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. 2012 Oct 30;109(44):18198-203.
doi: 10.1073/pnas.1216199109. Epub 2012 Oct 15.

Arabidopsis proline-rich protein important for development and abiotic stress tolerance is involved in microRNA biogenesis

Xiangqiang Zhan  1 , Bangshing Wang, Hongjiang Li, Renyi Liu, Rajwant K Kalia, Jian-Kang Zhu, Viswanathan Chinnusamy
Affiliations

Arabidopsis proline-rich protein important for development and abiotic stress tolerance is involved in microRNA biogenesis

Xiangqiang Zhan et al. Proc Natl Acad Sci U S A. .

Abstract

MicroRNAs (miRNAs) are important for plant development and stress responses. However, factors regulating miRNA metabolism are not completely understood. SICKLE (SIC), a proline-rich protein critical for development and abiotic stress tolerance of Arabidopsis, was identified in this study. Loss-of-function sic-1 mutant plants exhibited a serrated, sickle-like leaf margin, reduced height, delayed flowering, and abnormal inflorescence phyllotaxy, which are common characteristics of mutants involved in miRNA biogenesis. The sic-1 mutant plants accumulated lower levels of a subset of miRNAs and transacting siRNAs but higher levels of corresponding primary miRNAs than the WT. The SIC protein colocalizes with the miRNA biogenesis component HYL1 in distinct subnuclear bodies. sic-1 mutant plants also accumulated higher levels of introns from hundreds of loci. In addition, sic-1 mutant plants are hypersensitive to chilling and salt stresses. These results suggest that SIC is a unique factor required for the biogenesis of some miRNAs and degradation of some spliced introns and important for plant development and abiotic stress responses.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Effect of sic-1 on leaf and inflorescence morphology. (A) Leaf morphology. Rosette and cauline leaves of sic-1 have serrated sickle-like margins, in contrast to those of WT. The leaf morphology of sic-1 is similar to that of se-1 and abh1-285. (B) SIC rescues the leaf-shape defect of sic-1. Rosette leaves of WT, sic-1, and sic-1 genetically transformed with native promoter-driven SIC gene (sic-1:SIC-Flag and sic-1:SIC-GFP). (C) Architecture of silique arrangements in WT, sic-1, se-1, and abh1-285. Arrow shows clustering of more than one silique on the same node in sic-1, se-1, and abh1-285.
Fig. 2.
Fig. 2.
Effect of sic-1 on the accumulation of miRNAs, ta-siRNAs, and pri-miRNAs. (A) Northern analysis of miRNA accumulation in WT, sic-1, se-1, and abh1-285 of Arabidopsis seedling and inflorescence. (B) Northern analysis of ta-siRNA in WT and sic-1 in Arabidopsis. (C) RT-PCR analysis of pri-miRNA accumulation in WT, sic-1, se-1, and abh1-285 of Arabidopsis (WT, sic-1, se-1, abh1 = RT-PCR, DNA = Columbia genomic DNA was used as template for PCR). (D) Quantitative RT-PCR analysis of pri-miRNA accumulation in WT and sic-1 in seedlings.
Fig. 3.
Fig. 3.
Effect of sic-1 on the accumulation of spliced introns. Accumulation of mRNA and spliced intron of CAX1 (CATION EXCHANGER 1/RARE COLD INDUCIBLE 4) and CRB4 (CHLOROPLAST RNA BINDING) genes was determined by Northern analysis.
Fig. 4.
Fig. 4.
Colocalization of SIC and HYL1 in distinct nuclear bodies. (A) Nuclear localization of the SIC-sGFP. Confocal microscope images of root cells of SIC-sGFP transgenic Arabidopsis plants. GFP, GFP fluorescence; FM4-64, fluorescence of FM4-64-stained cells; Merged, GFP+FM4-64 superimposed images. (B) Colocalization on SIC and HYL1 in distinct nuclear bodies. DAPI, DAPI-stained nuclei; HYL1, immunolocalization of HYL1 with anti-HYL antibody; SIC-FLAG, immunolocalization of SIC-FLAG with anti-FLAG antibody; Merged, merged field images of DAPI, HYL1, and SIC-FLAG.
Fig. 5.
Fig. 5.
Abiotic stress responses of the sic-1 mutant plants. (A) Chilling stress response of WT and sic-1. Seeds were germinated and grown on MS medium supplemented with 3% sucrose at 22 °C for 3 wk (Upper) or at 4 °C for 5 wk (Lower). (B) WT and sic-1 seeds were germinated and grown in soil at 22 °C for 3 wk, then transferred to and grown at 4 °C for 2 mo. Inset: Top view of the sic-1 under cold stress. (C) Chilling stress response of sic-1, se-1, and abh1-285. (D) NaCl stress response of sic-1, se-1, and abh1-285. Six-day-old seedlings were transplanted on to MS medium supplemented with 3% sucrose (Left) or with 3% sucrose with 150 mM NaCl (Right) and grown for 3 wk.
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