Progress 07/15/03 to 01/14/07
Outputs
Our objectives were: 1. Determine the importance of urease in ureide degradation in whole plants of Maple Arrow (drought-tolerant) and Williams 82 (drought-sensitive) grown under N-fixation. 2. Define the allantoate degradation routes in Maple Arrow and Williams 82. 3. Compare Mn uptake, translocation and availability in each cultivar. Results: 1. This problem was attacked because different routes for ureide degradation had been claimed, part of a model explaining the greater drought tolerance of N-fixation in Maple Arrow vs Williams 82. We showed that there was no difference in the route of conversion of allantoin to glyoxylate and ammonia between the varieties: i. allantoinase, ii. allantoate amidohydrolase (direct release of ammonia), iii. a putative ureiglycine amidohydrolase, iv. a ureidoglycolate urea-lyase. Thus, urea AND ammonia are released. Maple Arrow and Williams 82 both performed well under fixation when urease activity was inhibited by
phenylphosphorodiamidate, indicating that urea was not the exclusive N compound released from ureides. 2. We cloned an Arabidopsis allantoate amidohydrolase ortholog to a soybean gene. T-DNA disruption of the Arabidopsis gene blocked the ability to grow on allantoin as N source. Expression of the Arabidopsis gene in a yeast dal2 mutant (deficient in yeast allantoate amidinohydrolase) partially restored the ability to utilize allantoin N. The corrected yeast mutants accumulated a yellow compound-- apparently a conjugate of an allantoate derivative (ureidoglycine?). The compound may be partially toxic (its production in wild type using ammonia slows its growth). Also, it represents a sequestering of three of four N atoms in allantoin not available to yeast, another reason why the plant enzyme does not fully 'complement' the dal2 mutant. 3. Since Mn appeared to overcome drought sensitivity in N-fixation we examined Mn use efficiency in each cultivar. Maple Arrow and Williams 82
accumulated different levels of total leaf Mn when grown under non-fixing conditions. In greenhouse dry-down experiments we found that across all levels of Mn nutrition, leaf Mn concentrations of N-fixing Maple Arrow plants were higher than those of Williams 82. The difference ranged from 2.6-fold at the lowest, to 5.3-fold at the highest Mn treatment. Maple Arrow plants were also more responsive to Mn treatment, and increased in leaf Mn concentration from 1.8 to 4.3 mg/gDW, compared to Williams 0.6 to 0.8 mg/gDW. Leaf ureide levels in both cultivars decreased with higher leaf Mn content, indicating similar ureide breakdown. However, despite the difference in leaf Mn content, leaf ureide levels in Maple Arrow leaves were close to those in Williams 82, suggesting that the accumulated Mn in Maple Arrow leaves might be sequestered.
Impacts
The economic importance of soybean is growing. For example, its oil is being developed as a biofuel, and its protein is increasingly used in both human and animal nutrition. The ability to grow soybean with energy-intensive applications of nitrogenous fertilizer adds to soybean's profitability. Knowledge of the factors that allow fixation under drought-stress is important to improve soybean performance in states that may experience less rainfall if warming trends continue. Thus, our identification of the steps in assimilation of fixed nitrogen, and identification of the responsible genes can provide targets for breeding improved performance under water-deficit. Manganese (Mn) is a mineral that is important to virtually all the enzymes (catalytic proteins) that breakdown ureides, the chemical family bearing fixed nitrogen delivered to green tissues from the roots. Others have shown that Mn applications to the soil improve soybean fixation under water-stress, and our
results have shown that a 'drought-tolerant' variety (Maple Arrow) accumulates Mn to higher levels than a 'drought-sensitive' variety (Williams 82).
