The dicing activity of DCL3 and DCL4 is negatively affected by flavonoids.

KEY MESSAGE: The dicing activities of DCL3 and DCL4 are inhibited by accumulated metabolites in soybean leaves. Epicatechin and 7,4'-dihydroxyflavone inhibited Arabidopsis DCL3 and DCL4 in vitro. Flavonoids are major secondary metabolites in plants, and soybean (Glycine max L.) is a representative plant that accumulates flavonoids, including isoflavonoids, to high levels. Naturally-occurring RNA interference (RNAi) against the chalcone synthase (CHS) gene represses flavonoid (anthocyanin) biosynthesis in an organ-specific manner, resulting in a colorless (yellow) seed coat in many soybean cultivars. To better understand seed coat-specific naturally-occurring RNAi in soybean, we characterized soybean Dicer-like (DCL) 3 and 4, which play critical roles in RNAi. Using a previously established dicing assay, two dicing activities producing 24- and 21-nt siRNAs, corresponding to DCL3 and DCL4, respectively, were detected in soybean. Dicing activity was detected in colorless seed coats where RNAi against CHS genes was found, but no dicing activity was detected in leaves where CHS expression was prevalent. Biochemical analysis revealed that soybean leaves contained two types of inhibitors effective for Arabidopsis Dicers (AtDCL3 and AtDCL4), one of which was a heat-labile high molecular weight compound of 50 to 100 kD while another was a low molecular weight substance. We found that some flavonoids, such as epicatechin and 7,4'-dihydroxyflavone, inhibited both AtDCL3 and AtDCL4, but AtDCL4 was more sensitive to these flavonoids than AtDCL3. These results suggest that flavonoids inhibit the dicing activity of DCL4 and thereby attenuate RNAi in soybean leaves.

Post-Translational Regulation of the Dicing Activities of Arabidopsis DICER-LIKE 3 and 4 by Inorganic Phosphate and the Redox State.

In Arabidopsis thaliana, small interfering RNAs (siRNAs) generated by two Dicer isoforms, DCL3 and DCL4, function in distinct epigenetic processes, i.e. RNA-directed DNA methylation and post-transcriptional gene silencing, respectively. Plants often respond to their environment by producing a distinct set of small RNAs; however, the mechanism for controlling the production of different siRNAs from the same dsRNA substrate remains unclear. We established a simple biochemical method to visualize the dsRNA-cleaving activities of DCL3 and DCL4 in cell-free extracts prepared from Arabidopsis seedlings. Here, we demonstrate that different nutrient statuses of a host plant affect the post-translational regulation of the dicing activity of DCL3 and DCL4. Phosphate deficiency inhibited DCL3, and the activity of DCL3 was directly activated by inorganic phosphate. Sulfur deficiency inhibited DCL4 but not DCL3, and the activity of DCL4 was recovered by supplementation of the cell-free extracts with reductants containing a thiol group. Immunopurified DCL4 was activated by recombinant Arabidopsis thioredoxin-h1 with dithiothreitol. Therefore, DCL4 is subject to redox regulation. These results demonstrate that post-translational regulation of DCL activities fine-tunes the balance between branches of the gene silencing pathway according to the growth environment.

Regulation of a maize HD-ZIP IV transcription factor by a non-conventional RDR2-dependent small RNA.

Small non-coding RNAs are versatile riboregulators that control gene expression at the transcriptional or post-transcriptional level, governing many facets of plant development. Here we present evidence for the existence of a 24 nt small RNA (named small1) that is complementary to the 3' UTR of OCL1 (Outer Cell Layer1), the founding member of the maize HD-ZIP IV gene family encoding plant-specific transcription factors that are mainly involved in epidermis differentiation and specialization. The biogenesis of small1 depends on DICER-like 3 (DCL3), RNA-dependent RNA polymerase 2 (RDR2) and RNA polymerase IV, components that are usually required for RNA-dependent DNA-methylation. Unexpectedly, GFP sensor experiments in transient and stable transformation systems revealed that small1 may regulate its target at the post-transcriptional level, mainly through translational repression. This translational repression is attenuated in an rdr2 mutant background in which small1 does not accumulate. Our experiments further showed the possible involvement of a secondary stem-loop structure present in the 3' UTR of OCL1 for efficient target repression, suggesting the existence of several regulatory mechanisms affecting OCL1 mRNA stability and translation.

The RNA-binding domain of DCL3 is required for long-distance RNAi signaling.

Small RNA (sRNA)-mediated RNA silencing (also known as RNA interference, or RNAi) is a conserved mechanism in eukaryotes that includes RNA degradation, DNA methylation, heterochromatin formation and protein translation repression. In plants, sRNAs can move either cell-to-cell or systemically, thereby acting as mobile silencing signals to trigger noncell autonomous silencing. However, whether and what proteins are also involved in noncell autonomous silencing have not been elucidated. In this study, we utilized a previously reported inducible RNAi plant, PDSi, which can induce systemic silencing of the endogenous PDS gene, and we demonstrated that DCL3 is involved in systemic PDS silencing through its RNA binding activity. We confirmed that the C-terminus of DCL3, including the predicted RNA-binding domain, is capable of binding short RNAs. Mutations affecting RNA binding, but not processing activity, reduced systemic PDS silencing, indicating that DCL3 binding to RNAs is required for the induction of systemic silencing. Cucumber mosaic virus infection assays showed that the RNA-binding activity of DCL3 is required for antiviral RNAi in systemically noninoculated leaves. Our findings demonstrate that DCL3 acts as a signaling agent involved in noncell autonomous silencing and an antiviral effect in addition to its previously known function in the generation of 24-nucleotide sRNAs. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-023-00124-6.

