Abstract
Key message
Overexpression of Gshdz4 or GsNAC019 enhanced alkaline tolerance in transgenic Arabidopsis. We proved that Gshdz4 up-regulated both GsNAC019 and GsRD29B but GsNAC019 may repress the GsRD29B expression under alkaline stress.
Abstract
Wild soybean (Glycine soja) has a high tolerance to environmental challenges. It is a model species for dissecting the molecular mechanisms of salt-alkaline stresses. Although many NAC transcription factors play important roles in response to multiple abiotic stresses, such as salt, osmotic and cold, their mode of action in alkaline stress resistance is largely unknown. In our study, we identified a G. soja NAC gene, GsNAC019, which is a homolog of the Arabidopsis AtNAC019 gene. GsNAC019 was highly up-regulated by 50 mM NaHCO3 treatment in the roots of wild soybean. Further investigation showed that a well-characterized transcription factor, Gshdz4 protein, bound the cis-acting element sequences (CAATA/TA), which are located in the promoter of the AtNAC019/GsNAC019 genes. Overexpression of Gshdz4 positively regulated AtNAC019 expression in transgenic Arabidopsis, implying that AtNAC019/GsNAC019 may be the target genes of Gshdz4. GsNAC019 was demonstrated to be a nuclear-localized protein in onion epidermal cells and possessed transactivation activity in yeast cells. Moreover, overexpression of GsNAC019 in Arabidopsis resulted in enhanced tolerance to alkaline stress at the seedling and mature stages, but reduced ABA sensitivity. The closest Arabidopsis homolog mutant plants of Gshdz4, GsNAC019 and GsRD29B containing athb40, atnac019 and atrd29b were sensitive to alkaline stress. Overexpression or the closest Arabidopsis homolog mutant plants of the GsNAC019 gene in Arabidopsis positively or negatively regulated the expression of stress-related genes, such as AHA2, RD29A/B and KIN1. Moreover, this mutation could phenotypically promoted or compromised plant growth under alkaline stress, implying that GsNAC019 may contribute to alkaline stress tolerance via the ABA signal transduction pathway and regulate expression of the downstream stress-related genes.
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Abbreviations
- ABA:
-
Abscisic acid
- COR47:
-
Cold-regulated gene 47
- GFP:
-
Green fluorescent protein
- His:
-
Histidine
- KIN1 :
-
Kinase 1
- NACBS:
-
NAC binding sites
- NADP-ME :
-
NADP-dependent malic enzyme
- RD22:
-
Responsive to dehydration 22
- RD29A:
-
Responsive to dehydration 29A
- RD29B:
-
Responsive to dehydration 29B
- SD:
-
Synthetically defined medium
- USER:
-
Uracil-specific excision reagent
- WT:
-
Wild type
- OX:
-
Overexpression
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Acknowledgements
We would like to thank Dr. Guanzheng Qu for the service of confocal laser-scanning microscope in Northeast Forestry University.
Funding
This work was supported by the National Natural Science Foundation of China (31670272 to XD) and the Technological Innovation Team Building Program of the College of Heilongjiang Province (2011TD005) and NEAU starting grant to XD. This work was also supported by the National Natural Science Foundation of China (31501331).
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Experiment design LC, YZ. Experiment performance and data analyses LC, FY, YY, DZ, XS, XD, KY. Manuscript drafting LC, BL, XD.
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11103_2017_643_MOESM1_ESM.tif
Supplementary Fig. 1 Temporal and spatial expression patterns of GsNAC019 and GsRD29B in wild soybean roots under 200 mM NaCl treatment. (a) GsNAC019; (b) GsRD29B. The values represent the means of three biological replicates; the error bars indicate the SD. (TIF 11663 KB)
11103_2017_643_MOESM2_ESM.tif
Supplementary Fig. 2 Germination rates and Growth of Gshdz4 overexpression lines (#14, #20 and #23) and WT plants under 0, 0.4 μM, 0.6 μM, 0.8 μM or 1.0 μM ABA treatments at the germination stage. (TIF 23046 KB)
11103_2017_643_MOESM3_ESM.tif
Supplementary Fig. 3 Growth and root lengths of the Gshdz4 overexpression lines (#14, #20 and #23) and WT plants under 0, 30 μM, or 40μM ABA treatments at the seedling stage. Five plants were treated in one plate. The three plates were treated as independent experiments, and at least three independent experiments were conducted. The values represent the means of three biological replicates; the error bars indicate the SD. Significant differences are denoted with one or two stars if P < 0.05 or P < 0.01 by Student’s t-test. (TIF 11543 KB)
11103_2017_643_MOESM4_ESM.tif
Supplementary Fig. 4 Growth and root lengths of the mutant lines (athb40, atnac019 and atrd29b) and WT plants under 0, 30 μM, or 40μM ABA treatments at the seedling stage. Five plants were treated in one plate. The three plates were treated as independent experiments, and at least three independent experiments were conducted. The values represent the means of three biological replicates; the error bars indicate the SD. Significant differences are denoted with one or two stars if P < 0.05 or P < 0.01 by Student’s t-test. (TIF 18330 KB)
11103_2017_643_MOESM5_ESM.tif
Supplementary Fig. 5 Temporal and spatial expression patterns of GsRD29B in wild soybean under 50 mM NaHCO3 treatment. (a) Roots; (b) leaves. The values represent the means of three biological replicates; the error bars indicate the SD. (TIF 12970 KB)
11103_2017_643_MOESM6_ESM.tif
Supplementary Fig. 6 Gshdz4 up-regulated AtRD29B expression. (a) AtRD29B expression levels were up-regulated in the Gshdz4 overexpression lines (#20 and #23) under alkaline stress. (b) The AtRD29B expression levels were down-regulated in athb40 mutant plants. The values represent the means of three biological replicates; the error bars indicate the SD. Significant differences are denoted with one or two stars if P < 0.05 or P < 0.01 by Student’s t-test. (TIF 3168 KB)
11103_2017_643_MOESM7_ESM.tif
Supplementary Fig. 7 Temporal and spatial expression patterns of Gshdz4. (a) Gshdz4 relative expression analysis in wild soybean leaves. (b) Gshdz4 relative expression analysis in wild soybean roots. Samples were taken from 3-week wild soybean seedlings that were subjected to a 50 μM ABA treatment. The values represent the means of three biological replicates; the error bars indicate the SD. (TIF 13650 KB)
11103_2017_643_MOESM8_ESM.tif
Supplementary Fig. 8 Temporal and spatial expression patterns of GsNAC019. (a) GsNAC019 relative expression analysis in wild soybean leaves. (b) GsNAC019 relative expression analysis in wild soybean roots. Samples were taken from 3-week wild soybean seedlings that were subjected to a 50 μM ABA treatment. The values represent the means of three biological replicates; the error bars indicate the SD. (TIF 12860 KB)
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Cao, L., Yu, Y., Ding, X. et al. The Glycine soja NAC transcription factor GsNAC019 mediates the regulation of plant alkaline tolerance and ABA sensitivity. Plant Mol Biol 95, 253–268 (2017). https://doi.org/10.1007/s11103-017-0643-3
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DOI: https://doi.org/10.1007/s11103-017-0643-3