5-Methylcytosine (mC) is a well-characterized DNA modification, and is also predominantly reported in abundant non-coding RNAs in both prokaryotes and eukaryotes. However, the distribution and biological functions of mC in plant mRNAs remain largely unknown. Here, we report transcriptome-wide profiling of RNA mC in Arabidopsis thaliana by applying mC RNA immunoprecipitation followed by a deep-sequencing approach (mC-RIP-seq). LC-MS/MS and dot blot analyses reveal a dynamic pattern of mC mRNA modification in various tissues and at different developmental stages. mC-RIP-seq analysis identified 6045 mC peaks in 4465 expressed genes in young seedlings. We found that mC is enriched in coding sequences with two peaks located immediately after start codons and before stop codons, and is associated with mRNAs with low translation activity. We further demonstrated that an RNA (cytosine-5)-methyltransferase, tRNA-specific methyltransferase 4B (TRM4B), exhibits mC RNA methyltransferase activity. Mutations in TRM4B display defects in root development and decreased mC peaks. TRM4B affects the transcript levels of the genes involved in root development, which is positively correlated with their mRNA stability and mC levels. Our results suggest that mC in mRNA is a new epitranscriptome marker inArabidopsis, and that regulation of this modification is an integral part of gene regulatory networks underlying plant development.

DNA methylation on N-adenine (6mA) has recently been found to be a potentially epigenetic mark in several unicellular and multicellular eukaryotes. However, its distribution patterns and potential functions in land plants, which are primary producers for most ecosystems, remain largely unknown. Here we report global profiling of 6mA sites at single-nucleotide resolution in the genome of Arabidopsis thaliana at different developmental stages using single-molecule real-time sequencing. 6mA sites are widely distributed across the Arabidopsis genome and enriched over the pericentromeric heterochromatin regions. 6mA occurs more frequently in gene bodies than intergenic regions. Analysis of 6mA methylomes and RNA sequencing data demonstrates that 6mA frequency positively correlates with the gene expression level and the transition from vegetative to reproductive growth in Arabidopsis. Our results uncover 6mA as a DNA mark associated with actively expressed genes in Arabidopsis, suggesting that 6mA serves as a hitherto unknown epigenetic mark in land plants.

N 6 -Methyladenine (6mA) DNA methylation has recently been implicated as a potential new epigenetic marker in eukaryotes, including the dicot model Arabidopsis thaliana. However, the conservation and divergence of 6mA distribution patterns and functions in plants remain elusive. Here we report high-quality 6mA methylomes at single-nucleotide resolution in rice based on substantially improved genome sequences of two rice cultivars, Nipponbare (Nip; Japonica) and 93-11 (Indica). Analysis of 6mA genomic distribution and its association with transcription suggest that 6mA distribution and function is rather conserved between rice and Arabidopsis. We found that 6mA levels are positively correlated with the expression of key stressrelated genes, which may be responsible for the difference in stress tolerance between Nip and 93-11. Moreover, we showed that mutations in DDM1 cause defects in plant growth and decreased 6mA level. Our results reveal that 6mA is a conserved DNA modification that is positively associated with gene expression and contributes to key agronomic traits in plants.

Although the multiple organellar RNA editing factors (MORFs) in the plastids of Arabidopsis thaliana have been extensively studied, molecular details underlying how MORFs affect plant development in other species, particularly in rice, remain largely unknown. Here we describe the characterization of wsp1, a rice mutant with white-stripe leaves and panicles. Notably, wsp1 exhibited nearly white immature panicles at the heading stage. Transmission electron microscopy analysis and chlorophyll content measurement revealed a chloroplast developmental defect and reduced chlorophyll accumulation in wsp1. Positional cloning of WSP1 found a point mutation in Os04g51280, whose putative product shares high sequence similarity with MORF proteins. Complementation experiments demonstrated that WSP1 was responsible for the variegated phenotypes of wsp1. WSP1 is localized to chloroplasts and the point mutation in wsp1 affected the editing of multiple organellar RNA sites. Owing to the defect in plastid RNA editing, chloroplast ribosome biogenesis and ndhA splicing were also impaired in wsp1, which may affect normal chloroplast development in the leaves and panicles at the heading stage. Together, our results demonstrate the importance of rice WSP1 protein in chloroplast development and broaden our knowledge about MORF family members in rice.

