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In rice (Oryza sativa), the lemma and palea protect the internal organs of the floret，provide nutrients for seed development, and determine grain size. We previously revealed that a trans-acting small interfering RNA targeting AUXIN RESPONSE FACTORS (tasiR-ARF) regulates lemma polarity establishment via post-transcriptional repression of AUXIN RESPONSE FACTORS (ARFs) in rice. TasiR-ARF formation requires RNA-DEPENDENT RNA POLYMERASE 6 (RDR6). However, the underlying molecular mechanism of the tasiR-ARF-ARF regulon in lemma development remains unclear. Here, by genetic screening for suppressors of the thermosensitive mutant osrdr6-1, we identified three suppressors, huifu 1 (hf1), hf9, and hf17. Mapping-by-sequencing revealed that HF1 encodes a MYB transcription factor belonging to the KANADI1 family. The hf1 mutation partially rescued the osrdr6-1 lemma defect but not the defect in tasiR-ARF levels. DNA affinity purification sequencing analysis identified 17 725 OsKANADI1-associated sites, most of which contain the SPBP-box binding motif (RGAATAWW) and are located in the promoter, protein-coding, intron, and intergenic regions. Moreover, we found that OsKANADI1 could directly bind to the intron of OsARF3a in vitro and in vivo and promote OsARF3a expression at the transcriptional level. In addition, hf9 and hf17 are intragenic suppressors containing mutations in OsRDR6 that partially rescue tasiR-ARF levels by restoring OsRDR6 protein levels. Collectively, our results demonstrate that OsKANADI1 and tasiR-ARFs synergistically maintain the proper expression of OsARF3a and thus contribute to rice lemma development.

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PREMISE: Leaves are traditionally classified into microphylls and megaphylls, and recently have been regarded as independently originating in lycophytes, ferns, and seed plants. The developmental genetics of leaf dorsoventrality, a synapomorphy in vascular plants, has been extensively studied in flowering plants. AUXIN RESPONSE FACTOR4 (ARF4) genes are key to leaf abaxial identity in flowering plants, but whether they exist in ferns is still an open question. METHODS: ARF4 genes from Ceratopteris pteridoides, Cyrtomium guizhouense, and Parathelypteris nipponica were mined from transcriptomes and investigated in terms of evolutionary phylogeny and sequence motifs, with a focus on the tasiR-ARF binding site. In situ hybridization was used to localize expression of CpARF4 in Ceratopteris pteridoides. 5'RNA ligase-mediated-RACE was employed to verify whether CpARF4 transcripts were sliced by tasiR-ARF. RESULTS: ARF4 genes exist in ferns, and this lineage originates from a gene duplication in the common ancestor of ferns and seed plants. ARF4 genes are of a single copy in the ferns studied here, and they contain divergent and, at most, one tasiR-ARF binding site. CpARF4 is expressed in the abaxial but not the adaxial domain of leaf primordia at various developmental stages. Transcript slicing guided by tasiR-ARF is active in C. pteridoides, but CpARF4 probably has not been affected by it. CONCLUSIONS: Fern ARF4 genes differ in copy number and tasiR-ARF regulation relative to flowering plants, though they can be similarly expressed in the abaxial domain of leaves, revealing a key role for ARF4 genes in the evolution of leaf dorsoventrality of vascular plants.

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During in vitro maize plant regeneration somatic cells change their normal fate and undergo restructuring to generate pluripotent cells able to originate new plants. Auxins are essential to achieve such plasticity. Their physiological effects are mediated by auxin response factors (ARFs) that bind auxin responsive elements within gene promoters. Small trans-acting (ta)-siRNAs, originated from miR390-guided TAS3 primary transcript cleavage, target ARF3/4 class (tasiR-ARFs). Here we found that TAS3b precursor as well as derived tasiR-ARFbD5 and tasiR-ARFbD6 display significantly lower levels in non-embryogenic callus (NEC), while TAS3g, miR390 and tasiR-ARFg are more abundant in the same tissue. However, Argonaute (AGO7) and leafbladeless 1 (LBLl) required for tasiR-ARF biogenesis showed significantly higher transcript levels in EC suggesting limited tasiR-ARF biogenesis in NEC. The five maize ARFs targeted by tasiR-ARFs were also significantly enriched in EC and accompanied by higher auxin accumulation with punctuate patterns in this tissue. At hormone half-reduction and photoperiod implementation, plant regeneration initiated from EC with transient TAS3g, miR390 and tasiR-ARFg increase. Upon complete hormone depletion, TAS3b became abundant and derived tasiR-ARFs gradually increased at further regeneration stages. ZmARF transcripts targeted by tasiR-ARFs, as well as AGO7 and LBL1 showed significantly lower levels during regeneration than in EC. These results indicate a dynamic tasiR-ARF mediated regulation throughout maize in vitro plant regeneration.

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In most sexually reproducing plants, a single somatic, sub-epidermal cell in an ovule is selected to differentiate into a megaspore mother cell, which is committed to giving rise to the female germline. However, it remains unclear how intercellular signaling among somatic cells results in only one cell in the sub-epidermal layer differentiating into the megaspore mother cell. Here we uncovered a role of the THO complex in restricting the megaspore mother cell fate to a single cell. Mutations in TEX1, HPR1, and THO6, components of the THO/TREX complex, led to the formation of multiple megaspore mother cells, which were able to initiate gametogenesis. We demonstrated that TEX1 repressed the megaspore mother cell fate by promoting the biogenesis of TAS3-derived trans-acting small interfering RNA (ta-siRNA), which represses ARF3 expression. The TEX1 protein was present in epidermal cells, but not in the germline, and, through TAS3-derived ta-siRNA, restricted ARF3 expression to the medio domain of ovule primordia. Expansion of ARF3 expression into lateral epidermal cells in a TAS3 ta-siRNA-insensitive mutant led to the formation of supernumerary megaspore mother cells, suggesting that TEX1- and TAS3-mediated restriction of ARF3 expression limits excessive megaspore mother cell formation non-cell-autonomously. Our findings reveal the role of a small-RNA pathway in the regulation of female germline specification in Arabidopsis.

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