3D-multiple immunoimaging using whole male organs in rice

Abstract

The spatiotemporal regulation of proteins and RNAs is essential for the precise development of reproductive tissues in many organisms. The anther, a significant part of the male reproductive organ in plants, contains the somatic cell layers named the anther wall and germ cells located inside. The cell-to-cell communication between the soma and germ cells in the development of male and female tissues is reported, although the mechanism remains unknown in plants. Here, we successfully developed a simple 3D-organ-immunoimaging technique, which distinguishes each single cell from the four somatic cell layers and germ cells without the need for transformation, embedding, sectioning, or clearing in rice. The 3D-immunostaining method is also applicable to the intracellular localization of meiosis-specific proteins in meiocytes-namely MEL1, a germ cell-specific Argonaute in the cytoplasm, and ZEP1, a pachytene marker on meiotic chromosomes. Our study suggests that the 3D-multiple immunostaining method with single-cell and intracellular resolution will contribute to the organ-level elucidation of comprehensive molecular mechanisms and non-cell-autonomous systems.

Spatial control of ARGONAUTE-mediated RNA silencing in anther development

“…2a; Supplementary Table1). Recently, we developed the 3D-anther immunostaining system at subcellular and single-cell resolution 30 . To investigate the localization of these AGO1b/d proteins in anthers, we performed the 3D multiple immunostaining against AGO1b and AGO1d using whole-mount anthers at early meiosis.…”

“…3D-immuno imaging was performed as described 30 . Anthers fixed in 4% paraformaldehyde were transferred to PME buffer (50 mM PIPES, 5 mM EGTA and 5 mM MgSO 4 , pH 6.9) on a MAS-coated microscope slide, cut in distilled water using a scalpel, and incubated for 30 min at 25 °C.…”