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Arabidopsis floral initiator SKB1 confers high salt tolerance by regulating transcription and pre-mRNA splicing through altering histone H4R3 and small nuclear ribonucleoprotein LSM4 methylation - PubMed

. 2011 Jan;23(1):396-411.

doi: 10.1105/tpc.110.081356. Epub 2011 Jan 21.

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Arabidopsis floral initiator SKB1 confers high salt tolerance by regulating transcription and pre-mRNA splicing through altering histone H4R3 and small nuclear ribonucleoprotein LSM4 methylation

Zhaoliang Zhang et al. Plant Cell. 2011 Jan.

Abstract

Plants adapt their growth and development in response to perceived salt stress. Although DELLA-dependent growth restraint is thought to be an integration of the plant's response to salt stress, little is known about how histone modification confers salt stress and, in turn, affects development. Here, we report that floral initiator Shk1 kinase binding protein1 (SKB1) and histone4 arginine3 (H4R3) symmetric dimethylation (H4R3sme2) integrate responses to plant developmental progress and salt stress. Mutation of SKB1 results in salt hypersensitivity, late flowering, and growth retardation. SKB1 associates with chromatin and thereby increases the H4R3sme2 level to suppress the transcription of FLOWERING LOCUS C (FLC) and a number of stress-responsive genes. During salt stress, the H4R3sme2 level is reduced, as a consequence of SKB1 disassociating from chromatin to induce the expression of FLC and the stress-responsive genes but increasing the methylation of small nuclear ribonucleoprotein Sm-like4 (LSM4). Splicing defects are observed in the skb1 and lsm4 mutants, which are sensitive to salt. We propose that SKB1 mediates plant development and the salt response by altering the methylation status of H4R3sme2 and LSM4 and linking transcription to pre-mRNA splicing.

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Figures

Figure 1.
Figure 1.

Phenotypic Analysis of skb1-1 Mutant Plants Exposed to Salt Stress. (A) and (B) Comparison of the survival rate of the wild type (Col-0) and the skb1-1 mutant grown on MS medium containing the indicated concentration of NaCl. Photographs were taken and the survival rate was determined 30 d after seedling transfer to the treatment medium. Data represent means ± (

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of three independent experiments (n > 50). (C) and (D) Comparison of root growth of wild type (Col-0), skb1-1, and 35S:SKB1 skb1-1 plants grown on MS medium with 100 mM NaCl. Four-day-old seedlings were transferred from MS medium to MS medium with 0 or 100 mM NaCl. Root growth was measured relative to controls 10 d after seedling transfer onto the treatment medium. More than 24 roots were measured for each data point. Data represent means ±

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of three independent experiments. (E) and (F) Comparison of the root growth of the wild type (Col-0) and skb1-1 mutant grown on MS medium with 100 mM KCl, 100 mM KNO3, or 10 mM LiCl. Primary root length was measured and root growth relative to controls was analyzed 10 d after seedling transfer to the treatment medium. More than 24 roots were measured for each data point. Data represent means ±

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of three independent experiments. (G) Relative electrolyte leakage of leaves from wild-type (Col-0), skb1-1, and 35S:SKB1 Col-0 plants after exposure to 100 mM NaCl. Data represent means ±

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of three independent experiments.

Figure 2.
Figure 2.

The Sensitivity of Mutants of Genes That Regulate Flowering Time to Salt Stress. (A) and (B) Similar to skb1-1, the skb1-1 flc-3 double mutant is hypersensitive to salt stress, and the sensitivity of flc-3 to salt stress is similar to that of the wild type. (C) The survival rate of the other mutants of genes that regulate flowering time, including fca, fve, fld4, flk, fy, ld, prmt4a prmt4b, prmt10, and sdg8. Four-day-old seedlings were transferred from MS medium to MS medium with 0, 80, 120, or 160 mM NaCl. Survival rate was determined 30 d after seedling transfer to the treatment medium. Data represent means ±

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of three independent experiments (n > 40). (D) Flowering times of the wild type (Col-0) and the skb1-1 mutant treated on MS medium with 0, 25, 50, or 75 mM NaCl for 5 d before transfer to soil. [See online article for color version of this figure.]

Figure 3.
Figure 3.

Loss of SKB1 Leads to Hypersensitivity to ABA. (A) and (B) Seed germination of the wild type (Col-0), skb1-1, 35S:SKB1 skb1-1, and 35S:SKB1 Col-0. Seeds harvested on the same day were planted on MS medium containing 0, 0.2, 0.3, 0.5, or 1 μM ABA. Plates were transferred to a growth chamber after stratification at 4°C for 3 d. Photographs were taken, and germination rate was determined 3 d after the transfer. Data represent means ±

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of three independent experiments (n = 96). (C) and (D) Cotyledon greening of the wild type (Col-0), skb1-1, 35S:SKB1 skb1-1, and 35S:SKB1/Col-0. Photographs were taken and cotyledon greening rate was determined 6 d after transfer to the growth chamber. Data represent means ±

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of three independent experiments (n = 96). (E) Real-time quantitative RT-PCR analysis of the expression of salt stress and ABA-responsive genes in the wild type (Col-0) and skb1-1 and normalized with 18S rRNA expression. Error bars indicate the relative

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of three independent experiments. Template RNA isolated from 11-d-old seedlings growing on MS agar medium was treated with 200 mM NaCl for 6 h or 100 μM ABA for 3 h.

Figure 4.
Figure 4.

