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. 2020 Mar 26;21(7):2288.
doi: 10.3390/ijms21072288.

Knockout of the OsNAC006 Transcription Factor Causes Drought and Heat Sensitivity in Rice

Bo Wang  1   2 , Zhaohui Zhong  2 , Xia Wang  1 , Xiangyan Han  1 , Deshui Yu  1 , Chunguo Wang  1 , Wenqin Song  1 , Xuelian Zheng  2 , Chengbin Chen  1 , Yong Zhang  2   3
Affiliations

Knockout of the OsNAC006 Transcription Factor Causes Drought and Heat Sensitivity in Rice

Bo Wang et al. Int J Mol Sci. .

Abstract

Rice (Oryza sativa) responds to various abiotic stresses during growth. Plant-specific NAM, ATAF1/2, and CUC2 (NAC) transcription factors (TFs) play an important role in controlling numerous vital growth and developmental processes. To date, 170 NAC TFs have been reported in rice, but their roles remain largely unknown. Herein, we discovered that the TF OsNAC006 is constitutively expressed in rice, and regulated by H2O2, cold, heat, abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellin (GA), NaCl, and polyethylene glycol (PEG) 6000 treatments. Furthermore, knockout of OsNAC006 using the CRISPR-Cas9 system resulted in drought and heat sensitivity. RNA sequencing (RNA-seq) transcriptome analysis revealed that OsNAC006 regulates the expression of genes mainly involved in response to stimuli, oxidoreductase activity, cofactor binding, and membrane-related pathways. Our findings elucidate the important role of OsNAC006 in drought responses, and provide valuable information for genetic manipulation to enhance stress tolerance in future plant breeding programs.

Keywords: CRISPR-Cas9; NAC transcription factor; abiotic stresses; rice; transcriptome analysis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression profile analysis of OsNAC006. (A) Detection of OsNAC006 expression in various tissues and organs of rice using RT-qPCR. Four-week-old seedlings were used to harvest root, sheath and leaf samples at the seedling stage. Plants in stages before the heading stage were used to harvest root, stem, sheath, leaf and panicle samples at the reproductive growth stage. Error bars indicate the standard error (SE) based on three biological replicates. (B), Nuclear localization of OsNAC006 protein in the rice protoplast. NLS, Nuclear localization signal. Scale bar = 20 µm. (C) Expression levels of OsNAC006 under various abiotic stresses and hormone treatments. Four-week-old seedlings were subjected to treatment with cold (4 °C), heat (42 °C), PEG 6000 (20%, w/v), NaCl (200 mm), H2O2 (1%), IAA (100 μm), ABA (100 μm) and GA3 (100 μm). The relative expression level of OsNAC006 was measured by RT-qPCR at the indicated times. Error bars indicate SE based on three independent biological replicates.
Figure 2
Figure 2
Using the CRISPR-Cas9 system to create mutants. (A) Design of sgRNA sites for OsNAC006 exons. (B) Single-strand conformation polymorphism analysis of 11 independent OsNAC006-sgRNA01 T0 lines. M, Markers; WT, Wild-type. (C) Sanger sequencing of the target site in OsNAC006-sgRNA01 T0 lines. (D) Phenotypic analysis of OsNAC006 T0 mutant lines.
Figure 3
Figure 3
The drought-sensitive phenotype of osnac006 mutants. (A) Phenotypic analysis of OsNAC006 T1 mutant lines under drought stress. (B) Levels of O2– and H2O2 in WT and OsNAC006 T1 mutant lines subjected to drought stress. Drought-stressed leaf samples were incubated in nitro-blue tetrazolium (NBT) or diaminobenzidine (DAB) solution. (C) Chlorophyll content after 20-day salt stress. Superoxide dismutase (SOD) activity after 20-day drought stress. Catalase (CAT) activity after 20-day drought stress. Peroxidase (POD) activity after 20-day drought stress. Malondialdehyde (MDA) content after 20-day drought stress. H2O2 content after 20-day drought stress. O2– production rate after 20-day drought stress. Bars represent the mean ± SE of three independent experiments. (D) Phenotypic analysis of OsNAC006 T1 mutant lines under heat stress. (E) Levels of O2– and H2O2 in WT and OsNAC006 T1 mutant lines subjected to heat stress. Heat-stressed leaf samples were incubated in nitro-blue tetrazolium (NBT) or diaminobenzidine (DAB) solution. (F) Chlorophyll content after 4-day heat stress. Superoxide dismutase (SOD) activity after 4-day heat stress. Catalase (CAT) activity after 20-day salt stress. Peroxidase (POD) activity after 4-day heat stress. Malondialdehyde (MDA) content after 20-day drought stress. H2O2 content after 4-day heat stress. O2– production rate after 4-day heat stress. Bars represent the mean ± SE of three independent experiments. ∗ and ∗ ∗ represent significant differences at p < 0.05 and p < 0.01 compared to WT.
Figure 4
Figure 4
Global gene expression changes in knockout OsNAC006 rice. (A) The most significant clustering analysis of differentially expressed genes (DEGs) in WT and osnac006 T1 mutant lines. Targeted knockout of osnac006 resulted in profound changes to gene expression, physiology, and development compared with WT and controls without drought stress treatment. The colour scale corresponds to log2 (FPKM) values of the genes. (B) Number of DEGs in WT, osnac006_1 and osnac006_2 T1 mutant lines, based on expression profiles obtained by RNA-Seq. Total RNA was extracted from mixed samples from three separate plants. (C) DEGs shared by WT and osnac006_1 and WT and osnac006_2 lines before drought stress. (D) DEGs shared by WT and osnac006_1 and WT and osnac006_2 lines after drought stress. (E) Gene ontology (GO) classification of DEGs shared by WT and osnac006_1 and WT and osnac006_2 mutant lines under normal and drought stress conditions. The x-axis shows user-selected GO terms, and the y-axis shows the percentage of genes (number of a particular gene divided by the number of total genes).
Figure 5
Figure 5
Transcriptome analysis of genes systemically regulated in WT and osnac006 T1 mutant lines in response to drought stress. (A) Response to stimuli. (B) Oxidoreductase activity. (C) Membrane part. (D) Cofactor binding. Log2 fold change (FC) values for DEGs in WT and osnac006_1 and osnac006_2 mutant lines are shown before (drought−) and after (drought+) drought treatment.
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References

