Fig 1.
Isolation of NUP85 as a suppressor of sic-1 in response to ABA and salt stress.
(A) Luminescence images of wild type, sic-1 and sic-1 nup85 double mutant seedlings under control, ABA and high salt treatments. (B) Quantification of the luminescence intensities of Fig 1A. (C) Diagram of NUP85 genomic sequence and the three nup85 mutants used in this study. The sic-1 nup85 double mutant was caused by a G to A substitution which led to a pre-mature stop codon. (D) The expression of luciferase (LUC) in WT, sic-1 and sic-1 nup85 seedlings under control and ABA treatment. (E) The expression of RD29A in WT, sic-1 and sic-1 nup85 seedlings under control and ABA treatment. Total RNA was extracted from 7-day-old seedlings with mock or 50 μM ABA treatment. The relative transcript levels were normalized to Arabidopsis Actin2 (ACT2) and the normalized expression level of WT at 0 h was set to 1. Data are present as mean value ± SD (n = 3).
Fig 2.
The reduced expression of RD29A-LUC in response to ABA and salt stress was rescued in NUP85 complementation lines.
The Luminescence images of WT, sic-1, sic-1 nup85 double mutant and two independent NUP85 complementation lines with mock treatment (A), 200 mM NaCl treatment (B) and 100 μM ABA treatment (C). At least 20 seedlings of each genotype were treated with 100 μM ABA or 200 mM NaCl. Quantification of the luminescence intensities in A to C is present in (D). Error bars indicate SD. The experiments were conducted three independent times with similar results.
Fig 3.
The attenuated expression of stress responsive genes in nup85 mutants in response to ABA and salt stress.
(A) The expression of stress responsive genes RD29A, COR15A and COR47 in Col-0 wild type and two alleles of nup85 mutants under mock and 50 μM ABA treatments for 3 h. The RNA was extracted from 7-day-old seedlings which were treated with mock or 50 μM ABA for 3 h. Data are mean values ±SD of three independent replicates. Asterisks indicate significant differences compared to WT Col under the same treatments. (B) The expression of RD29A, COR15A and COR47 in Col-0 wild type and two mutant alleles of nup85. The RNA was extracted from 7-day-old seedlings which were treated with mock or 0.3 M NaCl for 5 h. Data are mean values ±SD of three independent replicates. Significance between mean values were analyzed by student’s t test (* P< 0.05). Asterisks indicate significant differences compared to WT Col under the same treatments. (C) Gene ontology enrichment analysis of the differentially expressed (DE) genes regulated by NUP85 under mock condition (p value < 0.05). (D) Heat map depiction of the NUP85 regulated ABA-responsive genes in Col-0 wild type and nup85-1 mutants under mock and ABA treatments.
Fig 4.
The hypersensitivity of nup85, hos1 and nup160 plants in response to ABA and salt stress.
(A) The phenotypes of Col-0 wild type and two mutant alleles of nup85 in response to ABA and NaCl. (B) Quantification of the primary root length of wild type, nup85-1 and nup85-2 at 7 days after transfer to the indicated media. (C) The ABA and NaCl inhibition of seedling growth of wild type, hos1 and nup160 mutants. (D) Quantification of the primary root length of wild type, hos1 and nup160 at 7 days after transfer to the indicated media. Phenotypes were documented at 7 days after the seedling transfer to ½ MS plates, 20 μM ABA or 100 mM NaCl containing medium. The experiments were repeated at least three independent times with consistent results. Values indicate means ± SD (n = 36). Significance between the mean values were analyzed with Student’s t test (* P< 0.05). Asterisks indicate significant differences compared to WT Col under the same treatments.
Fig 5.
The ABA and salt hypersensitivity of nup85-1 nup160 and nup85-1 hos1 double mutants.
(A)The root growth of Col-0 wild type, nup85-1, nup160, hos1 single mutants and nup85-1 nup160 as well as nup85-1 hos1 double mutants on control ½ MS and ABA or NaCl-containing MS medium. (B)Quantification of the primary root length of indicated genotypes at 7 days after transfer to control and ABA or NaCl- containing MS medium. Three-day-old seedlings from each genotype were transferred to the indicated media and primary root length was measured after 7 days. Values indicate means ± SD (n = 24). Asterisks indicate significant differences compared to WT Col under the same treatments. Significance between the mean values were analyzed with Student’s t test (* P< 0.05, ** P< 0.01).
Fig 6.
The impaired expression of stress responsive genes in nup160 and hos1 mutants in response to ABA and salt stress.
(A)The expression of stress responsive genes RD29A, COR15A and COR47 in Col-0 wild type, nup160 and hos1 mutants under mock and ABA treatment. (B)The expression of RD29A, COR15A and COR47 in Col-0 wild type, nup160 and hos1 mutants under mock and salt treatments. RNA was extracted from 7-day-old seedlings which were treated with mock, 50 μM ABA or 0.3 M NaCl. Data are mean values ±SD of three independent replicates. Significance between mean values were analyzed by student’s t test (* P< 0.05, ** P< 0.01). Asterisks indicate significant differences compared to WT Col under the same treatments.
Fig 7.
The interaction between NUP85 and MED18.
(A) Split luciferase complementation assay in Arabidopsis protoplasts showing the interaction between NUP85 and MED18. Empty Cluc and PYL1-Cluc were used as negative controls. Approximately 1×104 protoplasts per sample were co-transformed with indicated plasmids. The split-LUC complementation assay was repeated three independent times with consistent results. (B) Co-immunoprecipitation assays in Arabidopsis protoplasts confirming the interaction between NUP85 and MED18. CDK8-HA was used as a control. (C) The relative gene expression of RD29A, COR15A and COR47 in wild type and med18 mutants under mock or ABA treatments. (D)The relative gene expression of RD29A, COR15A and COR47 in wild type and med18 mutants under mock or salt treatments (0.3 M NaCl). In C and D, values indicate means ± SD (n = 3). Significance between the mean values were analyzed with Student’s t test (* P< 0.05).
Fig 8.
The hypersensitivity of med18 in response to ABA and salt stress.
(A) The root growth of Col-0 wild type and med18 mutants on control ½ MS and ABA or NaCl-containing MS medium at 7days after transfer. (B) Quantification of the primary root length of Col-0 wild type and med18 mutants at 7 days after transfer to control and ABA or NaCl- containing MS medium. Three-day-old seedlings were transferred to the ½ MS or ABA/NaCl containing media and primary root length was measured after 7 days. Values indicate means ± SD (n = 20). Asterisks indicate significant differences compared to WT Col under the same treatments. Significance between the mean values were analyzed with Student’s t test (* P< 0.05).