No abstract

Summary DNA methylation is a conserved epigenetic mark in plants and mammals. In Arabidopsis, DNA methylation can be triggered by small interfering RNAs (siRNAs) through an RNA-directed DNA methylation (RdDM) pathway. Here we report the identification of a new RdDM effector, RDM3/KTF1. Loss-of-function mutations in RDM3/KTF1 reduce DNA methylation and release the silencing of RdDM target loci without abolishing the siRNA triggers. KTF1 has similarity to the transcription elongation factor SPT5 and contains a C-terminal extension rich in GW/WG repeats. KTF1 colocalizes with ARGONAUTE 4 (AGO4) in punctate nuclear foci, and binds AGO4 and RNA transcripts. Our results suggest KTF1 as an adaptor protein that binds scaffold transcripts generated by Pol V and recruits AGO4 and AGO4-bound siRNAs to form an RdDM effector complex. The dual interaction of an effector protein with AGO and small RNA target transcripts may be a general feature of RNA silencing effector complexes.

Glu receptors are known to function as Glu-activated ion channels that mediate mostly excitatory neurotransmission in animals. Glu receptor–like genes have also been reported in higher plants, although their function is largely unknown. We have identified a rice (Oryza sativa) Glu receptor–like gene, designated GLR3.1, in which mutation by T-DNA insertion caused a short-root mutant phenotype. Histology and DNA synthesis analyses revealed that the mutant root meristematic activity is distorted and is accompanied by enhanced programmed cell death. Our results supply genetic evidence that a plant Glu receptor–like gene, rice GLR3.1, is essential for the maintenance of cell division and individual cell survival in the root apical meristem at the early seedling stage.

RNA-directed DNA methylation (RdDM) is a conserved mechanism for epigenetic silencing of transposons and other repetitive elements. We report that the rdm4 (RNA-directed DNA Methylation4) mutation not only impairs RdDM, but also causes pleiotropic developmental defects in Arabidopsis. Both RNA polymerase II (Pol II)-and Pol V-dependent transcripts are affected in the rdm4 mutant. RDM4 encodes a novel protein that is conserved from yeast to humans and interacts with Pol II and Pol V in plants. Our results suggest that RDM4 functions in epigenetic regulation and plant development by serving as a transcriptional regulator for RNA Pol V and Pol II, respectively. DNA methylation and histone modifications are important epigenetic silencing mechanisms in eukaryotic cells (Chan et al. 2005;Matzke and Birchler 2005). In plants, DNA methylation is catalyzed by DNA methytransferase MET1, CMT3, and DRM2 (Chan et al. 2005). MET1 and CMT3 mainly function in maintaining DNA hypermethylation at CG and CHG sites (H is A, T, or C) during DNA duplication, while the function of DRM2 is involved in de novo DNA methylation at CHH sites directed by RNA (Chan et al. 2005). RNA-directed DNA methylation (RdDM) was first discovered in plants (Wassenegger et al. 1994), and plays important roles in transgene silencing, genome integrity, and transposon stability (Matzke et al. 2009).In the RdDM pathway, both 24-nucleotide (nt) siRNAs and long noncoding RNA transcripts are essential for de novo DNA methylation (Wierzbicki et al. 2008). The 24-nt siRNAs are generated in a pathway involving the putative DNA-directed RNA polymerase IV (Pol IV), RDR2 (RNA-dependent RNA polymerase 2), and DCL3 (Dicer-like RNA Pol IV and Pol V have distinct largest subunits-NRPD1 and NRPE1, respectively-but share some common subunits such as NRPD/E2 and NRPD/E4 (He et al. 2009b). Some subunits of Pol IV and Pol V are also shared with Pol II (Huang et al. 2009;Ream et al. 2009). It appears that the plant-specific RNA Pol IV and Pol V evolved from an ancestral RNA polymerase to function specifically in RdDM. The transcription activity of Pol II is tightly regulated with the help of a series of general transcription factors (Kornberg 2007). Although many general transcription factors of Pol II have been studied extensively, nothing is known about the regulation of transcription by Pol IV and Pol V.In the present study, we carried out a forward genetic screen for second site suppressors of the DNA demethylase mutant ros1, and identified a transcription factor, RDM4 (RNA-directed DNA Methylation4), that is required for RdDM in Arabidopsis. Interestingly, unlike other RdDM mutants, the rdm4 mutant plants display pleiotropic developmental phenotypes. RDM4 encodes a novel protein that is conserved in eukaryotic organisms. We found that RDM4 interacts with Pol II and Pol V in plants. Our results suggest that RDM4 is a regulator of Pol II and Pol V transcription, and thereby contributes to both development and RdDM. Results and DiscussionThe rdm4 mutation suppresses transcr...