SKB1 Expression Profile and H4R3sme2 Level in Global and Specific Sites in Response to Salt Stress and ABA Treatments. (A) RT-PCR analysis of the expression of SKB1 in the wild type (Col-0) in response to salt stress and ABA treatment. Template RNA isolated from 11-d-old seedlings grown on MS medium was treated with 200 mM NaCl for 6 h or 100 μM ABA for 3 h. TUBULIN was the loading control. Three biological replicates were performed with similar results. (B) Immunoblot analysis of SKB1 expression in response to salt stress and ABA treatment. Wild-type (Col-0) and skb1-1 plants were treated as in (A). (C) Immunoblot analysis of Arg symmetric dimethylation modification of the H4R3sme2 level in response to salt stress and ABA treatment, using histone-enriched whole protein extract and an H4R3sme2-specific antibody. Wild-type (Col-0) and skb1-1 seedlings were treated as in (A). (D), (F), and (K) Diagrams of the RD29A and RD29B (D), FLC (F), and HAB1 (K) genes structure, with bars representing the regions examined by ChIP shown in (E), (H), and (L), respectively. White boxes represent exons or 5′ or 3′ untranslated regions, and black lines represent introns. (E), (H), and (L) ChIP assay for RD29B and RD29A (E), FLC (H), and HAB1 (L) performed with anti-SKB1 and -H4R3sme2 antibodies. Chromatin extracted from 11-d-old seedlings grown on MS medium was treated with 200 mM NaCl for 6 h or 100 μM ABA for 3 h, untreated as a control. Data represent triplicate multiquantitative PCR measurements of immunoprecipitated DNA, and the input represents chromatin before immunoprecipitation. Error bars represent relative

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of ChIP data. (G) Quantitative RT-PCR analysis of the expression of FLC in response to salt stress. Template RNA isolated from 11-d-old seedlings grown on MS medium was treated with 200 mM NaCl for 6 h. The experiment was performed independently three biological replicates and normalized by TUBULIN expression. Error bars indicate the relative

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of three independent experiments. (I) Venn diagram of genes upregulated in the skb1-1 mutant in standard conditions (a) and in the wild type after treatment with 200 mM NaCl for 6 h (b), as determined by microarray analysis. (J) Real-time quantitative RT-PCR analysis of the expression of salt stress–induced genes in the wild type and skb1-1 mutant in standard and salt stress conditions. Template RNA, experiments, and data analysis are indicated in (G).

Figure 5.
Figure 5.

SKB1 Methylates the U snRNP Core Component LSM4. (A) Immunoblot analysis of Arg symmetric dimethylation modification with the SYM11 antibody in response to salt stress and ABA treatments. Total protein was extracted from 11-d-old wild-type (Col-0) or skb1-1 seedlings treated with 200 mM for 6 h or 100 μM ABA for 3 h. The experiment was performed independently three times with similar results. (B) Total protein from wild-type seedlings treated with ABA for 5 h was immunoprecipitated with SYM11. Protein bands (7 to 17 kD) were identified by mass spectrometry and are indicated at the right. (C) and (E) In vitro methylation assay. GST-SM or GST-LSM proteins and GST-SKB1 were purified from E. coli and incubated in the presence of [3H]-SAM. Methylated proteins were monitored by autoradiography. (D) Full-length protein sequence of Arabidopsis LSM4, the location of three RG boxes, and the truncations in LSM4 (d1), LSM4 (d2), and LSM4 (d3).

Figure 6.
Figure 6.

RNA Splicing Analysis. (A) RT-PCR analysis of the pre-mRNA splicing pattern of RD22 and AT1G13350 in wild-type (1), skb1-1 (2), and 35S:SKB1 skb1-1 (3) plants. Eleven-day-old seedlings grown on MS medium were treated with 200 mM NaCl for 6 h or 100 μM ABA for 3 h. Three biological replicates were performed with similar results. The splicing pattern of each alternative splicing isoform is shown at the right. Gray square, exon; black line, unspliced intron; bent line, spliced intron. Arrowheads at the right show primer locations. (B) and (C) Statistical chart of alternative splicing (AS) genes and events. ES, exon skipping; IR, intron retention; A5′S, alternative 5′ splicing; A3′S, alternative 3′ splicing. (D) The splicing pattern of genes in the wild type and skb1-1 mutant. Candidate genes were selected that were identified as having different splicing patterns in the wild type and skb1 mutant by transcriptome RNA sequencing.

Figure 7.
Figure 7.

LSM4 Structure and Pleiotropic Phenotypes of the lsm4 Mutant. (A) LSM4 structure and the T-DNA insertion site of the lsm4 mutant (Salk_063398). White boxes represent exons or 5′ or 3′ untranslated regins, and black lines represent introns. (B) RT-PCR analysis of LSM4 expression in 7-d-old wild-type (Col-0) and lsm4 seedlings. TUBULIN was used as a loading control. (C) The phenotypes of 4-week-old wild-type (Col-0) and lsm4 mutant seedlings. (D) The sensitivity of LSM4+/− F2 seedlings to ABA and salt stress. Photographs were taken after 8 d of culture at 23°C. Red arrows show nongreening seedlings or ungerminated seeds. (E) Quantitative analysis of wild-type (Col-0) and LSM4+/− F2 greening cotyledon rate in MS medium containing 0.5 μM ABA and of germination rate in MS medium containing 100 mM NaCl, as shown in (D), n = 120. (F) and (G) The pre-mRNA splicing pattern of genes, whose pre-mRNA splicing was affected by SKB1 mutation, in the lsm4 mutant.

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