DNA methylation is an important epigenetic mark in many eukaryotes1-5. In plants, 24-nt small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4) can direct de novo DNA methylation by the methyltransferase DRM22,4-6. Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nt siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that appears to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II, AGO4 and DRM2 in the nucleus. Our results suggest that RDM1 is a component of the RdDM effector complex and may play a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also suggest that although RDM1 and Pol V may function together at some RdDM target sites in the peri-nucleolar siRNA processing center, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm.

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.cold stress | hydroxyproline-rich glycoprotein | intron decay | mRNA stability M icroRNAs (miRNAs) are a class of endogenous small RNAs that function in gene regulation by guiding mRNA cleavage and translational repression and are critical for plant development and stress responses (1-9). The core components involved in miRNA biogenesis have been identified in plants. RNA polymerase II transcribes MIR genes; a 5′ 7-methyl guanosine cap and a 3′ poly(A) tail are added to produce primary miRNA (primiRNA) transcripts, which form imperfect stem-loop secondary structures by Watson-Crick base pairing between self-complementary foldback regions. In the Arabidopsis nucleus, the stemloop structure of the pri-miRNA is processed by the RNase III enzyme DICER-LIKE1 (DCL1) to produce a pre-miRNA, which is further processed to generate a 21-nt-long miRNA/miRNA* duplex. For accurate dicing, DCL1 requires the help of HYPO-NASTIC LEAVES1 (HYL1, a dsRNA-binding protein) and SERRATE (SE, a C2H2zinc-finger protein) (10). The HUA EN-HANCER 1 (HEN1) methyltransferase catalyzes 2'-O-methylation of the ribose sugar in the 3′ termini of miRNA/miRNA* duplexes (11). HASTY (HST), a homolog of mammalian EXPORTIN 5, helps export methylated miRNA/miRNA* duplexes from the nucleus to the cytosol (12). The mature miRNA is incorporated into ARGONAUTE1 (AGO1), forming an RNA-induced silencing complex, which scans for miRNA-complementary mRNAs and directs the cleavage or translational repression of the target mRNAs (1). miR173 and miRNA390 direct the biogenesis of transacting siRNAs (ta-siRNAs). Noncoding transcripts from TRANS-ACT-ING siRNA genes (TAS) are cleaved by the miRNA-containing AGO1/AGO7 complex (13, 14). The cleaved transcripts are converted into dsRNA by RDR6, and these dsRNAs are processed by DCL4 to yield ∼21-nt ta-siRNAs. Like miRNAs, tasiRNAs negatively regulate gene expression posttranscriptionally (15-18).N...

The phytohormone abscisic acid (ABA) is important for growth, development and stress responses in plants. Recent research has identified ABA receptors and signalling components that regulate seed germination and stomatal closure. However, proteins that regulate ABA signalling remain poorly understood. Here we use a forward-genetic screen to identify rbm25-1 and rbm25-2, two Arabidopsis mutants with increased sensitivity to growth inhibition by ABA. Using RNA-seq, we found that RBM25 controls the splicing of many pre-mRNAs. The protein phosphatase 2C HAB1, a critical component in ABA signalling, shows a dramatic defect in pre-mRNA splicing in rbm25 mutants. Ectopic expression of a HAB1 complementary DNA derived from wild-type mRNAs partially suppresses the rbm25-2 mutant phenotype. We suggest that RNA splicing is of particular importance for plant response to ABA and that the splicing factor RBM25 has a critical role in this response.

We report the identification and characterization of an Arabidopsis mutant, hos10-1 (for high expression of osmotically responsive genes), in which the expression of RD29A and other stress-responsive genes is activated to higher levels or more rapidly activated than in wild-type by low temperature, exogenous abscisic acid (ABA), or salt stress (NaCl). The hos10-1 plants are extremely sensitive to freezing temperatures, completely unable to acclimate to the cold, and are hypersensitive to NaCl. Induction of NCED3 (the gene that encodes the rate-limiting enzyme in ABA biosynthesis) by polyethylene glycol-mediated dehydration and ABA accumulation are reduced by this mutation. Detached shoots from the mutant plants display an increased transpiration rate compared with wild-type plants. The hos10-1 plants exhibit several developmental alterations, such as reduced size, early flowering, and reduced fertility. The HOS10 gene encodes a putative R2R3-type MYB transcription factor that is localized to the nucleus. Together, these results indicate that HOS10 is an important coordinating factor for responses to abiotic stress and for growth and development

The advent of clustered regularly interspaced short palindromic repeat (CRISPR) has had a profound impact on plant biology, and crop improvement. In this review, we summarize the state-of-the-art development of CRISPR technologies and their applications in plants, from the initial introduction of random small indel (insertion or deletion) mutations at target genomic loci to precision editing such as base editing, prime editing and gene targeting. We describe advances in the use of class 2, types II, V, and VI systems for gene disruption as well as for precise sequence alterations, gene transcription, and epigenome control.

