An <i>Arabidopsis</i> Nucleoporin <i>NUP85</i> modulates plant responses to ABA and salt stress

<div><p>Several nucleoporins in the nuclear pore complex (NPC) have been reported to be involved in abiotic stress responses in plants. However, the molecular mechanism of how NPC regulates abiotic stress responses, especially the expression of stress responsive genes remains poorly understood. From a forward genetics screen using an abiotic stress-responsive luciferase reporter (<i>RD29A-LUC</i>) in the <i>sickle-1</i> (<i>sic-1</i>) mutant background, we identified a suppressor caused by a mutation in <i>NUCLEOPORIN 85</i> (<i>NUP85</i>), which exhibited reduced expression of <i>RD29A-LUC</i> in response to ABA and salt stress. Consistently, the ABA and salinity induced expression of several stress responsive genes such as <i>RD29A</i>, <i>COR15A</i> and <i>COR47</i> was significantly compromised in <i>nup85</i> mutants and other nucleoporin mutants such as <i>nup160</i> and <i>hos1</i>. Subsequently, Immunoprecipitation and mass spectrometry analysis revealed that NUP85 is potentially associated with HOS1 and other nucleoporins within the nup107-160 complex, along with several mediator subunits. We further showed that there is a direct physical interaction between MED18 and NUP85. Similar to <i>NUP85</i> mutations, <i>MED18</i> mutation was also found to attenuate expression of stress responsive genes. Taken together, we not only revealed the involvement of <i>NUP85</i> and other nucleoporins in regulating ABA and salt stress responses, but also uncovered a potential relation between NPC and mediator complex in modulating the gene expression in plants.</p></div

The hypersensitivity of <i>nup85</i>, <i>hos1</i> and <i>nup160</i> plants in response to ABA and salt stress.

<p>(A) The phenotypes of Col-0 wild type and two mutant alleles of <i>nup85</i> in response to ABA and NaCl. (B) Quantification of the primary root length of wild type, <i>nup85-1</i> and <i>nup85-2</i> at 7 days after transfer to the indicated media. (C) The ABA and NaCl inhibition of seedling growth of wild type, <i>hos1</i> and <i>nup160</i> mutants. (D) Quantification of the primary root length of wild type, <i>hos1</i> and <i>nup160</i> 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 <i>t</i> test (* P< 0.05). Asterisks indicate significant differences compared to WT Col under the same treatments.</p

The hypersensitivity of <i>med18</i> in response to ABA and salt stress.

<p>(A) The root growth of Col-0 wild type and <i>med18</i> 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 <i>med18</i> 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 <i>t</i> test (* P< 0.05).</p

The attenuated expression of stress responsive genes in <i>nup85</i> mutants in response to ABA and salt stress.

<p>(A) The expression of stress responsive genes <i>RD29A</i>, <i>COR15A</i> and <i>COR47</i> in Col-0 wild type and two alleles of <i>nup85</i> 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 <i>RD29A</i>, <i>COR15A</i> and <i>COR47</i> in Col-0 wild type and two mutant alleles of <i>nup85</i>. 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 <i>t</i> 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 <i>NUP85</i> under mock condition (p value < 0.05). (D) Heat map depiction of the <i>NUP85</i> regulated ABA-responsive genes in Col-0 wild type and <i>nup85-1</i> mutants under mock and ABA treatments.</p

The reduced expression of <i>RD29A-LUC</i> in response to ABA and salt stress was rescued in <i>NUP85</i> complementation lines.

<p>The Luminescence images of WT, <i>sic-1</i>, <i>sic-1 nup85</i> 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.</p

The ABA and salt hypersensitivity of <i>nup85-1 nup160</i> and <i>nup85-1 hos1</i> double mutants.

<p>(A)The root growth of Col-0 wild type, <i>nup85-1</i>, <i>nup160</i>, <i>hos1</i> single mutants and <i>nup85-1 nup160</i> as well as <i>nup85-1 hos1</i> 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 <i>t</i> test (* P< 0.05, ** P< 0.01).</p

Isolation of <i>NUP85</i> as a suppressor of <i>sic-1</i> in response to ABA and salt stress.

<p>(A) Luminescence images of wild type, <i>sic-1</i> and <i>sic-1 nup85</i> double mutant seedlings under control, ABA and high salt treatments. (B) Quantification of the luminescence intensities of Fig 1A. (C) Diagram of <i>NUP85</i> genomic sequence and the three <i>nup85</i> mutants used in this study. The <i>sic-1 nup85</i> double mutant was caused by a G to A substitution which led to a pre-mature stop codon. (D) The expression of luciferase (<i>LUC</i>) in WT, <i>sic-1</i> and <i>sic-1 nup85</i> seedlings under control and ABA treatment. (E) The expression of <i>RD29A</i> in WT, <i>sic-1</i> and <i>sic-1 nup85</i> 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 <i>Arabidopsis Actin2</i> (<i>ACT2</i>) and the normalized expression level of WT at 0 h was set to 1. Data are present as mean value ± SD (n = 3).</p

The impaired expression of stress responsive genes in <i>nup160</i> and <i>hos1</i> mutants in response to ABA and salt stress.

<p>(A)The expression of stress responsive genes <i>RD29A</i>, <i>COR15A</i> and <i>COR47</i> in Col-0 wild type, <i>nup160</i> and <i>hos1</i> mutants under mock and ABA treatment. (B)The expression of <i>RD29A</i>, <i>COR15A</i> and <i>COR47</i> in Col-0 wild type, <i>nup160</i> and <i>hos1</i> 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 <i>t</i> test (* P< 0.05, ** P< 0.01). Asterisks indicate significant differences compared to WT Col under the same treatments.</p