Identification of Chimeric Repressors that Confer Salt and Osmotic Stress Tolerance in Arabidopsis

Abstract

We produced transgenic Arabidopsis plants that express chimeric genes for transcription factors converted to dominant repressors, using Chimeric REpressor gene-Silencing Technology (CRES-T), and evaluated the salt tolerance of each line. The seeds of the CRES-T lines for ADA2b, Msantd, DDF1, DREB26, AtGeBP, and ATHB23 exhibited higher germination rates than Wild type (WT) and developed rosette plants under up to 200 mM NaCl or 400 mM mannitol. WT plants did not grow under these conditions. In these CRES-T lines, the expression patterns of stress-related genes such as RD29A, RD22, DREB1A, and P5CS differed from those in WT plants, suggesting the involvement of the six transcription factors identified here in the stress response pathways regulated by the products of these stress-related genes. Our results demonstrate additional proof that CRES-T is a superior tool for revealing the function of transcription factors.

Keywords: CRES-T; osmotic stress; repressor; salt stress; stress tolerance; transcription factors.

Figure 1

Germination rates of Wild type (WT) and CRES-T lines (ADA2b-SRDX, Msantd-SRDX, DDF1-SRDX, DREB26-SRDX, AtGeBP-SRDX, and ATHB23-SRDX) were monitored on medium supplemented with 0 (a), 150 (b), 175 (c), and 200 (d) mM NaCl and 400 mM mannitol (e). A seed was regarded as germinated when the radicle protruded through the seed coat. Similar experiments were performed three times with similar results. Error bars indicate the standard error of the mean. Ten seeds were used for each triplet replication test. * Significantly different from WT under non-stress condition at p < 0.05.

Figure 2

Seedlings of CRES-T and WT plants on medium supplemented with different concentrations of NaCl and mannitol. (a) Photograph of plants on control (non-stress) medium at 4 days after sowing; (b) Seedlings on medium supplemented with 150, 175, and 200 mM NaCl and 400 mM mannitol at 7 days after sowing; (c) Root development of CRES-T and WT plants on control (non-stress) medium at 4 days after sowing; (d) Comparison of root development between CRES-T lines and WT plants on medium supplemented with 150, 175, and 200 mM NaCl and 400 mM mannitol at 7 days after sowing; (e) Appearances of CRES-T and WT seedlings on medium supplemented with 400 mM mannitol at 4 weeks after sowing. Scale = 1 cm.

Figure 3

Effect of salt and osmotic stress on seedling growth of CRES-T plants. Seeds of WT and CRES-T lines (ADA2b-SRDX, Msantd-SRDX, DDF1-SRDX, DREB26-SRDX, AtGeBP-SRDX, and ATHB23-SRDX) were germinated on medium supplemented with 150 (a), 175 (b), and 200 (c) mM NaCl and 400 mM mannitol (d), and the numbers of seedlings with visible cotyledons were scored. Similar experiments were performed three times with similar results. Error bars indicate the standard error of the mean. Ten seeds were used for each triplet replication test. Scale = 1 cm.

Figure 4

Effect of salt and osmotic stress on root growth of CRES-T plants. Seeds of WT and CRES-T lines (ADA2b-SRDX, Msantd-SRDX, DDF1-SRDX, DREB26-SRDX, AtGeBP-SRDX, and ATHB23-SRDX) were germinated on medium supplemented with 0 (a), 150 (b), 175 (c), and 200 (d) mM NaCl and 400 mM mannitol (e), and the root length of seedlings was scored. Similar experiments were performed three times with similar results. Error bars indicate the standard error of the mean. * Significantly different from WT under non-stress condition at p < 0.05. n = 10.

Figure 5

Expression levels of genes for six transcription factor and their chimeric repressor in CRES-T lines and WT plants was detected by RT-PCR. UBQ5 was used as an internal control. NC; negative control; PCR using mix primers to detect transcripts for all the six transcription factors and RNAs without RT reaction as templates. The figures in parentheses are the PCR cycle numbers to detect the products.

Figure 6

Expression of four salt tolerance-related genes, (a) RD29A (AT5G52310.1); (b) RD22 (AT5G25610.1); (c) DREB1A (AT4G25480.1); and (d) P5CS (AT2G39800.1) in CRES-T lines and WT plants, was monitored by quantitative real-time RT-PCR. Ten-day-old seedlings of the CRES-T lines and WT on 1/2 MS plates were transplanted onto plates containing 1/2 MS medium supplemented with or without 150 mM NaCl. The seedlings were harvested at 24 h after transplantation followed by RNA extraction and quantitative real-time RT-PCR. Expression levels relative to WT under non-stress conditions (1.0) are shown. Error bars indicate the standard error of the mean. * Significantly different from WT under non-stress condition at p < 0.05. n = 3.

Figure A1

Screening of salt tolerant CRES-T lines. Seeds of CRES-T lines and WT were germinated on 1/2 MS medium supplemented with 175 mM NaCl for 14 days. Representative photographs, in which three DREB26-SRDX and Msantd-SRDX lines were used, are shown. Scale = 2 cm.

Figure A2

Salt tolerance of CRES-T lines. (a) Germination rate of CRES-T lines and WT was determined at 4 days after sowing on 1/2 MS medium supplemented with 175 mM NaCl; (b) Frequency of seedlings with expanded cotyledons was determined at 5 days after sowing on 1/2 MS medium supplemented with 175 mM NaCl. Ten seeds were used for each quadruple replication test. * and **: Significantly different from WT under non-stress condition at p < 0.05 and 0.01, respectively. ADA2b-SRDX1, Msantd-SRDX1, DDF1-SRDX1, DREB26-SRDX1, AtGeBPSRDX1, and ATHB23-SRDX2 were used for further experiments.

Figure A3

Growth of CRES-T lines and WT plants in pots with vermiculite under non-stressed conditions at 20 days after sowing. Scale = 2 cm.

Figure A4

Salt tolerance test in rosette-stage plants. Three-week old CRES-T lines and WT plants planted in pots with vermiculite were subjected to 400 mM NaCl solutions for 7 days, and followed by treatment with salt-free water for a further 7 days. (a) WT, (b) ADA2b-SRDX, (c) Msantd-SRDX, (d) DDF1-SRDX, (e) DREB26-SRDX, (f) AtGeBP-SRDX, and (g) ATHB23-SRDX. Scale = 2 cm.

Figure A5

Expression of Msantd, DREB26, and ATHB23 in WT plants. Absolute expression levels of each transcription factor gene in WT plants under salt (150 mM NaCl) and osmotic (300 mM mannitol) stress conditions were extracted from the AtGenExpress Visualization Tool and plotted as a graph. (a) Expression of DREB26 in WT roots; (b) expression of Msantd in WT roots; and (c) expression of ATHB23 in WT roots.