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.
|
Progress 01/01/05 to 12/31/05
Outputs
Warm season N2-fixing legumes move fixed N from the nodules to the aerial portions of the plant primarily in the form of ureides, allantoin and allantoate, oxidation products of purines synthesized de novo in the nodule. Ureides are also products of purine turnover in senescing tissues, such as seedling cotyledons. A combination of biochemical and molecular approaches in both crop and model species has shed new light on the metabolic pathways involved in both the synthesis and degradation of allantoin. Improved understanding of ureide biochemistry includes two additional enzymatic steps in the conversion of uric acid to allantoin in the nodule and the mechanism of allantoin and allantoate breakdown in leaf tissue. Ureide accumulation and metabolism in leaves have also been implicated in feedback inhibition of N2-fixation under water limitation. Sensitivity to water deficit differs among soybean cultivars. We presented a discussion (Todd et al. 2006) of the potential
roles for ureides in feedback inhibition of N2-fixation under water limitation, and conclude that since the pathways of ureide degradation are identical in sensitive and tolerant varieties, that there must be some other underlying cause for the sensitivity of N2 fixation to water-deficit. Manganese supplementation has been shown to modify relative susceptibility or tolerance to this process in a cultivar-dependent manner. Our working hypothesis at present is that Mn use efficiency is an underlying factor in drought-tolerant N2-fixation. Indeed, the tolerant cultivar, Maple Arrow, accumulates almost 10X more Mn than does drought-sensitive Williams 81. We report the identification and cloning of an allantoate amidohydrolase (allantoate deiminase, EC 3.5.3.9) cDNA from Arabidopsis thaliana (L.) Heynh. This sequence, which we term AtAAH (Arabidopsis thaliana Allantoate Amidohydrolase), was shown to be functional by complementation of Saccharomyces cerevisiae dal2 mutants, blocked in
allantoate degradation. Following transfer to a medium containing allantoin as the sole nitrogen source, Ataah T-DNA insertion mutants were severely impaired and eventually died. Ataah mutants demonstrated higher allantoate levels than wild-type plants in the presence and absence of exogenous ureides, supporting a block in allantoate catabolism. AtAAH transcript was detected in all tissues examined by RT-PCR, consistent with a function in purine turnover in Arabidopsis. To our knowledge this is the first allantoate amidohydrolase gene identified in any plant species.
Impacts
Determining the ureide degradation route provides the first step in understanding why some cultivars are subject to ureide-induced inhibition of N-fixation under water deficit. While our results indicate that all soybean cultivars probably use the same degradation route, some enzymes may more efficiently incorporate Mn under drought conditions. (All ureide-degrading enzymes characterized to date require Mn.) Now that we have clones for at least two of the enzymes, allantoinase and allantoate urea-lyase (amidohydrolase), these could be developed as DNA markers to assess their linkage/identity to drought-tolerant QTL's.
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.
- Todd, CD, Tipton PA, Blevins DG, Pineda M, Piedras P, Polacco JC (2006) Update on ureide degradation in legumes. J Exp Botany 57: 5-12.
- Todd CD, Polacco JC (2006) AtAAH, an allantoate amidohydrolase from Arabidopsis thaliana. Planta 223: 1108-113.
|
Progress 01/01/04 to 12/31/04
Outputs
Our first goal was to define the ureide degradation routes in whole plants of `Maple Arrow' (drought-tolerant) and `Williams 82' (drought-sensitive) grown under N-fixation. This problem was attacked because different routes for ureide degradation had been invoked by others for these two varieties, and was part of a model explaining the greater drought tolerance of N-fixation in Maple Arrow vs Williams 82. We showed that there was no difference in the route of conversion of allantoin to glyoxylate and ammonia between the varieties: beyond allantoinase, the second enzyme (allantoate amidohydrolase) releases ammonia directly from allantoate. The 'third' enzyme, catalyzes the release of urea from ureidoglycolate. More recently, we have cloned soybean and Arabidopsis allantoate amidohydrolase orthologs. A T-DNA disruption of the Arabidopsis gene blocks the ability to grow on allantoin as N source. Expression of the Arabidopsis gene in a yeast dal2 mutant (deficient in
yeast allantoate amidinohydrolase) partially restores the ability to utilize allantoin N. The corrected yeast mutants accumulate a yellow compound which appears to be a conjugate of an allantoate derivative (ureidoglycine?). While the compound is not toxic, it represents a sequestering of three of four N atoms in allantoin not available to yeast, and thus explains in part why the plant enzyme does not fully 'complement' the dal2 mutant. Since Mn appeared to overcome drought sensitivity in N-fixation we examined Mn use efficiency in each cultivar. Our initial observations indicated that Maple Arrow and Williams 82 accumulate different levels of total leaf Mn when grown under non-fixing conditions. In greenhouse dry-down experiments, we analyzed the effect of different Mn regimes on leaf Mn content, as well as on ureide accumulation and breakdown in N2-fixing Maple Arrow and Williams 82 under water deficit. We found that across all levels of Mn nutrition, Mn concentrations of Maple
Arrow leaves were higher than Williams 82. The difference ranged from 2.6-fold at the lowest, to 5.3-fold at the highest Mn treatment. Maple Arrow plants were also more responsive to Mn treatment, and increased in leaf Mn concentration from 150 to 330 ug/gDW, compared to Williams 50 to 70 ug/gDW. Leaf ureide levels in both cultivars went down with higher leaf Mn content, indicating similar ureide breakdown. However, despite the difference in leaf Mn content, leaf ureide levels in Maple Arrow were close to those in Williams 82, suggesting that the accumulated Mn in Maple Arrow leaves might be sequestered.
