In a genetic screen for Arabidopsis mutants displaying pleiotropic alterations in vegetative development and stress responses we have identified a T-DNA insertion mutation in the Cap Binding Protein 20 (CBP20) gene, that encodes the 20kDa subunit of the nuclear mRNA cap binding complex (nCBC). Plants homozygous for the recessive cbp20 mutation show mild developmental abnormalities, such as serrated rosette leaves, delayed development and slightly reduced stature. Loss of the cbp20 function also confers hypersensitivity to abscisic acid during germination, significant reduction of stomatal conductance and greatly enhanced tolerance to drought. Expression of the wild type cDNA by CaMV35S promoter provides full genetic complementation of the pleiotropic cbp20 phenotype. Phenotypic characteristics of the cbp20 mutant are very similar to those of recently described abh1 mutant that is defective in the 80kDa subunit of nCBC. Our data thus confirm that both genes are dedicated to the same function. CBP20 provides a new target for breeding efforts that aim at the improvement of drought tolerance in plants. Our results also show that screening for pleiotropic phenotypes in mutant plant populations may be a fruitful strategy to isolate genes for agronomically important traits.
RNA interference (RNAi) is mediated by small, 20-24-nt-long, non-coding regulatory (s)RNAs such as micro (mi) and small interfering (si) RNAs via the action of ARGONAUTE (AGO) proteins. High-throughput sequencing of size-separated sRNA pools of plant crude extracts revealed that the majority of the canonical miRNAs were associated with high molecular weight RNA-induced silencing complexes co-migrating with AGO1 (HMW RISC). In contrast, the majority of 24-nt-long siRNAs were found in association with low molecular weight complexes co-migrating with AGO4 (LMW RISC). Intriguingly, we identified a large set of cytoplasmic sRNAs, including mature miRNA sequences, in the low molecular size range corresponding to protein-unbound sRNAs. By comparing the RISC-loaded and protein-unbound pools of miRNAs, we identified miRNAs with highly different loading efficiencies. Expression of selected miRNAs in transient and transgenic systems validated their altered loading abilities implying that this process is controlled by information associated with the diverse miRNA precursors. We also showed that the availability of AGO proteins is a limiting factor determining the loading efficiency of miRNAs. Our data reveal the existence of a regulatory checkpoint determining the RISC-loading efficiencies of various miRNAs by sorting only a subset of the produced miRNAs into the biologically active RISCs.
Barley (Hordeum vulgare L.) is an economically important crop cultivated in temperate climates all over the world. Adverse environmental factors negatively affect its survival and productivity. RNA silencing is a conserved pathway involved in the regulation of growth, development and stress responses. The key components of RNA silencing are the Dicer-like proteins (DCLs), Argonautes (AGOs) and RNA-dependent RNA polymerases (RDRs). Despite its economic importance, there is no available comprehensive report on barley RNA silencing machinery and its regulation. In this study, we in silico identified five DCL (HvDCL), eleven AGO (HvAGO) and seven RDR (HvRDR) genes in the barley genome. Genomic localization, phylogenetic analysis, domain organization and functional/catalytic motif identification were also performed. To understand the regulation of RNA silencing, we experimentally analysed the transcriptional changes in response to moderate, persistent or gradient heat stress treatments: transcriptional accumulation of siRNA- but not miRNA-based silencing factor was consistently detected. These results suggest that RNA silencing is dynamically regulated and may be involved in the coordination of development and environmental adaptation in barley. In summary, our work provides information about barley RNA silencing components and will be a ground for the selection of candidate factors and in-depth functional/mechanistic analyses.
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