drought is often a yield constraint for crops worldwide . a major benefit of producing soybean [ glycine max ( l. ) merr . ] is that it grows symbiotically with bradyrhizobium japonicum and reduces ( fixes ) atmospheric nitrogen to nitrogen - compounds that the plant can utilize without the need for expensive nitrogen fertilizer . however , n2 fixation is sensitive to water - deficit stress thereby reducing the plant 's ability to fix and utilize nitrogen for biomass accumulation and seed production ( see reviews by serraj et al . , 1999 ; although n2 fixation decreases during drought , the products of n2 fixation , ureides ( allantoin and allantoate ) , increase in leaves of drought - sensitive soybean genotypes ( king and purcell , 2001 ) . experiments have shown that increases in shoot ureide concentration are associated with the inhibition of n2 fixation in the nodules and , subsequently , drought sensitivity ( purcell et al . , 2000 ) . drought - sensitive n2-fixation genotypes subjected to water - deficit stress ( i.e. williams , biloxi , and ks4895 ) exhibit a reduction in nitrogenase activity , a decrease in ureide degradation , and an increase in shoot ureide concentrations relative to jackson , a genotype with drought - tolerant n2-fixation ( purcell et al . , 2000 ; vadez and sinclair , 2001 ) . therefore , lower ureide concentrations in leaves during water - deficit stress appears to prolong n2 fixation and thereby to confer drought tolerance ( purcell et al . , 2000 ; king and purcell , 2005 ) . in addition , physiological studies have shown that increases in mn availability to the plant during water - deficit stress increases ureide degradation in leaves and prolongs n2 fixation ( purcell et al . , 2000 ; an enzyme responsible for allantoate breakdown , allantoate amidohydrolase ( aah ; ec 3.5.3.9 ) , requires mn as a co - factor for activity ( lukaszewski et al . , 1992;werner et al . , 2008 ) and this enzyme has greater activity in jackson than in the drought - sensitive genotype ks4895 ( purcell et al . , 2000 ) . therefore , aah is a likely candidate for an important role conferring drought tolerance in soybean . allantoate amidohydrolase is important in purine salvage , and is found in many plant species that do not fix nitrogen . todd and polacco ( 2006 ) demonstrated the functional complementation of the arabidopsis gene in a yeast strain unable to utilize ureides . the yeast strain transformed with ataah was able to utilize allantoin as the sole nitrogen source , whereas the untransformed mutant yeast strain grew poorly in the presence of allantoin . furthermore , todd and polacco ( 2006 ) showed that an arabidopsis mutant lacking the functional aah was not able to breakdown ureides . they demonstrated that the aah protein is involved in allantoate catabolism in vivo and that mn is required for optimal activity . it is hypothesized that aah gene expression in soybean determines the shoot ureide concentrations during water - deficit stress and is responsible for the differential sensitivities of the n2-fixation response to drought among soybean genotypes . from this hypothesis , it is predicted that aah mrna levels in shoots are ( i ) higher in drought - tolerant versus drought - sensitive soybean genotypes , ( ii ) differentially expressed in response to water - deficit stress , and ( iii ) inversely correlated with shoot ureide concentrations . therefore , the objectives were to examine aah mrna levels and ureide concentrations in shoots during well - watered and water - deficit stress for two soybean genotypes differing in drought tolerance . drought - tolerant ( jackson ; pi 548657 ) and drought - sensitive ( williams ; pi 548631 ) soybean genotypes were planted in 1.0 l volume pots containing 2 parts n - free peat - perlite - vermiculite mixture ( lb2 , sungro horticulture inc . , bellevue , wa ) to 1 part captina silt loam ( fine - silty , siliceous , mesic typic fragiudult ) soil collected from the university of arkansas experiment station near fayetteville , ar . the soil was saturated with 1.0 l of a n - free nutrient solution ( desilva et al . , 1996 ) , drained one seed was planted per pot and soil inoculated with bradyrhyizobium japonicum ( strain usda 110 ) . growth chamber conditions included day / night temperature of 25/23 c and a 16 h photoperiod beginning at 06.00 h with metal halide lamps providing approximately 300 mol m s photosynthetically active radiation at plant