Plant roots move through the soil by elongation. This is vital to their ability to anchor the plant and acquire water and minerals from the soil. In order to identify new genes involved in root elongation in rice, we screened an ethyl methane sulfonate (EMS)-mutagenized rice library, and isolated a short root mutant, Osglu3-1. The map-based cloning results showed that the mutant was due to a point mutation in OsGLU3, which encodes a putative membrane-bound endo-1,4-β-glucanase. Osglu3-1 displayed less crystalline cellulose content in its root cell wall, shorter root cell length, and a slightly smaller root meristem as visualized by restricted expression of OsCYCB1,1:GUS. Exogenous application of glucose can suppress both the lower root cell wall cellulose content and short root phenotypes of Osglu3-1. Consistently, OsGLU3 is ubiquitously expressed in various tissues with strong expression in root tip, lateral root, and crown root primodia. The fully functional OsGLU3-GFP was detected in plasma membrane, and FM4-64-labeled compartments in the root meristem and elongation zones. We also found that phosphate starvation, an environmental stress, altered cell wall cellulose content to modulate root elongation in a OsGLU3-dependant way.

The involvement of OsKASI in FA synthesis is found to play a critical role in root development of rice. The root system plays important roles in plant nutrient and water acquisition. However, mechanisms of root development and molecular regulation in rice are still poorly understood. Here, we characterized a rice (Oryza sativa L.) mutant with shortened roots due to a defect in cell elongation. Map-based cloning revealed that the mutation occurred in a putative 3-oxoacyl-synthase, an ortholog of β-ketoacyl-[acyl carrier protein] synthase I (KASI) in Arabidopsis, thus designated as OsKASI. OsKASI was found to be ubiquitously expressed in various tissues throughout the plant and OsKASI protein was localized in the plastid. In addition, OsKASI deficiency resulted in reduced fertility and a remarkable change in fatty acid (FA) composition and contents in roots and seeds. Our results demonstrate that involvement of OsKASI in FA synthesis is required for root development in rice.

Cytokinins play important roles in regulating plant development, including shoot and root meristems, leaf longevity, and grain yield. However, the in planta functions of rice cytokinin receptors have not been genetically characterized yet. Here we isolated a rice mutant, Osckt1, with enhanced tolerance to cytokinin treatment. Further analysis showed that Osckt1 was insensitive to aromatic cytokinins but responded normally to isoprenoid and phenylurea-type cytokinins. Map-based cloning revealed that the mutation occurred in a putative cytokinin receptor gene, histidine kinase 6 (OsHK6). OsCKT1 was found to be expressed in various tissues throughout the plant and the protein was located in the endoplasmic reticulum. In addition, whole-genome gene expression profiling analysis showed that OsCKT1 was involved in cytokinin regulation of a number of biological processes, including secondary metabolism, sucrose and starch metabolism, chlorophyll synthesis, and photosynthesis. Our results demonstrate that OsCKT1 plays important roles in cytokinin perception and control of root development in rice.

Partial sequences of the mitochondrial DNA cytochrome c oxidase subunit I gene (COI) of seven Tegillarca granosa populations, which were collected from China's coastal areas, were amplified by polymerase chain reaction (PCR). The length of COI gene of 38 Tegillarca granosa individuals from seven populations was all 660bp. One hundred and three variable sites were detected in the nucleotide sequences of 660 bp, and 17 different haplotypes were identified. The result showed that the seven populations could be divided into two groups based on the the genetic distance and phylogenetic analysis of their COI gene sequences. The two groups were classified as Group in the Northern Fujian (including Fujian) and Group in the Southern Fujian. Group in the Northern Fujian was composed of five populations and the genetic distance was 0.0016. Group in the Southern Fujian was composed of two populations and the genetic distance was 0.0006. However, the genetic distance between the two groups was significantly high (0.1529), which suggested significant genetic differentiation between the two groups. It suggested that Group in the North of Fujian (including Fujian) and Group in the South of Fujian should be the same species, but they were different subspecies.