Endocytosis is essential for the maintenance of protein and lipid compositions in the plasma membrane and for the acquisition of materials from the extracellular space. Clathrin-dependent and -independent endocytic processes are well established in yeast and animals; however, endocytic pathways involved in cargo internalization and intracellular trafficking remain to be fully elucidated for plants. Here, we used transgenic green fluorescent protein–flotillin1 (GFP-Flot1) Arabidopsis thaliana plants in combination with confocal microscopy analysis and transmission electron microscopy immunogold labeling to study the spatial and dynamic aspects of GFP-Flot1–positive vesicle formation. Vesicle size, as outlined by the gold particles, was ∼100 nm, which is larger than the 30-nm size of clathrin-coated vesicles. GFP-Flot1 also did not colocalize with clathrin light chain-mOrange. Variable-angle total internal reflection fluorescence microscopy also revealed that the dynamic behavior of GFP-Flot1–positive puncta was different from that of clathrin light chain-mOrange puncta. Furthermore, disruption of membrane microdomains caused a significant alteration in the dynamics of Flot1-positive puncta. Analysis of artificial microRNA Flot1 transgenic Arabidopsis lines established that a reduction in Flot1 transcript levels gave rise to a reduction in shoot and root meristem size plus retardation in seedling growth. Taken together, these findings support the hypothesis that, in plant cells, Flot1 is involved in a clathrin-independent endocytic pathway and functions in seedling development.

Arabidopsis thaliana respiratory burst oxidase homolog D (RbohD) functions as an essential regulator of reactive oxygen species (ROS). However, our understanding of the regulation of RbohD remains limited. By variable-angle total internal reflection fluorescence microscopy, we demonstrate that green fluorescent protein (GFP)-RbohD organizes into dynamic spots at the plasma membrane. These RbohD spots have heterogeneous diffusion coefficients and oligomerization states, as measured by photobleaching techniques. Stimulation with ionomycin and calyculin A, which activate the ROS-producing enzymatic activity of RbohD, increases the diffusion and oligomerization of RbohD. Abscisic acid and flg22 treatments also increase the diffusion coefficient and clustering of GFP-RbohD. Single-particle analysis in clathrin heavy chain2 mutants and a Flotillin1 artificial microRNA line demonstrated that clathrin- and microdomain-dependent endocytic pathways cooperatively regulate RbohD dynamics. Under salt stress, GFP-RbohD assembles into clusters and then internalizes into the cytoplasm. Dual-color fluorescence cross-correlation spectroscopy analysis further showed that salt stress stimulates RbohD endocytosis via membrane microdomains. We demonstrate that microdomain-associated RbohD spots diffuse at the membrane with high heterogeneity, and these dynamics closely relate to RbohD activity. Our results provide insight into the regulation of RbohD activity by clustering and endocytosis, which facilitate the activation of redox signaling pathways.

Summary: Fruit ripening is a developmental process that is spatio‐temporally tuned at multiple levels. Molecular dissections of the mechanisms underlying the ripening process have revealed a network encompassed by hormones, transcriptional regulators, epigenomic modifications and other regulatory elements that directly determine fruit quality and the postharvest commodity of fresh produce. Many studies have addressed the important roles of ethylene, abscisic acid (ABA) and other hormones in regulating fruit ripening. Recent studies have shown that some spontaneous mutants for tomato transcription factors (TFs) have resulted from loss‐of‐function or dominant‐negative mutations. Unlike in DNA methylation variation, the histone mark H3K27me3 may be conserved and prevents the transcriptional feedback circuit from generating autocatalytic ethylene. These observations of a network of partially redundant component indicate the need to improve our current understanding. Here, we focussed on the recent advances and future challenges in investigations of the molecular mechanisms of fruit ripening. We also identified several issues that still need to be addressed in future studies.

Reactive oxygen species (ROS) are small molecules with high oxidative activity, and are usually produced as byproducts of metabolic processes in organisms. ROS play an important role during the interaction between plant hosts and pathogenic fungi. Phytopathogenic fungi have evolved sophisticated ROS producing and scavenging systems to achieve redox homeostasis. Emerging evidences suggest that ROS derived from fungi are involved in various important aspects of the development and pathogenesis, including formation of conidia, sclerotia, conidial anastomosis tubes (CATs) and infectious structures. In this mini-review, we summarize the research progress on the redox homeostasis systems, the versatile functions of ROS in the development and pathogenesis of phytopathogenic fungi, and the regulation effects of exogenous factors on intercellular ROS and virulence of the fungal pathogens.