Analysis of alanine aminotransferase in various organs of soybean (Glycine max) and in dependence of different nitrogen fertilisers during hypoxic stress

Abstract

Alanine aminotransferase (AlaAT) catalyses the reversible conversion of pyruvate and glutamate into alanine and oxoglutarate. In soybean, two subclasses were identified, each represented by two highly similar members. To investigate the role of AlaAT during hypoxic stress in soybean, changes in transcript level of both subclasses were analysed together with the enzyme activity and alanine content of the tissue. Moreover, the dependency of AlaAT activity and gene expression was investigated in relation to the source of nitrogen supplied to the plants. Using semi-quantitative PCR, GmAlaAT genes were determined to be highest expressed in roots and nodules. Under normal growth conditions, enzyme activity of AlaAT was detected in all organs tested, with lowest activity in the roots. Upon waterlogging-induced hypoxia, AlaAT activity increased strongly. Concomitantly, alanine accumulated. During re-oxygenation, AlaAT activity remained high, but the transcript level and the alanine content decreased. Our results show a role for AlaAT in the catabolism of alanine during the initial period of re-oxygenation following hypoxia. GmAlaAT also responded to nitrogen availability in the solution during waterlogging. Ammonium as nitrogen source induced both gene expression and enzyme activity of AlaAT more than when nitrate was supplied in the nutrient solution. The work presented here indicates that AlaAT might not only be important during hypoxia, but also during the recovery phase after waterlogging, when oxygen is available to the tissue again.

Fig. 1

Sequence comparison of the four AlaATs from soybean. a Comparison of the intron and exon structure of the four AlaAT genomic sequences identified in soybean. The striking similarity between Glyma07g05130 and Glyma16g01630 or Glyma02g04320 and Glyma01g03260 from both coding and non-coding sequences is explained from genomic duplication events. Data are extracted from the Phytozome database at http://www.phytozome.net. b Alignment of the translated amino acid sequences from GmAlaAT1 (Glyma07g05130), GmAlaAT2 (Glyma02g04320), GmAlaAT3 (Glyma01g03260) and GmAlaAT4 (Glyma16g01630)

Fig. 2

Phylogenetic tree of the AlaAT enzyme family. Unrooted neighbour-joining sequence comparison was performed using all fully sequenced coding sequences known to date for AlaAT genes in plants Chlamydomonas reinhardtii and Physcomitrella patens. Translated protein sequences were used for sequence alignment. A division into two subfamilies, A and B, can be observed. The full list with NCBI accession codes is given in “Materials and methods”. The four genes identified in soybean are marked in bold

Fig. 3

Analysis of alanine content, AlaAT transcript levels and enzyme activity in various organs of soybean. a Alanine content in pods, leaves, roots and nodules (nod) of nodulated and non-nodulated soybean plants. b AlaAT enzyme activity measured in pods, leaves, roots and nodules (nod) of nodulated and non-nodulated soybean plants. c Semi-quantitive RT-PCR analysis of GmAlaAT transcript levels in pods, leaves, roots and nodules (nod) of nodulated and non-nodulated soybean plants. GmAlaAT-A represents transcript levels from the genes belonging to subfamily A (GmAlaAT1 and -4), whereas GmALaAT-B indicate transcripts from subfamily B (GmAlaAT2 and -3). Due to the very high sequence similarity, it was not possible to distinguish between individual genes within each subfamily. The constitutively expressed actin gene was used as loading control to normalise the samples. The bars indicate mean value ± SD (n = 4). Values that differ significantly according to a one-way ANOVA (P < 0.05) and Tukey posthoc test are marked with different letters. Small letters are used to indicate differences between tissues from either nodulated or non-nodulated plants. Capital letters indicate differences within the same kind of tissue from either nodulated or non-nodulated plants

Fig. 4

Hypoxic response of alanine accumulation and AlaAT enzyme activity and transcript level in relation to different nitrogen fertilisation conditions of non-nodulated and nodulated soybean plants. Alanine content (a, d), AlaAT activity (b, e) and AlaAT transcript levels as determined by semiquantitative RT-PCR (c, f) in roots or nodules of soybean plants without nodules (a–c) or with nodules (d–f). PCR conditions and sequence specificity are as described for Fig. 3. Material was analysed before the waterlogging treatment (C control) and after plants were waterlogged for 3 days (W). The bars indicate mean value ± SD as determined from four biological replicates. Values that differ significantly according to a one-way ANOVA (P < 0.05) and Tukey posthoc test are marked with different letters. Small letters are used to indicate differences induced by normoxia or hypoxia within the same nitrogen treatment. Capital letters indicate differences induced by the nitrogen treatment within either normoxic or hypoxic material

Fig. 5

Changes in alanine content, AlaAT activity and transcript levels in soybean tissues before and during a 3-day waterlogging treatment and subsequent recovery. a The amount of alanine as determined in roots and nodules of nodulated and non-nodulated soybean plants. b The activity of AlaAT as measured in extracts from nodulated and non-nodulated soybean plants. c Semiquantitive RT-PCR analysis of GmAlaAT transcript levels in roots and nodules of soybean. PCR conditions and sequence specificity are as described in Fig. 3. Samples were taken before waterlogging (C control), after 3 days of waterlogging (W), and 3 days after the waterlogging treatment was ceased (R recovery). The bars indicate mean value ± SD as determined from four biological replicates. Small letters indicate differences between the different treatments, C, W and R, within the same tissue. Capital letters indicate differences between nodulated and non-nodulated tissue within C, W or R treated samples