Rice Science ›› 2021, Vol. 28 ›› Issue (6): 547-556.DOI: 10.1016/j.rsci.2021.09.003
• Research Paper • Previous Articles Next Articles
Dan Zeng1, Chunchao Wang1, Junpin Xie2, Fan Zhang1, Jialing Lu1, Xiaorong Shi2, Yingyao Shi2, Yongli Zhou1()
Received:
2020-07-01
Accepted:
2020-10-12
Online:
2021-11-28
Published:
2021-11-28
Dan Zeng, Chunchao Wang, Junpin Xie, Fan Zhang, Jialing Lu, Xiaorong Shi, Yingyao Shi, Yongli Zhou. Stress-Activated Protein Kinase OsSAPK7 Regulates Salt- Stress Tolerance by Modulating Diverse Stress-Defensive Responses in Rice[J]. Rice Science, 2021, 28(6): 547-556.
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Fig. 1. Domain structure and subcellular localization of OsSAPK7.A, Schematic representation of the domain structure of OsSAPK7. The highly conserved kinase region at the N-terminal and the Glu-rich motif at the C-terminal are shown.B, Green fluorescence protein (GFP) signals arising from the OsSAPK7- GFP fusion proteins expressed in tobacco leaves were detected using a confocal microscope. Red signals represent chloroplast auto-fluorescence.The only GFP signal expressed by pCAMBIA-1300 in tobacco leaves served as a negative control.
Fig. 2. Expression of OsSAPK7 in rice under salt-stress conditions.After 20-day-old rice seedlings were subjected to 100 mmol/L NaCl treatment, OsSAPK7 expression levels were assessed by qRT-PCR. Means and standard errors were obtained from three biological replicates. ** and *** indicate statistically significant differences at the 0.01 and 0.001 levels, respectively.
Fig. S1. Graphic representation of vector constructs and expression analysis of OsSAPK7 in transgenic plants.A, Positive OsSAPK7-overexpression transgenic plants was verified by PCR method. M, marker; +, positive; -, negative.B, Expression of OsSAPK7 in wild type 9804 and OsSAPK7-overexpression transgenic plants as determined by qRT-PCR. Actin gene was the control. Values are Mean ± SE (n = 3). ***, P < 0.001 according to the Student’s t-test.C, Protein expression of OsSAPK7 in 9804 and OsSAPK7-overexpressing transgenic plants as assessed by western blotting.D, A schematic description of the WT OsSAPK7 gene. Red letters represent the target sequence.E, Sanger sequencing results of mutations in OsSAPK7 knockouts. Above is the editing sequence of the target site, and below is the sequencing chromatogram.
Fig. 3. Phenotypic reactions of OsSAPK7-overexpression, OsSAPK7 knockout and wild type (WT) 9804 lines under salt-stress conditions.A, Phenotypic comparison of seedings grown under salt stress at the seedling stage. OsSAPK7-overexpression lines (OE11 and OE12), OsSAPK7 knockout lines (SA2 and SA23) and 9804 plants under normal conditions for 20-day-old seedlings were transferred to Hoagland's nutrient solution supplemented with 100 mmol/L NaCl for 10 d and then recovered for 7 d.B, Survival rates of OE11, OE12, SA2, SA23 and 9804 plants after recovered for 7 d. Values are Mean ± SE (n = 3). **, P < 0.05 according to the Student's t-test.
Fig. S2. Root growth and germination rates of OsSAPK7 transgenic lines and wild type 9804 under salt-stress conditions.A and B, Phenotype of OsSAPK7-overexpression (OE11 and OE12), OsSAPK7 knockout (SA2 and SA23) and wild type 9804 plants grown in distilled water (A) or in distilled water supplemented with 100 mmol/L NaCl solution (B).C and D, Root lengths of OsSAPK7-overexpression, OsSAPK7 knockout and 9804 plants grown in distilled water (C) or in distilled water supplemented with 100 mmol/L NaCl solution (D).E to H, Seed germination rates of OsSAPK7-overexpression, OsSAPK7 knockout and wild type 9804 plants grown in distilled water or in distilled water supplemented with 100 mmol/L NaCl solution.Values are Mean ± SE (n = 3). *, P < 0.05 according to the Student’s t-test.
Fig. 4. Responses of 20-day-old transgenic lines and wild type 9804 at 6 d after exposure to 100 mmol/L NaCl treatment.A, Na+/K+ ratio in roots. B, Na+/K+ ratio in shoots. C, Chlorophyll content in the leaves under control and salt-stress conditions for 6 d. D, Superoxide dismutase (SOD) activity in leaves. E, Catalase (CAT) activity in leaves. F, Malomdiadehyde (MDA) content of leaves. G, Proline content of leaves.OE11 and OE12 are the OsSAPK7-overexpression lines. SA2 and SA23 are the OsSAPK7 knockout lines. Values are Mean ± SE (n = 3). * and ** indicate significant differences at the 0.05 and 0.01 levels according to the Student's t-test compared with 9804, respectively.
Fig. 5. Transcriptome profiling analysis of OsSAPK7 knockout line SA23 and wild type (WT, 9804) under salt stress conditions.A, Venn diagram of differentially expressed genes (DEGs) in WT vs SA23 at 0, 12 and 36 h under salt-stress conditions.B, Hierarchical clustering of DEGs between WT and SA23 at 12 and 36 h under salt stress. The color scale represents log2 of the fragment per kilo base of exon model per million mapped fragments (FPKM).C, Gene Ontology analysis of DEGs in WT vs SA23 at 12 and 36 h under salt-stress conditions.D, Kyoto Encyclopedia of Genes and Genomes pathway analysis of DEGs in WT vs SA23.
Fig. 6. Expression patterns of differentially expressed genes (DEGs) in OsSAPK7 knockout line SA23 compared with wild type 9804.A, Hierarchical clustering of DEGs associated with electron carrier activity, photosynthesis and zeatin synthesis pathway regulated by OsSAPK7 after salt-stress treatment. Color scale represents the log2 of the fragment per kilo base of exon model per million mapped fragments (FPKM).B, Expression patterns of key genes in 9804 and SA23 assessed by qRT-PCR and FPKM. Values are Mean ± SE (n = 3).
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