The pollen wall is a specialized extracellular cell wall that protects male gametophytes from various environmental stresses and facilitates pollination. Here, we reported that bHLH010 and bHLH089 together are required for the development of the pollen wall by regulating their specific downstream transcriptional and metabolic networks. Both the exine and intine structures of bhlh010 bhlh089 pollen grains were severely defective. Further untargeted metabolomic and transcriptomic analyses revealed that the accumulation of pollen wall morphogenesis-related metabolites, including polysaccharides, glyceryl derivatives, and flavonols, were significantly changed, and the expression of such metabolic enzyme-encoding genes and transporter-encoding genes related to pollen wall morphogenesis was downregulated in bhlh010 bhlh089 mutants. Among these downstream target genes, CSLB03 is a novel target with no biological function being reported yet. We found that bHLH010 interacted with the two E-box sequences at the promoter of CSLB03 and directly activated the expression of CSLB03. The cslb03 mutant alleles showed bhlh010 bhlh089–like pollen developmental defects, with most of the pollen grains exhibiting defective pollen wall structures.

Gametophyte development in angiosperms occurs within diploid sporophytic structures and requires coordinated development; e.g., development of the male gametophyte pollen depends on the surrounding sporophytic tissue, the tapetum. The mechanisms underlying this interaction remain poorly characterized. The peptide CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 19 (CLE19) plays a “braking” role in preventing the harmful overexpression of tapetum transcriptional regulators to ensure normal pollen development in Arabidopsis. However, the CLE19 receptor is unknown. Here, we show that CLE19 interacts directly with the PXY-LIKE1 (PXL1) ectodomain and induces PXL1 phosphorylation. PXL1 is also required for the function of CLE19 in maintaining the tapetal transcriptional regulation of pollen exine genes. Additionally, CLE19 induces the interactions of PXL1 with SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptors required for pollen development. We propose that PXL1 and SERKs act as receptor and coreceptor, respectively, of the extracellular CLE19 signal, thereby regulating tapetum gene expression and pollen development.

Early anther morphogenesis is a crucial process for male fertility in plants, governed by the transcription factor SPL. While the involvement of AGAMOUS (AG) in SPL activation and microsporogenesis initiation is well established, our understanding of the mechanisms governing the spatial distribution and precise expression of SPL during anther cell fate determination remains limited. Here, we present novel findings on the abnormal phenotypes of two previously unreported SPL mutants, spl-4 and spl-5, during anther morphogenesis. Through comprehensive analysis, we identified ARF3 as a key upstream regulator of SPL. Our cytological experiments demonstrated that ARF3 plays a critical role in restricting SPL expression specifically in microsporocytes. Moreover, we revealed that ARF3 directly binds to two specific auxin response elements on the SPL promoter, effectively suppressing AG-mediated activation of SPL. Notably, the arf3 loss-of-function mutant exhibits phenotypic similarities to the SPL overexpression mutant (spl-5), characterized by defective adaxial anther lobes. Transcriptomic analysis revealed differential expression of the genes involved in the morphogenesis pathway in both arf3 and spl mutants, with ARF3 and SPL exhibited opposing regulatory effects on this pathway. Taken together, our study unveils the precise role of ARF3 in restricting the spatial expression and preventing aberrant SPL levels during early anther morphogenesis, thereby ensuring the fidelity of the critical developmental process in plants.