. Eu3 is directly involved in urease activation, since anti-Eu3 inhibited the in vitro activation of urease. Both Eu1 (embryo urease) and Eu3 accumulated in parallel in the developing embryo, and both proteins accumulate in response to developmental cues-- Eu1 is absent in vegetative tissues in which Eu3 is found at much lower levels . Apo-Eu1 is not necessary for the high embryonic level of Eu3. In the absence of Eu3, Eu1 levels are lower, perhaps because of destabilization in the absence of Ni. Under objective 2, arginase activity was detected at low levels during soybean embryo development while no transcript was detected. In contrast, arginase transcript levels increased sharply on germination, reaching a maximum at 2 to 4 DAG, in parallel with increases in arginase activity which
was localized largely in cotyledons. To assess arginase activity in vivo, a soybean urease-deficient mutant was used to detect urea accumulation. No urea was detected in developing seeds of the mutant, whereas in germinating seedlings, urea accumulation paralleled arginase transcript level and arginase specific activity. Cultured cotyledons, as their in vivo counterparts, did not accumulate urea when arginine was provided in the presence of other amino acids in a "mock seed coat exudate." However, when arginine was provided as sole N source, it was rapidly degraded to urea (and presumably ornithine) but did not support growth. Thus, there appears to be lack of recruitment of the low-level arginase activity to hydrolyze free arginine (up to 60% of total free amino acid nitrogen) in developing cotyledons, thus avoiding a futile urea cycle. Cotyledon arginase was shown to be mitochondrial. Mitochondrial uptake of arginine in vitro in preparations from developing and germinating
cotyledons was identical with respect to rate, apparent Km and insensitivity to respiratory uncouplers and ionophores.
Impacts
The four Ni enzymes in nature enzymes are extremely important in the conversions of urea, ammonia, methane and CO2. E.g., urease recycles ~10(exp11) lbs urea-N from biological and industrial sources annually. Thus, understanding the active processes for Ni incorporation into urease is important. Arginase is the major repository of soy seed protein N, and a precursor to polyamines, NO, proline and urea. It is crucial to understand how the plant segregates its synthesis from its breakdown/conversion to ultimate products.
Publications
- Polacco JC, Freyermuth SK, Gerendas J, Cianzio SR (1999) Soybean genes involved in nickel insertion into urease. J Exp Botany 50: 1149-1156.
- Gerendas J, Polacco JC, Freyermuth SK, Sattelmacher B (1999) Significance of Nickel for plant growth and metabolism J Plant Nutr Soil Sci 162: 241-256.
- Freyermuth SK, Bacanamwo M, Polacco JC (2000) The soybean Eu3 gene encodes a Ni-binding protein necessary for urease activity. The Plant J 21: 53-60.
- Goldraij A, Polacco JC (2000) Arginine degradation by arginase in mitochondria of soybean seedling cotyledons. Planta accepted
- Coello P, Polacco, JC (1999) ARR6, a response regulator from Arabidopsis is differentially regulated by plant nutritional status. Plant Science 143: 211-220.
- Goldraij A, Coello P, Polacco JC (1998) Nucleotide sequence of a soybean seedling arginase (Accession No. AF035671). Plant Physiol 116: 867-869.
- Goldraij A, Polacco JC (1999) Arginase is inoperative in developing soybean seeds. Plant Physiol 119: 297-304.
|
Progress 01/01/98 to 12/31/98
Outputs
We examined the production and degradation of urea in soybean embryos. Labeled precursors (2,7 C-14 allantoate and guanido C-14 arginine) applied a urease-negative mutant indicated that virtually all accumulated urea came from arginine and not ureides in soybean seedlings. Moveover, there was NO production of urea in developing embryos, in spite of their high Arg pools and measurable arginase activity. The urease-negative mutant is altered in the production of an accessory gene (called ureE/G because this Ni-binding protein contains domains of two bacterial urease accessory genes, ureE and ureG), essentially for Ni placement in the urease metallocenter. The null mutant employed appears to have a deletion in the gene encoding ureE/G. To test whether Co could functionally replace Ni in wild-type and ureE/G nulls, plants were grown under -Ni+Co vs -Ni-Co and +Ni-Co conditions. The first two treatments resulted in virtually no urease activity whereas the latter had full
activity, thus indicating that Co could not replace Ni in vivo, although we do not know whether an inactive urease had Co at the active site.
Impacts
(N/A)
Publications
- Gerendas, J., Polacco, J. C., Freyermuth, S. K. and Sattelmacher, B. 1998. Co does not replace Ni with respect to urease activity in zucchini (Cucurbita pepo convar. giromontiina) and soybean (Glycine max). Plant and Soil 203: 127-135.
- Freyermuth, S. K., Forde, B. G. and Polacco, J. C. 1999. Nucleotide Sequence of a cDNA Encoding an Arabidopsis Urease Accessory Protein (Accession No. AF109374). (PGR99-012). Plant Physiol. 119: 364.
|
Progress 01/01/97 to 12/31/97
Outputs
The developing soybean seed avoids a futile cycle by maintaining separate its large arginine pools and an active arginase. We confirmed this in in vitro cotyledon cultures fed arginine as part of a mock seed coat exudate: There was little or no urea release (which accumulates in the urease-negative mutants employed), the cotyledons exhibited increases in protein and dry weight. However, when arginine was fed ass sole N source, there was no net synthesis of protein but a large accumulation of urea (and lack of net increases in protein or dry weight). Cotyledon uptake of arginine under both conditions was identical. We are now trying to establish that the mitochondrial membrane is a selective barrier against arginine gaining access to arginase. The mutant cotyledon employed, a null allele of the Eu3 gene, appears to lack a protein showing homology a bacterial UreG, involved in the proper in vivo addition of Ni to the urease active site. We have established an in vitro
Ni activation system which detects complementation between negative alleles at two loci, Eu2 and Eu3. At present we have recovered two defective alleles at each locus and no phenotypically identical mutant (lacking the activities of both soybean urease isozymes) at any other locus.
