Unknown,Transcriptomics,Genomics,Proteomics

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Global and local perturbation of the tomato microRNA pathway by a trans-activated DICER-LIKE 1 mutant

ABSTRACT: DICER-like 1 (DCL1) is a major player in micro RNA (miRNA) biogenesis and accordingly, its few known loss-of-function mutants are either lethal or display arrested seedling development. Consequently, generation of dcl1 mutants by reverse genetics and functional analysis of DCL1 in late-developing organs are challenging. Here, these challenges were resolved through the unique use of trans-activated RNA interference. Global, as well as organ-specific tomato DCL1 (SlDCL1) silencing was induced by crossing the generated responder line (OP:SlDCL1IR) with the appropriate driver line. Constitutive trans-activation knocked down SlDCL1 levels by ~95%, resulting in severe abnormalities including post-germination growth arrest accompanied by decreased miRNA and 21-nucleotide small RNA levels, but prominently elevated levels of 22- nucleotide small RNAs. The elevation in the 22- nucleotide small RNAs was correlated with specific upregulation of SlDCL2b and SlDCL2d, which are likely involved in their biogenesis. Leaf- and flower-specific OP:SlDCL1IR trans-activation inhibited blade outgrowth, induced premature bud senescence and produced pale petals, respectively, emphasizing the importance of SlDCL1-dependent small RNAs in these processes. Together, our results establish OP:SlDCL1IR as an efficient tool for analyzing processes regulated by SlDCL1-mediated gene regulation in tomato. Examination of small RNA populations in control (35S:LhG4) and SlDCL1 silenced seedlings (35S>>SlDCL1IR) 2 biological replicates each

ORGANISM(S): Solanum lycopersicum

SUBMITTER: Tzahi Arazi

PROVIDER: E-GEOD-51959 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Abscisic acid (ABA) regulates plant development and adaptation to environmental conditions. The ABA biosynthesis pathway in plants has been thoroughly elucidated; however, very few transcription factors directly regulating the expression of ABA biosynthetic genes have been identified. Here we show that the tomato (Solanum lycopersicum) zinc finger transcription factor SlZFP2, which is mainly expressed in developing fruits and axillary buds, negatively regulates ABA biosynthesis. Overexpression of SlZFP2 resulted in multiple phenotypic changes, including more branches, early flowering, delayed fruit ripening, lighter seeds and faster seed germination, whereas gene silencing by RNA interference (RNAi) caused poor fruit set and inhibited seed germination. Gene expression analysis showed that SlZFP2 represses ABA biosynthesis mainly through downregulation of the ABA biosynthetic genes SITIENS (SIT), FLACCA (FLC) and aldehyde oxidase SlAO1. SlZFP2 delays the onset of ripening through suppression of the ripening regulator COLORLESS NON-RIPENING (CNR). Using bacterial one hybrid screening and a selected amplification and binding assay we identified the (A/T)(G/C)TT repeat as the core binding sequence of SlZFP2. We further identified a large number of tomato genes containing putative SlZFP2 binding sites in their promoter regions. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that SIT, FLC and SlAO1 are direct targets of SlZFP2 through binding to their promoter regions. We propose that SlZFP2 represents a novel negative regulator for fine tuning ABA biosynthesis during fruit development and provides a potentially valuable tool for dissecting the role of ABA in fruit ripening.To gain further insight on transcriptome changes regulated by SlZFP2, we sequenced a representative SlZFP2 RNAi line in LA1589 background and its nontransgenic sibling (WT) on a Miseq platform. The RNAi line 207 showed defected fruit set and ABA biosynthesis were chosen for profiling gene expression via RNA sequencing. Its nontransgenic sibling was served as controls. Three biological replicates were conducted.

Project description:Backgropund:In a major paradigm shift in the last decade, the knowledge about a whole class of non-coding RNAs known as miRNAs has emerged, which have proved these to be important regulators of a wide range of cellular processes by the way of modulation of gene expression. It is reported that some of these miRNAs are modified by addition or deletion of nucleotides at their ends, after biogenesis. However, the biogenesis and functions of these modifications are not well studied in eukaryotes, especially in plants. In this study, we examined the miRNA modifications in different tissues of the various plants, namely rice, tomato and Arabidopsis and identified some common features of such modifications. Results:We have analyzed different aspects of miRNA modifications in plants. To achieve this end, we developed a PERL script to find the modifications in the sequences using small RNA deep sequencing data. The modification occurs in both mature and passenger (star) strands, as well as at both the 5' and 3' ends of miRNAs. Interestingly, we found a position-specific nucleotide biased modification, as evident by increased number of modification at the 5' end with the presence of Cytosine (nucleotide 'C') at the 3âend of the miRNA sequence. The level of modifications is not strictly dependent on the abundance of miRNA. Our study showed that the modification events are independent of plant species, tissue and physiological conditions. Our analysis also indicates that the RNAi enzyme, namely, the RNA dependent RNA polymerase 6 (RDR6) may not have any role in Arabidopsis miRNA modifications. Some of these modified miRNAs are bound to AGO1, suggesting their possible roles in biological processes. Conclusions:This is a first report that reveals that 5' nucleotide additions are preferred for mature miRNA sequences with 3â terminal âCâ nucleotide. Our analysis also indicates that the miRNAs modifications involving addition of nucleotides to the 5â or 3â end are independent of RDR6 activity and are not restricted by plant species, physiological conditions and tissue types. The results also indicate that such modifications might be important for biological processes. small RNA profiles of Healthy and ToLCV infected tomato leaves were generated by deep sequencing using Illumina GAII.

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Global and local perturbation of the tomato microRNA pathway by a trans-activated DICER-LIKE 1 mutant

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