Expression profiling by high throughput sequencing
Summary
The anther-enriched phased, small interfering RNAs (phasiRNAs) play vital roles in regulating male fertility in grass species. The long non-coding precursors of phasiRNAs are products of RNA polymerase II and are likely regulated by transcription factors (TFs). A few putative transcriptional regulators of the 21- or 24-nucleotide phasiRNA loci (i.e., 21- or 24-PHAS loci) have been identified in maize (Zea mays), but whether any of the individual TFs or TF combinations are sufficient to activate the PHAS loci is unclear. Here, we identified the temporal gene coexpression networks (modules) associated with maize anther development, including two modules highly enriched for the 21- or 24-PHAS loci. Comparisons of these coexpression modules and gene sets dysregulated in several reported male sterile TF mutants revealed new insights into the timing of activities of the TFs, including antagonistic roles for OUTER CELL LAYER 4 and Male sterile 32. Trans-activation assays of individual TFs using bulk-protoplast RNA sequencing (RNA-seq) showed that two of the TFs coexpressed with 21-PHAS loci could activate several 21-nt phasiRNA pathway genes, but none was sufficient to activate 21-PHAS loci. Screens for combinatorial activities of these TFs and, separately, the recently reported putative transcriptional regulators of 24-PHAS loci using single-cell (protoplast) RNA-seq, did not detect reproducible activation of 21-PHAS or 24-PHAS loci. Collectively, our results suggest that the endogenous transcriptional machineries and/or chromatin states in the anthers are necessary to activate reproductive PHAS loci. Nonetheless, the single-cell RNA-seq approach shows potential for future studies of combinatorial activities of TFs.
Overall design
Gene expression profiling of maize leaf protoplasts ectopically expressing putative transcriptional regulators of anther-enriched, 21-nt reproductive phasiRNAs
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