Allantoate amidohydrolase transcript expression is independent of drought tolerance in soybean

Abstract

Drought is a limiting factor for N(2) fixation in soybean [Glycine max (L.) Merr.] thereby resulting in reduced biomass accumulation and yield. Drought-sensitive genotypes accumulate ureides, a product of N(2) fixation, during drought stress; however, drought-tolerant genotypes have lower shoot ureide concentrations, which appear to alleviate drought stress on N(2) 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 N(2)-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.

Fig. 1.

Shoot ureide concentrations versus water availability and cultivar. The experiment was conducted twice in a growth chamber. 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). Bars indicate the standard error of the mean.

Fig. 2.

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. RNA from five replications was combined at equal concentrations of 15 μg. The same membrane was exposed with all three probes independently. 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.

Fig. 3.

Response of DREB2 expression to severe 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.

Fig. 4.

Regression of AAH expression to shoot ureide concentrations. Each point represents an experimental unit from two independent experiments. Data collected from Jackson and Williams subjected to three water-availability treatments from well-watered control to severe water-deficit stress.