Summary Low temperature stress hinders plant growth and chloroplast development and can limit the geographic range of cultivars. In rice, japonica cultivars have greater chilling tolerance than indica cultivars, but the molecular mechanism underlying chilling tolerance is unclear. Here, we report an RNA‐binding protein, DUA1, cloned from the indica cultivar Dular, which exhibits a deficiency in chloroplast development at an early stage of development under low‐temperature conditions. DUA1 shares high sequence homology with the pentatricopeptide repeat family and functions in plastid RNA editing under low‐temperature conditions. Our data suggest that DUA1 can bind to the plastid‐encoded rps8‐182 transcript and disruption of DUA1 activity impairs editing. The RNA editing cofactor WSP1, a partner of DUA1, also participates in chloroplast development at low temperature. Western blot analysis indicates that WSP1 enhances DUA1 stability under low temperatures. DUA1 sequence analyses of rice core germplasm revealed that three major haplotypes of DUA1 and one haplotype showed substantial differences in chlorophyll content under low‐temperature conditions. Variation at DUA1 may play an important role in the adaptation of rice to different growing regions.

No abstract

The morphology of bulliform cells located on the upper epidermis of leaves is one of the most important cell structures affecting leaf shape. Although many mechanisms regulating the development of bulliform cells have been reported, the fine regulatory mechanisms governing this process have rarely been described. To identify novel components regulating rice leaf morphology, a mutant showing a constitutively rolling phenotype from the seedling stage to flowering, known as crm1-D, was selected for further analysis. Anatomical analyses in crm1-D were attributable to the size reduction of bulliform cells. The crm1-D was controlled by a single dominant nuclear gene. Map-based cloning revealed that Roc8, an HD zipper class IV family member, was responsible for the crm1-D phenotype. Notably, the 50-bp sequence in the 3 0untranslated region (3 0-UTR) of the Roc8 gene represses Roc8 at the translational level. Moreover, the roc8 knockdown lines notably increased the size of bulliform cells. A series of assays revealed that Roc8 negatively regulates the size of bulliform cells. Unexpectedly, Roc8 was also observed to positively mediate lignin biosynthesis without incurring a production penalty. The above results show that Roc8 may have a practical application in cultivating materials with high photosynthetic efficiency and low lignin content.

Rice is a facultative short-day plant (SDP), and the regulatory pathways for flowering time are conserved, but functionally modified, in Arabidopsis and rice. Heading date 1 (Hd1), an ortholog of Arabidopsis CONSTANS (CO), is a key regulator that suppresses flowering under long-day conditions (LDs), but promotes flowering under short-day conditions (SDs) by influencing the expression of the florigen gene Heading date 3a (Hd3a). Another key regulator, Early heading date 1 (Ehd1), is an evolutionarily unique gene with no orthologs in Arabidopsis, which acts as a flowering activator under both SD and LD by promoting the rice florigen genes Hd3a and RICE FLOWERING LOCUST 1 (RFT1). Here, we report the isolation and characterization of the flowering regulator Heading Date Repressor1 (HDR1) in rice. The hdr1 mutant exhibits an early flowering phenotype under natural LD in a paddy field in Beijing, China (39°54'N, 116°23'E), as well as under LD but not SD in a growth chamber, indicating that HDR1 may functionally regulate flowering time via the photoperiod-dependent pathway. HDR1 encodes a nuclear protein that is most active in leaves and floral organs and exhibits a typical diurnal expression pattern. We determined that HDR1 is a novel suppressor of flowering that upregulates Hd1 and downregulates Ehd1, leading to the downregulation of Hd3a and RFT1 under LDs. We have further identified an HDR1-interacting kinase, OsK4, another suppressor of rice flowering under LDs. OsK4 acts similarly to HDR1, suppressing flowering by upregulating Hd1 and downregulating Ehd1 under LDs, and OsK4 can phosphorylate HD1 with HDR1 presents. These results collectively reveal the transcriptional regulators of Hd1 for the day-length-dependent control of flowering time in rice.