Ultrastructure analysis reveals sporopollenin deposition and nexine formation at early stage of pollen wall development in Arabidopsis

In angiosperm, pollen wall formation is a critical step for male gametophyte development. Pollen wall constitutes of the outer layer exine and the inner layer intine. Exine is further divided into sexine and nexine. In Arabidopsis, the general process of pollen wall formation has been reported. However, the nexine formation has not been revealed. Here, we observed the process of pollen wall formation in Arabidopsis thaliana using transmission electron microscope. After callose wall is formed, the primexine is present between plasma membrane and the callose layer in the tetrad. With plasma membrane undulation, sporopollenin precursors accumulated on the peak of undulated membrane which is further developed into probacula. The primexine determines plasma membrane undulation and sporopollenin accumulation based on previous analysis of an undulation-deficient mutant. Some materials obviously different from sporopollenin are filled between the primexine and plasma membrane. These materials cover all the surface of plasma membrane and gradually develop into nexine. After microspore is released from tetrad, the nexine layer is formed and the probacula is further developed into sexine with continued accumulation of sporopollenin. Based on these observations, we proposed a developmental model of early pollen wall formation.