Project description:In plants, miR390 directs the production of tasiRNAs from TRANS-ACTING SIRNA 3 (TAS3) transcripts to regulate AUXIN RESPONSIVE FACTOR (ARF) genes, transcription factors critical for auxin signaling; these tasiRNAs are known as tasiARFs. This pathway is highly conserved, with the TAS3 as the only one noncoding gene present almost ubiquitously in land plants. To understand the evolution of this miR390-TAS3-ARF pathway, we characterized homologs of these three genes from thousands of plant species, from bryophytes to angiosperms. Both miR390 and TAS3 are present and functional in liverworts, confirming their ancestral role to regulate ARFs in land plants. We found the lower-stem region of MIR390 genes, critical for accurate DCL1 (DICER-LIKE 1) processing, is conserved in sequence in seed plants. We propose a model for the transition of functional tasiRNA sequences in TAS3 genes occurred at the emergence of vascular plants, in which the two miR390 target sites of TAS3 genes showed distinct pairing patterns in different plant lineages. Based on the cleavability of miR390 target sites and the distance between target site and tasiARF we inferred a potential bidirectional processing mechanism exists for some TAS3 genes. We also demonstrated a tight mutual selection between tasiARF and its target genes, and characterized unusual aspects and diversity of regulatory components of this pathway. Taken together, these data illuminate the evolutionary path of the miR390-TAS3-ARF pathway in land plants, and demonstrate the significant variation that occurs in the production of phasiRNAs in plants, even in the functionally important and archetypal miR390-TAS3-ARF regulatory circuit.
Project description:Pathways underlying miRNA biogenesis, degradation, and activity were established early in land plant evolution, but the 24-nt siRNA pathway that guides DNA methylation was incomplete in early land plants, especially lycophytes. We show that the functional diversification of key gene families such as DICER-LIKE and ARGONAUTE (AGO) as observed in angiosperms occurred early in land plants followed by parallel expansion of the AGO family in ferns and angiosperms. We uncovered an unexpected AGO family specific to lycophytes and ferns. Our phylogenetic analyses of miRNAs in lycophytes, bryophytes, ferns, and angiosperms refined the temporal origination of conserved miRNA families in land plants.
Project description:The microRNA390 targets the TAS3 transcript leading to the production of trans-acting small interference RNAs that subsequently post-transcriptionally repress the mRNAs encoding the Auxin Response Factors (ARF)2, ARF3, and ARF4. This module has been linked to the development of lateral organs in both aerial and underground tissues. Previously, we have shown that the miR390/TAS3/ARFs module mediates the control of lateral roots and symbiotic nodules in legumes. Here, we show that a member of the Lateral Organ Boundaries Domain (LBD) family of transcription factors, designated as MtLBD17/29a, is a target of the miR390/TAS3/ARFs module. Transcriptomic, RT-qPCR and promoter fusion analysis revealed that MtLBD17/29a is induced under symbiotic conditions, whereas no induction was observed in roots overexpressing miR390 or silenced in ARF2/3/4. ChIP-PCR experiments demonstrated that MtARF2 directly binds to an Auxin Response Element (ARE) present in the MtLBD17/29a promoter activating its expression. Knockdown of MtLBD17/29a reduced the length of primary and lateral roots and increasing the density of lateral roots, whereas overexpression of MtLBD17/29a produced the opposite phenotype. Interestingly, both knockdown and overexpression of MtLBD17/29a resulted in a significant reduction in the number of nodules and infection events, as well as impaired the induction of the symbiotic genes Nodulation Signaling Pathway (NSP) 1 and 2, reminiscent of the phenotype observed in ARF2/3/4 silenced roots. These results demonstrate that MtLBD17/29 is regulated by the miR390/TAS3/ARFs module and a direct target of ARF2, revealing a new lateral root regulatory hub recruited by legumes to act in the root nodule symbiotic program.
