Formation of intercellular gas space in the diaphragm during the development of aerenchyma in the leaf petiole of Sagittaria trifolia

The development of aerenchyma in the petiole of Sagittaria trifolia L. was studied by means of light-microscopy, scanning electron microscope, transmission electron microscope and immunofluorescence, focusing on the formation of intercellular spaces in diaphragms and its relationship with the organization of cortical microtubule arrays. A complex and organized honeycomb-like schizogenous aerenchyma formed by cylinders and vascular diaphragms was observed in the petiole of S. trifolia at different developmental stages. Cell division was the primary factor contributing to the increased volume of air spaces at early stages, while cell enlargement became the primary factor at later stages. The cortical microtubules localize at the sites where intercellular spaces and the secondary cell walls will be formed or deposited during the formation of intercellular spaces by the separation of diaphragm cells. Cortical microtubules were observed at the boundary of diaphragm cells and the fringes of intercellular spaces at later developmental stages where cell expansion occurs rapidly. These observations support the hypothesis that reorganization of cortical microtubule arrays might be related to the formation of air spaces in diaphragms and are involved in the deposition of secondary cell walls.