height . plants were watered as needed for 30 d after emergence to maintain 70% soil water capacity ( swc ) . the experiment consisted of a 23 factorial with randomized complete block design with five blocks for experiment one and 2 blocks for experiment two . two genotypes ( jackson and williams ) were examined over three water treatments on the basis of swc : well - watered at 70% ; moderate water deficit at 50% , and severe water deficit at 30% . to reach each percentage swc for simultaneous sampling the first interval was to allow all pots , except for the 70% swc treatment , to reach 50% swc . during the second interval , water was added to maintain corresponding pots at 70% and 50% swc , while the remaining pots were allowed to dry to 30% swc . the entire drying period was 4 d. at the v7 stage of development ( fehr and caviness , 1977 ) , fully expanded trifoliolate leaves at the fifth node were collected from each genotype , water treatment , and replication combination . three 1 cm diameter leaf punches were collected and combined into one sample per leaf . the tissue was dried at 55 c , and a 30 mg subsample was ground to pass a 1 mm sieve . ureide concentration was determined using the colorimetric procedure of young and conway ( 1942 ) as described by desilva et al . rna was collected from the remaining leaf tissue , frozen in liquid n2 , and stored at 80 c before extraction . total rna ( 15 g ) from each sample was separated on 1% agarose formaldehyde gels . the accumulation of transcripts by rna - blot hybridization was measured using standard techniques ( sambrook et al . , 1989 ) and p - labelled probes as described previously ( gomez et al . , 2005 ) . probes for aah and dreb2 were obtained through a standard pcr procedure using gene - specific primers ( charlson et al . , 2005 ; chen et al . , 2006 ) . the aah probe was amplified from williams est clone gm - c1019 - 6420 ( genbank no . aw704657 ) using primer sequences 5-caccaccacaaccaccatgtattctgccac-3 ( forward ) and 5-acagttaccatatcacttatgataggttttccag-3 ( reverse ) ( see supplementary fig . the soybean dreb2 gene was used as a positive control to validate that plants were responding differentially to water availability at the transcript level of gene expression ; dreb2 was amplified from genomic dna of jackson . dq208968 ) , primers were 5-atggaagaagcgggtttagg-3 ( forward ) and 5-atcttcaggtttgggatact-3 ( reverse ) and amplified a 480 bp amplicon probe starting at the start codon ( atg ) and ending near the stop codon ( tag ) . levels of gene expression were calculated as a ratio of aah or dreb2 to the 18s ribosomal rna from rice ( oryza sativa l. ) . the response of jackson ( drought - tolerant ) and williams ( drought - sensitive ) to water availability was as expected regarding ureide concentration between both experiments across water treatments . for well - watered and moderate - water stress , jackson and williams had similar ureide concentrations within an experiment : 10 m and 50 m mg dw for experiments one and two , respectively ( fig . 1 ) . as expected , williams had a 23-fold greater ureide concentration during severe - water stress relative to well - watered conditions , whereas jackson showed very little increase in ureide concentrations during severe - water stress . drought - tolerant ( jackson ) and drought - sensitive ( williams ) soybean genotypes were subjected to three water - availability treatments : well - watered control ( 70% soil water capacity , swc ) , moderate water - deficit stress ( 50% swc ) , and severe ( 30% swc ) water - deficit stress . data were averaged within treatment and cultivar for five replications in experiment one ( a ) and two replications in experiment two ( b ) . in addition to the observation of expected levels of ureides in water - stressed samples , expression patterns of a known drought - induced gene were also as anticipated . the dreb2 gene encodes a transcription factor that binds to a drought - responsive element found in the promoters of many plant genes thought to be active in water - stress responses ( sakuma et al . , 2002 ) . transcripts encoding dreb2 are strongly induced in response to water stress in arabidopsis ( liu et al . , therefore , dreb2 was a useful , positive control to validate that changes in gene expression resulting from water - deficit stress could be detected using our experimental approach . the transcript level of soybean dreb2 was similar between genotypes and 