Project description:Plant TRANS-ACTING SIRNA3 (TAS3)-derived short-interfering RNAs (siRNAs) include tasiR-ARFs, which are functionally conserved in targeting AUXIN RESPONSE FACTOR (ARF) genes, and a set of non-tasiR-ARF siRNAs, which have rarely been studied. In this study, TAS3 siRNAs were systematically characterized in rice (Oryza sative). Small RNA-seq results showed that an overwhelming majority of TAS3 siRNAs belong to the non-tasiR-ARF group, while tasiR-ARFs occupy a diminutive fraction. Phylogenetic analysis of TAS3 genes across dicot and monocot plants revealed that the siRNA-generating regions were highly conserved in grass species, especially in the oryzoideae. Target genes were identified for not only tasiR-ARFs but also non-tasiR-ARF siRNAs by analyzing rice degradome datasets, and some of these siRNA-target interactions were experimentally confirmed in rice tas3 mutants. Consistent with altered expression of target genes, phenotypic variations were observed for mutants in three TAS3 loci in comparison to wild-type rice. The regulatory role of ribosomes in the TAS3 siRNA-target interactions was further revealed by the fact that TAS3 siRNA-mediated target cleavage, in particular tasiR-ARFs targeting ARF2/3/14/15, occurred extensively in rice polysome samples. Altogether our study sheds new insights into TAS3 genes in plants and expand our knowledge about rice TAS3 siRNA-target interactions.
Project description:MicroRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) play an important role in plant stress responses but the small RNA (sRNA) repertoire is poorly characterized in extremphophiles. We report sRNA transcriptomes of leaf and flower tissues of two mangrove species Bruguiera gymnorrhiza and Kandelia candel (Rhizophoraceae) based on Illumina sequencing. A total of 32 known and 5 novel miRNA families were identified collectively.Besides the conserved miR390-TAS3-ARF pathway, B. gymnorrhiza possessed additional high-abundance tasiRNAs derived from the less-conserved regions of TAS3 transcripts with expanded potential targets.his study provides the first comprehensive sRNA transcriptome for mangroves B. gymnorrhiza and K. candel. Comparative analyses revealed that these two species systematically optimize sRNA expression levels, accompanied with rewiring of sRNA regulatory networks, as an evolutionarily strategy for stress adaptations. Furthermore, 24-nt sRNAs mapped to the telomeric repeats (CCCTAAA)n were highly abundant in both B. gymnorrhiza and K. candel.
Project description:Small non-coding RNAs (sRNAs) play key roles in plant development, growth and responses to biotic and abiotic stresses. At least four classes of sRNAs have been well characterized in plants, including repeat-associated siRNAs (rasiRNAs), microRNAs (miRNAs), trans-acting siRNAs (tasiRNAs) and natural antisense transcript-derived siRNAs. Chinese fir (Cunninghamia lanceolata) is one of the most important coniferous evergreen tree species in China. No sRNA from Chinese fir has been described to date. To obtain sRNAs in Chinese fir, we sequenced a sRNA library generated from seeds, seedlings, leaves, stems and calli, using Illumina high throughput sequencing technology. A comprehensive set of sRNAs were acquired, including conserved and novel miRNAs, rasiRNAs and tasiRNAs. With BLASTN and MIREAP we identified a total of 115 conserved miRNAs comprising 40 miRNA families and one novel miRNA with precursor sequence. The expressions of 16 conserved and one novel miRNAs and one tasiRNA were detected by RT-PCR. Utilizing real time RT-PCR, we revealed that four conserved and one novel miRNAs displayed developmental stage-specific expression patterns in Chinese fir. In addition, 209 unigenes were predicted to be targets of 30 Chinese fir miRNA families, of which five target genes were experimentally verified by 5' RACE, including a squamosa promoter-binding protein gene, a pentatricopeptide (PPR) repeat-containing protein gene, a BolA-like family protein gene, AGO1 and a gene of unknown function. We also demonstrated that the DCL3-dependent rasiRNA biogenesis pathway, which had been considered absent in conifers, existed in Chinese fir. Furthermore, the miR390-TAS3-ARF regulatory pathway was elucidated. We unveiled a complex population of sRNAs in Chinese fir through high throughput sequencing. This provides an insight into the composition and function of sRNAs in Chinese fir and sheds new light on land plant sRNA evolution.