Impacts
Determining the ureide degradation route provides the first step in understanding why some cultivars are subject to ureide-induced inhibition of N-fixation under water deficit. Outlining the biochemical and molecular regulatory mechanisms may provide a target plant modification or may identify beneficial management practices to eliminate yield losses associated with water-stress.
Publications
- No publications reported this period
|
Progress 01/01/03 to 12/31/03
Outputs
In the first year of the project we compared the two varieties, Maple Arrow and Williams 82, and determined that, contrary to the established model, both varieties used the same pathway to degrade ureides. Using a combination of in vitro, in vivo and whole plant studies we showed that both cultivars produced both ammonia and urea during ureide breakdown. Both Maple Arrow and Williams 82 appear to utilize an allantoate amidohydrolase to liberate ammonia and carbon dioxide from allantoate, followed by an ureidoglycolate urea-lyase which forms urea and glyoxylate. Using the urease inhibitor PPD we established that lack of a functional urease caused no dramatic detrimental effects on nitrogen assimilation in either cultivar, again contrary to what would be expected under the existing model. These results were presented in the publication indicated below. To confirm this, we have generated an RNAi construct designed to eliminate urease activity in Maple Arrow and in
conjunction with the MU Plant Transformation Core have begun to regenerate transgenic soybean. We are now testing a new model, one based on Mn availability, to determine why these two cultivars differ with respect to N-fixation under water limiting conditions. Our initial observations indicated that Maple Arrow and Williams 82 accumulate different levels of total leaf Mn when grown under non-fixing conditions. Using these results we have begun greenhouse dry-down experiments to analyze the effect of different Mn regimes on ureide accumulation and breakdown in both cultivars under water deficit. In an effort to isolate the genes responsible for allantoate degradation in soybean we have expressed candidate genes in a dal2 strain of S. cerevisiae, which is unable to metabolize allantoate, and selected for clones able to utilize ureides as a sole nitrogen source. We constructed a soybean cDNA library able to express the plant proteins in S. cerevisiae. We screened both this library and an
Arabidopsis cDNA library in the same host as the candidate genes, using the same selection. Using this method we have yet to identify a cDNA encoding a functional allantoate degrading enzyme. This might suggest that either the plant enzyme is composed of two or more subunits such that a single cDNA is unable to rescue the mutant phenotype in yeast. Alternatively, there may be post-translational requirements for either protein targeting or modification which do not allow the protein to functionally complement the yeast strain. We have identified Arabidopsis T-DNA knockout lines which are unable to utilize allantoin as a sole nitrogen source. Insertion of the T-DNA in the Arabidopsis allantoinase prevents the conversion of allantoin to allantoic acid. These lines accumulate allantoin when it is supplied in the nutrient solution. Insertion of the T-DNA in one of our candidate genes causes accumulation of allantoic acid when the plants are supplied with allantoin, suggesting that it is
defective in the release of nitrogen from allantoate. We have identified the corresponding cDNAs for both sequences in soybean for use as probes in comparing Maple Arrow and Williams 82.
Impacts
Determining the ureide degradation route provides the first step in understanding why some cultivars are subject to ureide-induced inhibition of N-fixation under water deficit. Outlining the biochemical and molecular regulatory mechanisms may provide a target plant modification or may identify beneficial management practices to eliminate yield losses associated with water-stress.
Publications
- Todd, C.D. and Polacco, J.C. (2004) Soybean cultivars `Williams 82' and `Maple Arrow' produce both urea and ammonia during ureide degradation. J. Exp. Bot. 55:867-877
|
|