| Summary: | 博士 === 國立中興大學 === 生物科技學研究所 === 104 === Chapter 1: AtRRP6L1, a homolog of conserved yeast exosomal Rrp6p, plays an important role in transcriptional gene silencing in Arabidopsis
RNA-directed DNA methylation (RdDM), the conserved de novo DNA methylation pathway in Arabidopsis, mediates epigenetic silencing of genes, transposons, and other repetitive elements. From genetic screening of thousands of T-DNA insertion lines of Arabidopsis, besides two known components (DTF1 and IDN2) of the RdDM pathway, a new regulator (AtRRP6L1) of DNA methylation was discovered, of which mutations reduced DNA methylation in a subset of RdDM target loci. The AtRRP6L1 (At1g54440) is a homolog to yeast Rrp6p. The phenotypes of atrrp6l1 and the DNA methylation levels at AtSN1 locus were recovered by complementation with 35S::AtRRP6L1 in atrrp6l1. Loss-of-function in AtRRP6L1 released the transcriptional gene silencing (TGS) of the RD29A-LUC transgene and endogenous RD29A, demonstrating the ability of AtRRP6L1 in regulating gene expression of RdDM target loci. DNA methylation of MEA-ISR was lower in rrp6l1-1 by bisulfite sequencing. Similarly, the levels of the HaeIII-digested rrp6l1-1 DNA fragments were also decreased at IGN5 by chop-PCR analysis, indicating the methylation levels at IGN5 were reduced. Quantitative real time-PCR analysis showed that the AtSN1and IG2 transcripts in rrp6l1-1 were significantly increased while AtMU1 transcript remained unaffected. The atrrp6l1 mutations also led to release of TGS from an endogenous RdDM locus, solo LTR, without affecting the DNA methylation status. Therefore, AtRRP6L1 exhibits both DNA methylation-dependent and -independent roles in TGS. In summary, the results demonstrate that AtRRP6L1 acts as an important epigenetic regulator through different silencing mechanisms.
Chapter 2: A WD40 protein, AtGHS40, negatively modulates abscisic acid degrading and signaling genes and may affect pre-ribosomal RNA processing during seedling growth under high glucose conditions
Sugars are not only as the primary source of fuel and energy but also play pivotal roles as signaling molecules for plants growth. We have screened thousands of T-DNA insertion lines to identify a glucose hypersensitive (ghs) 40-1 mutant in the presence of 3% glucose (Glc). The ghs40-1 mutant displayed severely impaired cotyledon greening and expansion and showed enhanced reduction in hypocotyl elongation of dark-grown seedlings when grown in Glc concentrations higher than 3%. The Glc-hypersensitivity of ghs40-1 was correlated with the hyposensitive phenotype of 35S::AtGHS40 seedlings. The impaired phenotypes of ghs40-1 were recovered by complementation with 35S::AtGHS40 in ghs40-1. The AtGHS40 (At5g11240) gene encodes a WD40 protein localized primarily in the nucleus and nucleolus using transient expression of AtGHS40-RFP in onion cells and of AtGHS40-EGFP and EGFP-AtGHS40 in Arabidopsis protoplasts. AtGHS40 was expressed in all Arabidopsis organs and its transcript was significantly induced by ABA, high Glc, high salt and drought. The ABA biosynthesis inhibitor fluridone markedly recovered seedling growth arrest under high Glc conditions, suggesting that the Glc hypersensitivity of ghs40-1 mainly functions through ABA accumulation, but not affects ethylene signaling. Quantitative real time-PCR (qRT-PCR) analysis showed that APL3 and CHS were significantly induced in ghs40-1 under 4.5% Glc conditions, whereas high Glc greatly repressed CAB1, PC2 and ASN1. AtGHS40 acts downstream of HXK1 and is activated by ABI4 while ABI4 expression is negatively modulated by AtGHS40 in the Glc signaling network. However, AtGHS40 may not affect ABI1 and SnRK2.6 gene expression. Given that AtGHS40 inhibited ABA degrading and signaling gene expression levels under high Glc conditions, a new circuit of fine-tuning modulation by which ABA and ABA signaling gene expression are modulated in balance, occurred in plants. The abnormal pre-rRNA precursors increased significantly in ghs40-1 in the absence/presence of 4.5% Glc, suggesting that the mutation of AtGHS40 affects ribosomal biogenesis. High Glc addition only also affects ribosomal biogenesis. The ghs40-1 mutant showed low germination percentage with the addition of a protein synthesis inhibitor, cycloheximide. The ghs40-1 exhibited better resistance to streptomycin and spectinomycin than WT, suggesting that mutation of AtGHS40 might affects 40S ribosome. High Glc conditions only may also affect 40S ribosome. AtGHS40 might involve in ABA-mediated Glc signaling during early seedling development and play a crucial role in rRNA biogenesis in Arabidopsis in response to sugar.
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