Chemical shift assignment of dsRBD1 and dsRBD2 of Arabidopsis thaliana DRB3, an essential protein involved in RNAi-mediated antiviral defense.

As sessile organisms, plants need to counteract different biotic and abiotic stresses to survive. RNA interference provides natural immunity against various plant pathogens, especially against viral infections via inhibition of viral genome replication or translation. In plants, DRB3, a multi-domain protein containing two N-terminal dsRNA binding domains (dsRBD), plays a vital role in RNA-directed DNA methylation of the geminiviral genome. Additionally, DRB3 arrests the replication of the viral genome in the viral replication complex of RNA viruses through a mechanism that has yet to be fully deciphered. Therefore, as a first step towards exploring the structural details of DRB3, we present a nearly complete backbone and side chain assignment of the two N-terminal dsRBD domains.

Transcriptome-wide identification and functional investigation of the RDR2- and DCL3-dependent small RNAs encoded by long non-coding RNAs in Arabidopsis thaliana.

The involvement of the long non-coding RNAs (lncRNAs) in small RNA (sRNA)-related pathways remains elusive. Taking advantage of the public sRNA sequencing data, we searched for RNA-dependent RNA polymerase 2 (RDR2)- and Dicer-like 3 (DCL3)-dependent sRNAs generated from the lncRNAs of Arabidopsis thaliana. First, 55,162 sRNAs were identified to be RDR2- and DCL3-dependent. These sRNAs were then mapped onto the lncRNAs. As a result, a total of 26,643 sRNAs found their loci on 3,834 lncRNAs, and 29,388 sRNAs found their loci on 4,174 reverse complementary (RC) sequences of the lncRNAs. To support the formation of the double-stranded precursors for sRNA generation, double-stranded RNA sequencing (dsRNA-seq) reads were mapped onto the sense and antisense strands of the lncRNAs with RDR2- and DCL3-dependent sRNA loci. As a result, 1,075 regions longer than 100 nt were identified to be covered by dsRNA-seq reads on 390 sense strands of the lncRNAs, and 1,352 regions were identified on 544 RC strands. Besides, 2,238 out of 3,211 dsRNA-seq read-covered sRNA loci were supported by degradome sequencing data on the sense strands of the lncRNAs. Interestingly, dozens of dsRNA-seq read-covered regions with AGO4-associated sRNA loci showed site-specific chromatin modification patterns. Thus, some of the lncRNAs were integrated into the RDR2- and DCL3-dependent sRNA biogenesis pathway. Moreover, our results indicated that the site-specific chromatin modifications mediated by the AGO4-associated sRNAs might play a regulatory role on the transcription activity of the lncRNA genes.

Generation and characterization of a tomato DCL3-silencing mutant.

DICER-like 3 (DCL3) is a major player in heterochromatic 24-nucleotide (nt) small RNA (sRNA) and long microRNA (lmiRNA) biogenesis, and higher plant DCL3 mutants have been characterized from Arabidopsis thaliana and rice. Here, a tomato DCL3 (SlDCL3) mutant was generated through the use of trans-activated artificial miRNA and characterized. Constitutive trans-activation knocked down SlDCL3 levels by ∼64%, resulting in dramatically decreased 24-nt sRNA levels and a significant increase in 21- and 22-nt sRNAs. The latter was correlated with specific upregulation of SlDCL4 and SlDCL2b, which function in the biogenesis of 21- and 22-nt sRNAs, respectively. Moreover, at the majority of sRNA-generating genomic loci, an almost complete overlap between small RNA signatures of control and silenced seedlings was observed, suggesting that the reductions in 24-nt sRNAs at these loci were compensated for by biogenesis of 21- and 22-nt sRNAs from the same double-stranded RNA substrates. In addition, bioinformatic analysis and reduced expression in SlDCL3-silenced seedlings identified four novel tomato lmiRNAs, two of which were found to be developmentally regulated. Taken together, these results establish the requirement of SlDCL3 for the biogenesis of 24-nt sRNAs and lmiRNAs in tomato and suggest SlDCL4 and SlDCL2b as surrogates for SlDCL3.

Transgenerational phenotypic and epigenetic changes in response to heat stress in Arabidopsis thaliana.

Exposure to heat stress causes physiological and epigenetic changes in plants, which may also be altered in the progeny. We compared the progeny of stressed and control Arabidopsis thaliana wild type and Dicer-like mutant dcl2, dcl3, and dcl4 plants for variations in physiology and molecular profile, including global genome methylation, mRNA levels, and histone modifications in the subset of differentially expressed genes at normal conditions and in response to heat stress. We found that the immediate progeny of heat-stressed plants had fewer, but larger leaves, and tended to bolt earlier. Transposon expression was elevated in the progeny of heat-stressed plants, and heat stress in the same generation tended to decrease global genome methylation. Progeny of stressed plants had increased expression of HSFA2, and reduction in MSH2, ROS1, and several SUVH genes. Gene expression positively correlated with permissive histone marks and negatively correlated with repressive marks. Overall, the progeny of heat stressed plants varied in both their physiology and epigenome and dcl2 and dcl3 mutants were partially deficient for these changes.