Impacts
(N/A)
Publications
- FREYERMUTH, S. K. et al. 1996. Metabolic aspects of plant interaction with commensal methylotrophs. (ME Lidstrom and RF Tabita, Eds). Microbial Growth on C1 Compounds. pp 277-284, Kluwer, Dordrecht, Netherlands.
|
Progress 01/01/96 to 12/30/96
Outputs
We have take a molecular genetic approach to examine arginine degradation by arginase and urease in soybean. Probing with an Arabidopsis cDNA for arginase (recovered by correction of an arginase-negative yeast mutant) we have recovered several seemingly identical arginase cDNA clones from soybean 3 DAG axes. Antibody raised against an E.coli-produced GST/Arabidopsis arginase fusion protein detected identical ca. 70 kDa proteins in soybean and Arabidopsis seedling extracts on denaturing gels. This is twice the size predicted from deduced protein sequence and may represent unusually strong disulfide linkages between subunits or some other covalent linkage. The mitochondrial location of arginase is being examined with respect to controlling substrate access. Its deduced N-terminus has a 29 residue transit sequence with a processing cleavage site at 16/17 according to the MitoProt application of Claros and Vincens. Cobalt could not replace Nickel in urease activation in
hydroponic soybean plants (wildtype or urease accessory gene mutants eu2/eu2 or eu3/eu3); however, it bound the Eu3 gene product, UreE/G, as well as Ni in a column binding assay, indicating that the specificity for Ni insertion resides elsewhere. The UreE/G protein has been purified to near homogeneity and two interactions are currently being examined: with apourease and with the Eu2 gene product in an in vitro urease activation assay. US Patent No. 5,512,069.
Impacts
(N/A)
Publications
- FREYERMUTH, S. K. et al. 1996. Metabolic aspects of plant interaction with commensal methylotrophs. (ME Lidstrom and RF Tabita, Eds). Microbial Growth on C1 Compounds. pp 277-284, Kluwer, Dordrecht, Netherlands.
|
Progress 01/01/95 to 12/30/95
Outputs
Urease-negative soybean mutants were used to demonstrate that inability to assimilate urea did not block the utilization of ureides in N2-fixing plants or in cell cultures utilizing ureides. Urea accumulation patterns pointed to arginine, rather than ureides, as being the predominant urea precursor in plants and cell culture (3). However, urease activity was essential for normal development of Arabidopsis germinated on water, based on seedling sensitivity to urease inhibitors (2). Apparently, the N recycling function of urease is critical in these small, N-limited seeds. There was a concomitant increase of both urease and arginase in Arabidopsis seedlings (2), suggesting that the arginase reaction was providing the urease substrate, in agreement with the lack of diminution of urea pools by allopurinol, an inhibitor of ureide formation from purines. Functional Arabidopsis arginase cDNA, recovered by complementation cloning in yeast (1), will be employed in
sense/antisense transgenics to evaluate the arginase role in seed N mobilization. One of the soybean mutants lacking both urease isozymes was shown to be defective in a gene coding for a urease accessory protein. This protein (sharing homology with two bacterial accessory proteins (4)), is missing in a null allele and is altered in a dominant allele mutant.
Impacts
(N/A)
Publications
- KRUMPELMAN PM, FREYERMUTH SK, CANNON JC, FINK GR, POLACCO JC 1995. Nucleotide sequence of Arabidopsis thaliana arginase expressed in yeast. Plant Physiol 107: 1479-1480.
- ZONIA LE, STEBBINS NE, POLACCO JC 1995. Essential Role of Urease in Germination of Nitrogen-limited Arabidopsis thaliana seeds. Plant Physiol107: 1097-1103.
- STEBBINS NE, POLACCO JC 1995. Urease is not essential for ureide degradation in soybean. Plant Physiol 109: 169-175.
|
Progress 01/01/94 to 12/30/94
Outputs
Ureides (allantoin and allantoic acid) are not catabolized to urea to a physiologically significant extent since variation of the ureide contribution to total N available to urease-negative (eu3-e1/eu3-e1) tissues resulted in no change in seed N, a small change in callus growth and little or no change in urea accumulation in seeds, seedlings and callus. N(subscript 2)-supported plants (35 DAG) had higher leaf ureide pools and accumulated seven times as much leaf urea as NH(subscript 4)NO(subscript 3)-supported plants. However, there was no difference in seed N yield between plants of either N regime. Lack of an active urease reduced callus growth on both arginine and allantoin as sole N. However, the reduction was greater on arginine (73%) than on allantoin (39%), consistent with greater urea accumulation in arginine-supported callus: Urease-negative cells accumulated 17 times more urea than urease-positive cells, an accumulation ratio which was only 1.8 in cells
utilizing allantoin. NH(subscript 4)NO(subscript 3)-supported callus generated much less urea than water-germinated 3 DAG seedlings (/<-/1.1 vs 40 (mu)moles-g dw(superscript -1) urea). When seeds were first imbibed in 1 mM allopurinol, there was a 90% decrease in seedling ureide pools but no significant decrease in urea.
Impacts
(N/A)
Publications
|
Progress 01/01/93 to 12/30/93
Outputs
See MO-BCHB0261.
Impacts
(N/A)
Publications
- No publications reported this period.
|
|