Project description:In plants, tasiRNAs form a class of endogenous secondary siRNAs produced through the action of RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6) upon microRNA-mediated cleavage of non-coding TAS RNAs. In Arabidopsis thaliana, TAS1, TAS2 and TAS4 tasiRNA production proceeds via a single cleavage event mediated by 22nt-long or/and asymmetric miRNAs in an ARGONAUTE-1 (AGO1)-dependent manner. By contrast, tasiRNA production from TAS3 seems to follow the so-called ‘two-hit’ process, where dual targeting of TAS3, specifically mediated by the 21nt-long, symmetric miR390, initiates AGO7-dependent tasiRNA production. Interestingly, features for TAS3 tasiRNA production differ in other plant species and we show here that such features also enable TAS3 tasiRNA biogenesis in Arabidopsis, and that a single miR390 targeting event is, in fact, sufficient for this process, suggesting that the ‘one-hit’ model underpins all the necessary rudiments of secondary siRNA biogenesis from plant TAS transcripts. Further results suggest that the two-hit configuration likely enhances the fidelity of tasiRNA production and, hence, the accuracy of downstream gene regulation. Finally, we show that a ‘non-cleavable one-hit’ process allows tasiRNA production from both TAS1 and TAS3 transcripts, indicating that RDR6 recruitment does not require miRNA cleavage, nor does the recruitment, as we further show, of SUPRRESSOR-OF-GENE-SILENCING-3, indispensable for tasiRNA generation.
Project description:The Streptophyta include unicellular and multicellular charophyte green algae and land plants. Colonization of the terrestrial habitat by land plants was a major evolutionary event that has transformed our planet. So far lack of genome information on unicellular charophyte algae hinders our understanding of the origin and the evolution from unicellular to multicellular life in Streptophyta. This work reports the high-quality reference genome and transcriptome of Mesostigma viride, a single-celled charophyte alga with a position at the base of Streptophyta. There are abundant segmental duplications and transposable elements in M. viride, which contribute to a relatively large genome with high gene content compared to other algae and early diverging land plants. This work identifies the origin of genetic tools that multicellular Streptophyta have inherited and key genetic innovations required for evolution of land plants from unicellular aquatic ancestors. The findings shed light on the age-old questions of the evolution of multicellularity and the origin of land plants.
Project description:The Streptophyta include unicellular and multicellular charophyte green algae and land plants. Colonization of the terrestrial habitat by land plants was a major evolutionary event that has transformed our planet. So far lack of genome information on unicellular charophyte algae hinders our understanding of the origin and the evolution from unicellular to multicellular life in Streptophyta. This work reports the high-quality reference genome and transcriptome of Mesostigma viride, a single-celled charophyte alga with a position at the base of Streptophyta. There are abundant segmental duplications and transposable elements in M. viride, which contribute to a relatively large genome with high gene content compared to other algae and early diverging land plants. This work identifies the origin of genetic tools that multicellular Streptophyta have inherited and key genetic innovations required for evolution of land plants from unicellular aquatic ancestors. The findings shed light on the age-old questions of the evolution of multicellularity and the origin of land plants.
Project description:The Streptophyta include unicellular and multicellular charophyte green algae and land plants. Colonization of the terrestrial habitat by land plants was a major evolutionary event that has transformed our planet. So far lack of genome information on unicellular charophyte algae hinders our understanding of the origin and the evolution from unicellular to multicellular life in Streptophyta. This work reports the high-quality reference genome and transcriptome of Mesostigma viride, a single-celled charophyte alga with a position at the base of Streptophyta. There are abundant segmental duplications and transposable elements in M. viride, which contribute to a relatively large genome with high gene content compared to other algae and early diverging land plants. This work identifies the origin of genetic tools that multicellular Streptophyta have inherited and key genetic innovations required for evolution of land plants from unicellular aquatic ancestors. The findings shed light on the age-old questions of the evolution of multicellularity and the origin of land plants.