Table 1.
Basic information on the MIOX family in Arabidopsis thaliana and G. max.
Fig 1.
The phylogenetic tree of GsMIOX1a and MIOX from different plants.
The analysis was based on a multiple amino acid sequence alignment. A neighbor-joining tree was generated using MEGA 5.0. The aligned protein sequences from GenBank (with accession numbers) included GsMIOX1 (Glycine soja, KHN39268), AtMIOX1 (Arabidopsis thaliana, NP_001154337), AtMIOX2 (Arabidopsis thaliana, NP_565459), AtMIOX4 (Arabidopsis thaliana, NP_194356), AtMIOX5 (Arabidopsis thaliana, NP_200475), MtMIOX (Medicago truncatula, KEH18252), CaMIOX (Cicer arietinum, XP_004510326), OsMIOX (Oryza sativa, NP_001057871), ZmMIOX (Zea mays, NP_001141330), PvMIOX (Phaseolus vulgaris, XP_007135612), GaMIOX (Gossypium arboretum, KHF98793), SiMIOX (Setaria italica, XP_004966088), GmMIOX1a (Glycine max, Glyma07g01660), GmMIOX1b (Glycine max, Glyma08g21300), GmMIOX2a (Glycine max, Glyma01g00840), GmMIOX2b (Glycine max, Glyma07g15190), GmMIOX4 (Glycine max, Glyma08g01690), SbMIOX (Sorghum bicolor, Sb10g022160), and RcMIOX (Ricinus communis, 29912.m005347) with gene ID from Phytozome v9.1.
Fig 2.
Expression patterns of GsMIOX1a in G. soja 07256.
(A) The accumulation of GsMIOX1a transcripts under 50 mM NaHCO3 (pH 8.5) in G. soja 07256 as determined by quantitative real-time PCR analysis. Root samples were collected at 0, 1, 3, 6, 12, and 24 h. (B) Expression levels of GsMIOX1a in different soybean varieties including G. soja 50109 (G5), G. max Suinong 28 (G28), G. soja 07256 (G7), and G. max Hefeng 55 (G55). Total RNA was extracted from the roots of 3-week-old seedlings of four soybean varieties whose roots had been submerged in nutrient solution containing 50 mM NaHCO3. (C) The tissue-specific expression analysis of GsMIOX1a as determined by quantitative real-time PCR. The tissues included young leaf (YL), root (R), stem (S), old leaf (OL), episperm (EM), epicotyl (EP), hypocotyl (H) and flower (F). GAPDH was used as an internal control. The experiment included three fully independent biological repeats, and three technical repeats and the mean value are presented. The error bar represents the ± S.E. (standard error, n = 3). Significant differences analyses were conducted using the T-test method and are denoted by one or two stars, corresponding to p<0.05 and p<0.01, respectively.
Fig 3.
Characterization of the GsMIOX1a OX and atmiox1 mutant lines.
(A) Schematic representation of the construct for GsMIOX1a gene overexpression in Arabidopsis. (B) GsMIOX1a gene expression in the WT and OX lines. The expression levels were analyzed by semi-quantitative RT-PCR, and the ACTIN2 gene was used as an internal standard. (C) T-DNA and the flanking sequence of the AtMIOX1 gene in the atmiox1 mutant. The FP and RP primer pair was used for homozygous analysis, and the LB and RP primer pair was used for T-DNA insertion analysis. (D) PCR identification of the atmiox1 mutant. (E) Semi-quantitative RT-PCR identification of the atmiox1 mutant.
Fig 4.
Effect of alkaline stress on the germination of the WT, atmiox1, and GsMIOX1a OX lines.
(A) Phenotypes of WT, atmiox1 and OX plants grown on 1/2 MS medium with or without 11 mM NaHCO3. Photographs were taken 3 days after germination. (B) The seed germination rates of the WT, atmiox1 and OX Arabidopsis lines. Seeds were considered to be germinated when the radicles completely penetrated the seed coats. A total of 120 seeds from each line were used for each experiment.
Fig 5.
Effect of alkaline stress on mature plants of the WT, atmiox1, and GsMIOX1a OX lines.
(A) Phenotypes of the WT, atmiox1 and OX plants in response to 100 mM NaHCO3 stress. Three-week-old Arabidopsis plants were irrigated with 100 mM NaHCO3 solution every 3 days for a total of 12 days. The photographs were taken on the 13th day after stress exposure. (B) The free proline content of the WT, atmiox1 and OX plants under control conditions or stress treatment. (C) The POD activity of the WT, atmiox1 and OX plants under control conditions or alkaline stress. (D-E) The AsA content in the leaf extracts of the GsMIOX1a OX Arabidopsis, atmiox1 and WT lines. The total amount of AsA comprised the reduced (AsA) and oxidized forms (DHA). AsA contents were measured in the leaves of 5-week-old GsMIOX1a OX Arabidopsis, atmiox1 and WT plants under normal and alkaline stress conditions in a growth chamber. Leaves of the same age and size were selected to minimize experimental error. Error bars represent the ± S.E. (n = 3). Significant differences were analyses using the T-test method. Significant differences from WT are denoted by one or two stars, corresponding to p<0.05 and p<0.01, respectively.
Fig 6.
Expression of marker genes in the WT, atmiox1 and GsMIOX1a OX seedlings under alkaline stress.
(A) H+-Ppase expression levels under alkaline stress. (B) KIN1 expression levels under alkaline stress. (C) NADP-ME expression levels under alkaline stress. (D) RD29B expression levels under alkaline stress. To explore the transcript levels of stress-responsive genes, 2-week-old WT, atmiox1 and OX seedlings (line #4) were treated with 1/2 MS solution containing 50 mM NaHCO3 (pH 8.5) for 0, 1, 3, 6, and 12 h. The relative transcript levels were determined by quantitative real-time PCR. The AtActin2 gene served as an internal reference, and transcript levels were normalized to WT at 0 h. The values represent the means of three independent biological replicates and three technological replicates for each. One or two stars correspond to p<0.05 and p<0.01, respectively.