UB2/UB3/TSH4-anchored transcriptional networks regulate early maize inflorescence development in response to simulated shade.

Increasing planting density has been adopted as an effective means to increase maize (Zea mays) yield. Competition for light from neighbors can trigger plant shade avoidance syndrome, which includes accelerated flowering. However, the regulatory networks of maize inflorescence development in response to high-density planting remain poorly understood. In this study, we showed that shade-mimicking treatments cause precocious development of the tassels and ears. Comparative transcriptome profiling analyses revealed the enrichment of phytohormone-related genes and transcriptional regulators among the genes co-regulated by developmental progression and simulated shade. Network analysis showed that three homologous Squamosa promoter binding protein (SBP)-like (SPL) transcription factors, Unbranched2 (UB2), Unbranched3 (UB3), and Tasselsheath4 (TSH4), individually exhibited connectivity to over 2,400 genes across the V3-to-V9 stages of tassel development. In addition, we showed that the ub2 ub3 double mutant and tsh4 single mutant were almost insensitive to simulated shade treatments. Moreover, we demonstrated that UB2/UB3/TSH4 could directly regulate the expression of Barren inflorescence2 (BIF2) and Zea mays teosinte branched1/cycloidea/proliferating cell factor30 (ZmTCP30). Furthermore, we functionally verified a role of ZmTCP30 in regulating tassel branching and ear development. Our results reveal a UB2/UB3/TSH4-anchored transcriptional regulatory network of maize inflorescence development and provide valuable targets for breeding shade-tolerant maize cultivars.

ZmELF3.1 integrates the RA2-TSH4 module to repress maize tassel branching.

Tassel branch number (TBN) is a key agronomic trait for adapting to high-density planting and grain yield in maize. However, the molecular regulatory mechanisms underlying tassel branching are still largely unknown. Here, we used molecular and genetic studies together to show that ZmELF3.1 plays a critical role in regulating TBN in maize. Previous studies showed that ZmELF3.1 forms the evening complex through interacting with ZmELF4 and ZmLUX to regulate flowering in maize and that RA2 and TSH4 (ZmSBP2) suppresses and promotes TBN in maize, respectively. In this study, we show that loss-of-function mutants of ZmELF3.1 exhibit a significant increase of TBN. We also show that RA2 directly binds to the promoter of TSH4 and represses its expression, thus leading to reduced TBN. We further demonstrate that ZmELF3.1 directly interacts with both RA2 and ZmELF4.2 to form tri-protein complexes that further enhance the binding of RA2 to the promoter of TSH4, leading to suppressed TSH4 expression and consequently decreased TBN. Our combined results establish a novel functional link between the ELF3-ELF4-RA2 complex and miR156-SPL regulatory module in regulating tassel branching and provide a valuable target for genetic improvement of tassel branching in maize.

ZmSPL10, ZmSPL14 and ZmSPL26 act together to promote stigmatic papilla formation in maize through regulating auxin signaling and ZmWOX3A expression.

Maize silk is a specialized type of stigma, covered with numerous papillae for pollen grain capture. However, the developmental process of stigmatic papillae and the underlying regulatory mechanisms have remained largely unknown. Here, we combined the cytological, genetic and molecular studies to demonstrate that three homologous genes ZmSPL10, ZmSPL14 and ZmSPL26 play a central role in promoting stigmatic papilla formation in maize. We show that their triple knockout mutants are nearly complete lack of stigmatic papilla, resulting in a severe reduction in kernel setting. Cellular examination reveals that stigmatic papilla is developed from a precursor cell, which is the smaller daughter cell resulting from asymmetric cell division of a silk epidermal cell. In situ hybridization shows that ZmSPL10, ZmSPL14 and their target genes SPI1, ZmPIN1b, ZmARF28 and ZmWOX3A are preferentially expressed in the precursor cells of stigmatic papillae. Moreover, ZmSPL10, ZmSPL14 and ZmSPL26 directly bind to the promoters of SPI1, ZmPIN1b, ZmARF28 and ZmWOX3A and promote their expression. Further, Zmwox3a knockout mutants display severe defects in stigmatic papilla formation and reduced seed setting. Collectively, our results demonstrate that ZmSPL10, ZmSPL14 and ZmSPL26 act together to promote stigmatic papilla development through regulating auxin signaling and ZmWOX3A expression.

DWARF53 interacts with transcription factors UB2/UB3/TSH4 to regulate maize tillering and tassel branching.

Strigolactones (SLs) are a recently identified class of phytohormones that regulate diverse developmental processes in land plants. However, the signaling mechanism of SLs in maize (Zea mays) remains largely unexplored. Here, we identified the maize gene DWARF 53 (ZmD53) and demonstrated that ZmD53 interacts with the SL receptors DWARF 14A/B (ZmD14A/B) in a rac-GR24-dependent manner. Transgenic maize plants expressing a gain-of-function mutant version of Zmd53 exhibited insensitivity to exogenous rac-GR24 treatment and a highly pleiotropic phenotype, including excess tillering and reduced tassel branching, indicating that ZmD53 functions as an authentic SL signaling repressor in maize. In addition, we showed that ZmD53 interacts with two homologous maize SPL transcription factors, UB3 and TSH4, and suppresses their transcriptional activation activity on TB1 to promote tillering. We also showed that UB2, UB3, and TSH4 can physically interact with each other and themselves, and that they can directly regulate the expression of TSH4, thus forming a positive feedback loop. Furthermore, we demonstrated that ZmD53 can repress the transcriptional activation activity of UB3 and TSH4 on their own promoters, thus decreasing tassel branch number. Our results reveal new insights into the integration of SL signaling and the miR156/SPL molecular module to coordinately regulate maize development.