In this study, melatonin (MEL)‐mediated plant resistance to tobacco mosaic virus (TMV) was examined to study local infection in Nicotiana glutinosa and systemic infection in Solanum lycopersicum. Exogenous application of 100 µm MEL increased anti‐virus infection activity to 37.4% in virus‐infected N. glutinosa plants. The same treatment significantly reduced relative levels of virus RNA analysed by qRT‐PCR and virus titres measured by dot‐ELISA, and increased the relative expression levels of the PR1 and PR5 genes analysed by qRT‐PCR, in virus‐infected S. lycopersicum. MEL treatment induced considerable accumulations of salicylic acid (SA) and nitric oxide (NO) but did not significantly affect production of hydrogen peroxide (H2O2) in the virus‐infected S. lycopersicum plants. Transgenic nahG N. tabacum was used to determine whether MEL‐induced TMV resistance was dependent on the SA pathway. The results showed that the relative RNA level of the TMV analysed by qRT‐PCR and virus titres analysed by dot‐ELISA were not reduced by the MEL treatment in the nahG transgenic N. tabacum seedlings treated twice with 100 µm MEL. The increased relative expression levels of PR1 and PR5 were greatly reduced when cPTIO, an NO scavenger, was included in the MEL treatment. A working model of MEL‐mediated plant resistance to TMV is proposed. MEL‐mediated plant resistance to viruses provides a new avenue to control plant viral diseases.

Low temperature is a major stress that severely affects plant development, growth, distribution, and productivity. Here, we examined the function of a 2-oxoglutarate-dependent dioxygenase-encoding gene, SlF3HL , in chilling stress responses in tomato ( Solanum lycopersicum cv. Alisa Craig [AC]). Knockdown (KD) of SlF3HL (through RNA interference) in tomato led to increased sensitivity to chilling stress as indicated by elevated levels of electrolyte leakage, malondialdehyde (MDA) and reactive oxygen species (ROS). In addition, the KD plants had decreased levels of proline and decreased activities of peroxisome and superoxide dismutase. The expression of four cold-responsive genes was substantially reduced in the KD plants. Furthermore, seedling growth was significantly greater in AC or SlF3HL -overexpression plants than in the KD plants under either normal growth conditions with methyl jasmonate (MeJA) or chilling stress conditions. SlF3HL appears to positively regulate JA accumulation and the expression of JA biosynthetic and signaling genes under chilling stress. Together, these results suggest that SlF3HL is a positive regulator of chilling stress tolerance and functions in the chilling stress tolerance pathways, possibly by regulating JA biosynthesis, JA signaling, and ROS levels.

GAMYBs are positive GA signaling factors that exhibit essential functions in reproductive development, particularly in anther and pollen development. However, there is no direct evidence of the regulation of any GAMYB in these biological processes in tomato (Solanum lycopersicum). Here, we identified a tomato GAMYB-like gene, SlMYB33, and characterized its specific roles. SlMYB33 is predominately expressed in the stamens and pistils. During flower development, high mRNA abundance of SlMYB33 is detected in both male and female organs, such as microspore mother cells, anthers, pollen grains, and ovules. Silencing of SlMYB33 leads to delayed flowering, aberrant pollen viability, and poor fertility in tomato. Histological analyses indicate that SlMYB33 exerts its function in pollen development in the mature stage. Further transcriptomic analyses imply that the knockdown of SlMYB33 significantly inhibits the expression of genes related to flowering in shoot apices, and alters the transcription of genes controlling sugar metabolism in anthers. Taken together, our study suggests that SlMYB33 regulates tomato flowering and pollen maturity, probably by modulating the expression of genes responsible for flowering and sugar metabolism, respectively.