2-fold greater at severe water - deficit stress compared with the control plants ( figs 2 , 3 ) . the data support that ureide concentration for jackson and williams and dreb2 responded to water availability as expected from previous research ( purcell et al . northern blot analysis of allantoate amidohydrolase ( aah ) , dreb2 , and 18s ribosomal rnas . gene expression between experiments was similar , therefore , only data from experiment one are shown . gene expression was examined for drought - tolerant ( jackson ) and drought - sensitive ( williams ) soybean genotypes over three water availability treatments ; well - watered control ( 70% soil water capacity , swc ) , moderate water - deficit stress ( 50% swc ) , and severe ( 30% swc ) water - deficit stress . gene expression between experiments was similar , therefore , only data from experiment one are shown . gene expression was examined for drought - tolerant ( jackson ) and drought - sensitive ( williams ) soybean genotypes over three water - availability treatments ; well - watered control ( 70% soil water capacity , swc ) , moderate water - deficit stress ( 50% swc ) , and severe ( 30% swc ) water - deficit stress . data were standardized over treatments for comparison by comparing all treatments to dreb2 expression for williams at well - watered conditions . relative expression for jackson at 70% swc was 1.1 , therefore values are only shown for the moderate and severe water - deficit stress treatments . although ureide concentrations were lower for jackson relative to williams ( fig . 1 ) , aah transcript levels were similar between the drought - tolerant and drought - sensitive soybean genotypes ( fig . in addition , and contrary to the observation of shoot ureide concentrations differing among water - deficit treatments , aah transcript levels remained stable regardless of water - deficit stress ( fig . , the gmaah probe was gene specific , as indicated by the detection of only one band from southern blot analysis ( data not shown ) . therefore , the disparity between transcript expression and ureide concentration may not be accounted for by a cross - hybridization of the probe with the expression of one or more homologous genes during northern blot analysis . since aah breaks down allantoate , a ureide , to a non - ureide ( ureidoglycolate ) , we expected to observe aah transcript levels increasing with a concurrent decrease in shoot ureide concentrations . linear regression analysis indicated that the response of ureide concentration to aah expression was significant ( p=0.02 ) , however , the relationship was weak ( r=0.15 ) ( fig . data collected from jackson and williams subjected to three water - availability treatments from well - watered control to severe water - deficit stress . soybean genotypes with drought tolerance have lesser shoot ureide concentrations relative to drought - sensitive genotypes during water - deficit stress ( king and purcell , 2001 ) , and aah is involved in ureide breakdown in shoots . therefore , the role of aah transcript expression relative to the drought - tolerance trait and ureide concentration in shoots was examined . the results of this study indicated that mrna levels of aah were weakly associated with shoot ureide concentration and independent of soybean response to water - deficit stress or the drought - tolerance trait in soybean . these data indicate that either aah does not play a role in drought tolerance or post - translational regulation is involved in aah activity . there are several lines of physiological and biochemical evidence supporting post - translational regulation of aah enzymatic activity . two enzymes are involved in ureide catabolism : allantoinase ( ec 3.5.2.5 ) and aah . during ureide catabolism , allantoinase breaks down allantoin to allantoate . 2008 ) demonstrated that both allantoinase and aah proteins are localized to the er in arabidopsis , supporting that ureide catabolism occurs in the er . 