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. 2006 Jul;18(7):1736-49.
doi: 10.1105/tpc.106.042184. Epub 2006 Jun 2.

STABILIZED1, a stress-upregulated nuclear protein, is required for pre-mRNA splicing, mRNA turnover, and stress tolerance in Arabidopsis

Byeong-ha Lee  1 , Avnish Kapoor, Jianhua Zhu, Jian-Kang Zhu
Affiliations

STABILIZED1, a stress-upregulated nuclear protein, is required for pre-mRNA splicing, mRNA turnover, and stress tolerance in Arabidopsis

Byeong-ha Lee et al. Plant Cell. 2006 Jul.

Abstract

In plants, many gene transcripts are very unstable, which is important for the tight control of their temporal and spatial expression patterns. To identify cellular factors controlling the stability of unstable mRNAs in plants, we used luciferase imaging in Arabidopsis thaliana to isolate a recessive mutant, stabilized1-1 (sta1-1), with enhanced stability of the normally unstable luciferase transcript. The sta1-1 mutation also causes the stabilization of some endogenous gene transcripts and has a range of developmental and stress response phenotypes. STA1 encodes a nuclear protein similar to the human U5 small ribonucleoprotein-associated 102-kD protein and to the yeast pre-mRNA splicing factors Prp1p and Prp6p. STA1 expression is upregulated by cold stress, and the sta1-1 mutant is defective in the splicing of the cold-induced COR15A gene. Our results show that STA1 is a pre-mRNA splicing factor required not only for splicing but also for the turnover of unstable transcripts and that it has an important role in plant responses to abiotic stresses.

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Figures

Figure 1.
Figure 1.
Comparison of Luminescence Images and Intensity between the Wild Type and sta1-1 under Stress. (A) Wild-type and sta1-1 seedlings on MS agar plates. (B) Luminescence images corresponding to plates in (A). (C) Quantification of luminescence intensities over the time periods indicated (n = 20 for cold stress and ABA, n = 10 for NaCl treatment; error bars indicate sd).
Figure 2.
Figure 2.
Comparison of Expression Levels between the Wild Type and sta1-1 of Endogenous RD29A and the RD29A-LUC Transgene by RNA Hybridization and Nuclear Run-On Analysis. (A) RNA hybridization with total RNA (20 μg) from samples treated with cold (72 h), ABA (3 h), or NaCl (3 h). (B) RNA hybridization with total RNA (20 μg) from samples treated with cold (0, 6, 24, or 72 h). (C) Nuclear run-on analysis with 72-h cold-treated samples.
Figure 3.
Figure 3.
sta1-1 Germination and Chilling Sensitivity. (A) Germination test of the wild type and sta1-1 on MS agar medium with ABA (0 or 0.1 μM). Photographs were taken at 4 d after imbibition. (B) Germination rates of the wild type and sta1-1 on MS medium with ABA (0, 0.1, 0.5, or 1 μM). Rates were scored at 7 d after imbibition. Data from three replicate experiments are shown. Error bars indicate se. (C) Chilling sensitivity test of the wild type and sta1-1 at 4°C. Four-day-old seedlings were transferred to 4°C, and the photograph was taken ∼6 months later.
Figure 4.
Figure 4.
sta1-1 Sensitivity to Various Salt and Osmotic Stress Conditions. (A) to (D) Comparisons of the wild type and sta1-1 in root growth on MS agar medium with ABA (A), NaCl (B), mannitol (C), and LiCl (D). Root growth was measured relative to controls. At least eight seedling roots were measured for each data point. Error bars indicate se. (E) and (F) Comparisons between the wild type and sta1-1 in seedling growth on MS agar medium with mannitol (Man) (E) or LiCl (F). Photographs were taken at 13 d after seedling transfer onto the treatment medium. All experiments were performed three times except for LiCl treatment (two times) with different seeds lots, and each time nearly identical results were obtained.
Figure 5.
Figure 5.
Developmental Phenotypes of sta1-1. (A) to (C) Morphology of wild-type and sta1-1 plants: 3 weeks old (A), 5 weeks old (B), and 6 weeks old (C). (D) Comparison of leaf morphology between the wild type and sta1-1. (E) Early-bolting phenotype of sta1-1. (F) Leaf number comparison between the wild type and sta1-1 upon bolting after different stratification periods.
Figure 6.
Figure 6.
Molecular Cloning of STA1 and Functional Complementation. (A) Positional cloning of STA1. Numbers of recombinations are from 308 F2 progeny seedlings with the phenotype conferred by sta1-1. Markers used at the recombination positions were, from left, T4I9-29K, F4C21-27K, F9H3-80K, F9H3-32K, and F9H3-3K. (B) Molecular complementation of the sta1-1 developmental defect with the wild-type STA1 gene. (C) and (D) Molecular complementation of the RD29A-LUC expression defect with the wild type STA1 gene. Shown are seedlings on an MS agar plate (C) and the corresponding luminescence image (D).
Figure 7.
Figure 7.
Characterization of STA1. (A) Predicted domain in the STA1 protein. The asterisk represents the mutation site in sta1-1. PRP1, PRP1 splicing factor N-terminal domain; NLS, nuclear localization signal; HAT, half a TPR; TPR, tetratricopeptide repeat; UBQ, ubiquitin. (B) to (D) Confocal microscopic images of an Arabidopsis root expressing the GFP-STA1 fusion protein. (E) A 4′,6-diamidino-2-phenylindole–stained root corresponding to the root in (D). (F) to (M) Expression of STA1 promoter–GUS in Arabidopsis. Expression in whole seedlings ([F] and [G]), root (H), leaf (I), flower (J), silique (K), guard cells (L), and trichome (M). For observation of guard cells and trichomes, the epidermal layer was peeled from leaves.
Figure 8.
Figure 8.
COR15A Expression in sta1-1. Total RNA (20 μg) from 14-d-old seedlings after cold, ABA, or NaCl treatment (A) or different cold stress durations (B) was subjected to RNA hybridization with the probes shown. Arrows indicate COR15A nonspliced transcript.
Figure 9.
Figure 9.
Expression of STF and STA1 in the Wild Type and sta1-1. (A) and (B) RNA gel blot analysis of wild-type and sta1-1 total RNA (20 μg) with STA1 and STF probes after different treatments (A) or different cold durations (B). (C) and (D) Nuclear run-on analysis with samples after 72 h of cold treatment. STA1 (C) and STF (D) were analyzed.
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