Three-dimensional reconstruction of Picea wilsonii Mast. pollen grains using automated electron microscopy.

Three-dimensional electron microscopy (3D-EM) has attracted considerable attention because of its ability to provide detailed information with respect to developmental analysis. However, large-scale high-resolution 3D reconstruction of biological samples remains challenging. Herein, we present a 3D view of a Picea wilsonii Mast. pollen grain with 100 nm axial and 38.57 nm lateral resolution using AutoCUTS-SEM (automatic collector of ultrathin sections-scanning electron microscopy). We established a library of 3,127 100 nm thick serial sections of pollen grains for preservation and observation, demonstrating that the protocol can be used to analyze large-volume samples. After obtaining the SEM images, we reconstructed an entire pollen grain comprising 734 serial sections. The images produced by 3D reconstruction clearly revealed the main components of the P. wilsonii pollen grain, i.e., two sacci and pollen corpus, tube cell, generative cell, and two degenerated prothallial cells, and their internal organization. In addition, we performed a quantitative analysis of the different pollen grain cells, including sacci, and found that there were 202 connections within a saccus SEM image. Thus, for the first time, this study provided a global 3D view of the entire pollen grain, which will be useful for analyzing pollen development and growth.

Nitric Oxide Enhances Cytotoxicity of Lead by Modulating the Generation of Reactive Oxygen Species and Is Involved in the Regulation of Pb2+ and Ca2+ Fluxes in Tobacco BY-2 Cells.

Lead is a heavy metal known to be toxic to both animals and plants. Nitric oxide (NO) was reported to participate in plant responses to different heavy metal stresses. In this study, we analyzed the function of exogenous and endogenous NO in Pb-induced toxicity in tobacco BY-2 cells, focusing on the role of NO in the generation of reactive oxygen species (ROS) as well as Pb2+ and Ca2+ fluxes using non-invasive micro-test technology (NMT). Pb treatment induced BY-2 cell death and rapid NO and ROS generation, while NO burst occurred earlier than ROS accumulation. The elimination of NO by 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) resulted in a decrease of ROS, and the supplementation of NO by sodium nitroprusside (SNP) caused an increased accumulation of ROS. Furthermore, the addition of exogenous NO stimulated Pb2+ influx, thus promoting Pb uptake in cells and aggravating Pb-induced toxicity in cells, whereas the removal of endogenous NO produced the opposite effect. Moreover, we also found that both exogenous and endogenous NO enhanced Pb-induced Ca2+ effluxes and calcium homeostasis disorder. These results suggest that exogenous and endogenous NO played a critical regulatory role in BY-2 cell death induced by Pb stress by promoting Pb2+ influx and accumulation and disturbing calcium homeostasis.

γ-Aminobutyric acid (GABA) homeostasis regulates pollen germination and polarized growth in Picea wilsonii.

γ-Aminobutyric acid (GABA) is a four-carbon non-protein amino acid found in a wide range of organisms. Recently, GABA accumulation has been shown to play a role in the stress response and cell growth in angiosperms. However, the effect of GABA deficiency on pollen tube development remains unclear. Here, we demonstrated that specific concentrations of exogenous GABA stimulated pollen tube growth in Picea wilsonii, while an overdose suppressed pollen tube elongation. The germination percentage of pollen grains and morphological variations in pollen tubes responded in a dose-dependent manner to treatment with 3-mercaptopropionic acid (3-MP), a glutamate decarboxylase inhibitor, while the inhibitory effects could be recovered in calcium-containing medium supplemented with GABA. Using immunofluorescence labeling, we found that the actin cables were disorganized in 3-MP treated cells, followed by the transition of endo/exocytosis activating sites from the apex to the whole tube shank. In addition, variations in the deposition of cell wall components were detected upon labeling with JIM5, JIM7, and aniline blue. Our results demonstrated that calcium-dependent GABA signaling regulates pollen germination and polarized tube growth in P. wilsonii by affecting actin filament patterns, vesicle trafficking, and the configuration and distribution of cell wall components.