EcINP1 role in Eschscholzia californica. EcINP1

Pollen grains show an enormous variety of aperture systems. What genes are involved in the aperture formation pathway and how conserved this pathway is in angiosperms remains largely unknown. INAPERTURATE POLLEN1 (INP1) encodes a protein of unknown function, essential for aperture formation in Arabidopsis, rice and maize. Yet, because INP1 sequences are quite divergent, it is unclear if their function is conserved across angiosperms. Here, we conducted a functional study of the INP1 orthologue from the basal eudicot Eschscholzia californica (EcINP1) using expression analyses, virus-induced gene silencing, pollen germination assay, and transcriptomics. We found that EcINP1 expression peaks at the tetrad stage of pollen development, consistent with its role in aperture formation, which occurs at that stage, and showed, via gene silencing, that the role of INP1 as an important aperture factor extends to basal eudicots. Using germination assays, we demonstrated that, in Eschscholzia, apertures are dispensable for pollen germination. Our comparative transcriptome analysis of wild-type and silenced plants identified over 900 differentially expressed genes, many of them potential candidates for the aperture pathway. Our study substantiates the importance of INP1 homologues for aperture formation across angiosperms and opens up new avenues for functional studies of other aperture candidate genes.

花粉粒的萌发孔系统（aperture systems）具有极为丰富的多样性。目前对于被子植物中参与花粉萌发孔形成通路的基因组成，以及该通路在被子植物中的保守性程度，仍知之甚少。无萌发孔花粉1（INAPERTURATE POLLEN1, INP1）编码一种功能未知的蛋白质，其在拟南芥、水稻和玉米的萌发孔形成过程中不可或缺。然而，由于不同物种的INP1序列差异显著，目前尚不清楚其功能在被子植物中是否保守。本研究以基部真双子叶植物花菱草（Eschscholzia californica）的INP1直系同源基因EcINP1为研究对象，通过表达分析、病毒诱导基因沉默、花粉萌发实验以及转录组学技术开展功能研究。研究发现，EcINP1的表达在花粉发育的四分体时期达到峰值，这与其在该时期参与萌发孔形成的功能相符；同时通过基因沉默实验证实，INP1作为重要的萌发孔调控因子的功能同样存在于基部真双子叶植物中。通过花粉萌发实验，本研究证实，在花菱草中，萌发孔并非花粉萌发所必需。对野生型植株与基因沉默植株的比较转录组分析显示，共鉴定出超过900个差异表达基因，其中多数为萌发孔形成通路的潜在候选基因。本研究证实了INP1同源基因在被子植物萌发孔形成过程中的重要性，并为其他萌发孔通路候选基因的功能研究开辟了全新方向。