bHLH cascade regulate 24-nt phasiRNA biogenesis in maize anther

Anthers are the male reproductive floral organs, but loss of the 24-nt class of phased small-interfering RNAs (phasiRNAs) may confer temperature-sensitive male sterility in maize. 24-nt phasiRNAs from 176 loci are abundant coordinately in fertile maize anthers after the last periclinal divisions are complete, all four wall layers are formed, and meiosis has initiated in the pollen mother cells. In this study, we found male sterile maize ms23, ms32, and bhlh122 mutants with various degrees of tapetal defects lack 24-PHAS mRNA precursors and 24-nt phasiRNAs, which were not affected in another male sterile mutant, bhlh51. Transcription from most of 24-PHAS loci depends on bHLH122 while bHLH122 and Dcl5 transcription depends on both Ms23 and Ms32. Multiple lines of evidence suggest that 24-nt phasiRNAs biogenesis is primarily controlled at multiple layers by MS23 and MS32, with distinctive action on 24-PHAS transcription by bHLH122, creating a transcription factor cascade in maize tapetal cells. Transcriptome and small RNA profiling of maize bhlh male sterile anthers by RNA- and sRNA-seq, comparing to fertile anthers花药是植物的雄性生殖花器官，但24核苷酸长度的阶段性小干扰RNA（phased small-interfering RNAs）这一类别的缺失，可能会导致玉米产生温敏雄性不育表型。在末次平周分裂完成、四层花药壁层形成且花粉母细胞启动减数分裂后，来自176个基因座的24-nt phasiRNAs会在可育玉米花药中协同大量积累。本研究发现，携带不同程度绒毡层缺陷的玉米雄性不育突变体ms23、ms32与bhlh122，均缺失24-PHAS mRNA前体与24-nt phasiRNAs；而另一雄性不育突变体bhlh51的相关产物则未受影响。绝大多数24-PHAS基因座的转录依赖bHLH122，而bHLH122与Dcl5的转录同时依赖Ms23和Ms32。多方面证据表明，24-nt phasiRNAs的生物合成主要受MS23与MS32在多个层面调控，且bHLH122对24-PHAS的转录具有特异性调控作用，由此在玉米绒毡层细胞中形成了一套转录因子级联调控通路。本研究通过RNA测序与小RNA测序，对玉米bhlh雄性不育花药与可育花药开展了转录组与小RNA组表达谱分析。