Coexpression network and trans-activation analyses of maize reproductive phasiRNA loci [scRNA-seq]

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. Single-cell ene expression profiling of maize leaf protoplasts ectopically expressing putative transcriptional regulators of anther-enriched eproductive phasiRNAs

花药富集的相位化小干扰RNA（phased small interfering RNAs，phasiRNAs）在禾本科物种的雄性育性调控中发挥关键作用。phasiRNAs的长链非编码前体由RNA聚合酶II催化合成，且极可能受转录因子（Transcription Factors, TFs）的调控。目前已在玉米（Zea mays）中鉴定出少量针对21核苷酸或24核苷酸phasiRNA基因座（即21-PHAS或24-PHAS基因座）的潜在转录调控因子，但尚不清楚单个TF或TF组合是否足以激活PHAS基因座。本研究鉴定出与玉米花药发育相关的时序基因共表达网络（模块），其中两个模块显著富集21-PHAS或24-PHAS基因座。通过对比上述共表达模块与已报道的多个雄性不育TF突变体中的失调基因集，本研究为TF的活性时序提供了新见解，包括揭示了外层细胞层4（OUTER CELL LAYER 4）与雄性不育32（Male sterile 32）的拮抗调控作用。利用批量原生质体RNA测序（bulk-protoplast RNA sequencing, RNA-seq）开展的单个TF反式激活实验显示，与21-PHAS基因座共表达的TF中有两个可激活多个21-nt phasiRNA通路基因，但均不足以激活21-PHAS基因座。本研究利用单细胞（原生质体）RNA测序，分别对这些TF以及近期报道的24-PHAS基因座潜在转录调控因子开展组合活性筛选，未检测到21-PHAS或24-PHAS基因座的可重复激活现象。综合来看，本研究结果表明，花药中的内源转录机器和/或染色质状态是激活生殖相关PHAS基因座的必要条件。尽管如此，单细胞RNA测序方法仍为后续TF组合活性研究提供了潜在可能。对异位表达花药富集型生殖phasiRNAs潜在转录调控因子的玉米叶片原生质体进行单细胞基因表达谱分析。