A cascade of bHLH-regulated pathways programs maize anther development

Abstract

The spatiotemporal development of somatic tissues of the anther lobe is necessary for successful fertile pollen production. This process is mediated by many transcription factors acting through complex, multi-layered networks. Here, our analysis of functional knockout mutants of interacting basic-helix-loop-helix genes Ms23, Ms32, bHLH122, and bHLH51 in maize (Zea mays) established that male fertility requires all four genes, expressed sequentially in the tapetum. Not only do they regulate each other, but they also encode proteins that form heterodimers that act collaboratively to guide many cellular processes at specific developmental stages. MS23 is confirmed to be the master factor, as the ms23 mutant showed the earliest developmental defect, cytologically visible in the tapetum, with the most drastic alterations in pre-meiotic gene expression observed in ms23 anthers. Notably, the male sterile ms23, ms32, and bhlh122-1 mutants lack 24-nt phased secondary small interfering RNAs (phasiRNAs) and the precursor transcripts from the corresponding 24-PHAS loci, while the bhlh51-1 mutant has wild-type levels of both precursors and small RNA products. Multiple lines of evidence suggest that 24-nt phasiRNA biogenesis primarily occurs downstream of MS23 and MS32, both of which directly activate Dcl5 and are required for most 24-PHAS transcription, with bHLH122 playing a distinct role in 24-PHAS transcription.

Genome-wide identification and expression analysis of bHLH transcription factors reveal their putative regulatory effects on petal nectar spur development in Aquilegia

“…The functions of bHLH transcription factors have been reported in animals and plants, including control of cell proliferation and specific cell lineages development (Heim et al, 2003; Toledo-Ortiz et al, 2003; Pillitteri et al, 2007; Pires and Dolan, 2010; Girin et al, 2011; Zhang et al, 2018; Zhang et al, 2021; Nan et al, 2022). Spur as a specific adjacent tissue, the pattern of cell division and cell lineages are different from the blade in Aquilegia (Shan et al, 2019; Yant et al, 2015).…”

Genetic Structure and Molecular Mechanisms Underlying the Formation of Tassel, Anther, and Pollen in the Male Inflorescence of Maize (Zea mays L.)

“…It is noteworthy that 15 new GMS genes have recently been identified and validated using the CRISPR/Cas9 system [ 68 , 150 , 151 ], showing the power of gene editing technologies in maize genomics and breeding. Here, a molecular regulatory network for maize male fertility is summarized and updated by considering the functional properties and sequential expression patterns of the reported maize GMS genes ( Figure 5 ).…”

“…Although the corresponding TFs (ZmMS32, ZmMS9, ZmbHLH51, ZmMYB80 and ZmMS7) are found to be encoded by GMS genes, whether a similar genetic pathway and regulatory network exist in maize needs to be investigated. Nan et al [ 68 , 150 , 151 ] compared the mutant phenotypes of four bHLH genes, i.e., ZmMs32 , ZmbHLH51 , ZmMs23 and ZmbHLH122 , and found that all four bhlh mutants have an aberrant tapetum. Combining several recent studies, a more complicated cascade of bHLH-regulated pathways was established ( Figure 5 ) [ 68 , 151 , 155 , 159 ].…”

“…Nan et al [ 68 , 150 , 151 ] compared the mutant phenotypes of four bHLH genes, i.e., ZmMs32 , ZmbHLH51 , ZmMs23 and ZmbHLH122 , and found that all four bhlh mutants have an aberrant tapetum. Combining several recent studies, a more complicated cascade of bHLH-regulated pathways was established ( Figure 5 ) [ 68 , 151 , 155 , 159 ]. Protein-protein interaction assays showed that ZmMS23 interacts with both ZmMS32 and ZmbHLH51, ZmbHLH122 interacts with both ZmMS32 and ZmbHLH51, and ZmbHLH51 forms homodimers.…”

“…Protein-protein interaction assays showed that ZmMS23 interacts with both ZmMS32 and ZmbHLH51, ZmbHLH122 interacts with both ZmMS32 and ZmbHLH51, and ZmbHLH51 forms homodimers. Supported further by cytological observation and gene expression analysis in each mutant, the order of essential actions is ZmMS23, ZmMS32, ZmbHLH122, and then ZmbHLH51 [ 68 , 151 , 155 , 159 ]. ZmbHLH122 directly activates ZmbHLH51 and interacts with ZmbHLH51 [ 68 ], but it appears that ZmbHLH122 is not a direct target of ZmMS23 or ZmMS32 [ 155 , 159 ].…”