    1. Zhang Q. Strategies for developing Green Super Rice. Proc. Natl. Acad. Sci. USA. 2007;104:16402–16409. doi: 10.1073/pnas.0708013104. - DOI - PMC - PubMed
    1. Hadiarto T., Tran L.S. Progress studies of drought-responsive genes in rice. Plant Cell Rep. 2011;30:297–310. doi: 10.1007/s00299-010-0956-z. - DOI - PubMed
    1. Nguyen H.M., Sako K., Matsui A., Suzuki Y., Mostofa M.G., Ha C.V., Tanaka M., Tran L.P., Habu Y., Seki M. Ethanol Enhances High-Salinity Stress Tolerance by Detoxifying Reactive Oxygen Species in Arabidopsis thaliana and Rice. Front. Plant Sci. 2017;8:1001. doi: 10.3389/fpls.2017.01001. - DOI - PMC - PubMed
    1. Xu N., Chu Y., Chen H., Li X., Wu Q., Jin L., Wang G., Huang J. Rice transcription factor OsMADS25 modulates root growth and confers salinity tolerance via the ABA-mediated regulatory pathway and ROS scavenging. PLoS Genet. 2018;14:e1007662. doi: 10.1371/journal.pgen.1007662. - DOI - PMC - PubMed
    1. Zhu M., Chen G., Dong T., Wang L., Zhang J., Zhao Z., Hu Z. SlDEAD31, a Putative DEAD-Box RNA Helicase Gene, Regulates Salt and Drought Tolerance and Stress-Related Genes in Tomato. PLoS ONE. 2015;10:e0133849. doi: 10.1371/journal.pone.0133849. - DOI - PMC - PubMed

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