1987 ) before transport to the er for ureide catabolism as proposed by werner et al . increases in ureide shoot concentrations during water - deficit stress may reflect increases in stored allantoin rather than of allantoate ( rice et al . in addition , aah mrna expression is not affected by allantoin concentrations in shoots ( todd and polacco , 2006 ; werner et al . , 2008 ) . furthermore , aah enzymatic activity is not affected by shoot allantoate levels ( rice et al . , the weak relationship between aah transcripts and shoot ureide concentrations may have resulted from the sequestration of allantoin in peroxisomes . however , if the process of ureide catabolism is maintained at a constant state in the er regardless of overall shoot ureide concentrations , then aah mrna levels would remain stable . the actual ratio of allantoin to allantoate during water - deficit stress deserves further consideration in the regulation of ureide catabolism . several reports show that increases in mn concentration in vitro and in planta result in increases in aah enzymatic activity ( lukaszewski et al . , 1992 ; purcell et al . , 2000 ; vadez et al . , 2000 ; vadez and sinclair , 2001 ) , and mn is an important co - factor for optimal aah enzymatic activity ( lukaszewski et al . the drought - tolerant genotype jackson acquires mn to a greater extent than ks4895 , a drought - sensitive genotype ( purcell et al . , 2000 ; king and purcell , 2005 ) . on the basis of the association of mn with shoot ureide concentrations , purcell et al . ( 2000 ) proposed that differences in shoot ureide concentrations between drought - tolerant and drought - sensitive soybean genotypes may result from regulation of aah enzymatic activity during water - deficit stress . however in this study , differences in drought tolerance were not associated with differential transcript expression of aah . this may indicate that differences in aah enzymatic activity due to mn availability are responsible for the differences in shoot ureide concentration during water - deficit stress . , 2007 ; werner et al . , 2008 ) . the role of mn in enzyme assembly is unknown . it is possible that the regulation of enzyme assembly may be regulated by mn availability , which might account for differences in the response of aah activity between drought - tolerant and drought - sensitive genotypes . for example , jackson may be more efficient at supplying mn to aah , which results in a greater metabolism of allantoate . greater demand for allantoate may drive the movement of stored allantoin from the peroxisomes to the er . therefore , ureide concentrations in the shoots would decrease as a result of increased ureide catabolism . our results , combined with other reports , support focusing attention on the importance of the aah enzyme in the regulation of shoot ureide concentrations and conferring of drought tolerance in soybean .
drought is a limiting factor for n2 fixation in soybean [ glycine max ( l. ) merr . ] thereby resulting in reduced biomass accumulation and yield . 
 drought - sensitive genotypes accumulate ureides , a product of n2 fixation , during drought stress ; however , drought - tolerant genotypes have lower shoot ureide concentrations , which appear to alleviate drought stress on n2 fixation . 
 a key enzyme involved in ureide breakdown in shoots is allantoate amidohydrolase ( aah ) . 
 it is hypothesized that aah gene expression in soybean determines shoot ureide concentrations during water - deficit stress and is responsible for the differential sensitivities of the n2-fixation response to drought among soybean genotypes . 
 the objectives were to examine the relationship between aah transcript levels and shoot ureide concentration and drought tolerance . 
 drought - tolerant ( jackson ) and drought - sensitive ( williams ) genotypes were subjected to three water - availability treatments : well - watered control , moderate water - deficit stress , and severe water - deficit stress . shoot ureide concentrations 
 were examined , in addition to gene expression of aah and dreb2 , a gene expressed during water - deficit stress . 
 as expected , dreb2 expression was detected only during severe water - deficit stress , and shoot ureide concentrations were greatest in the drought - sensitive genotype relative to the drought - tolerant genotype during water - deficit stress . 
 however , expression of aah transcripts was similar among water treatments and genotypes , indicating that aah mrna was not closely associated with drought tolerance . 
 ureide concentrations in shoots were weakly associated with aah mrna levels . 
 these results indicate that aah expression is probably not associated with the increased ureide catabolism observed in drought - tolerant genotypes , such as jackson . 
 further study of aah at the post - translational and enzymatic levels is warranted in order to dissect the potential role of this gene in drought tolerance .