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Flavonoids are the most important pigments for the coloration of flowers and seeds. In plant cells, flavonoids are synthesized by a multi-enzyme complex located on the cytosolic surface of the endoplasmic reticulum, and they accumulate in vacuoles. Two non-exclusive pathways have been proposed to mediate flavonoid transport to vacuoles: the membrane transporter-mediated pathway and the vesicle trafficking-mediated pathway. No molecules involved in the vesicle trafficking-mediated pathway have been identified, however. Here, we show that a membrane trafficking factor, GFS9, has a role in flavonoid accumulation in the vacuole. We screened a library of Arabidopsis thaliana mutants with defects in vesicle trafficking, and isolated the gfs9 mutant with abnormal pale tan-colored seeds caused by low flavonoid accumulation levels. gfs9 is allelic to the unidentified transparent testa mutant tt9. The responsible gene for these phenotypes encodes a previously uncharacterized protein containing a region that is conserved among eukaryotes. GFS9 is a peripheral membrane protein localized at the Golgi apparatus. GFS9 deficiency causes several membrane trafficking defects, including the mis-sorting of vacuolar proteins, vacuole fragmentation, the aggregation of enlarged vesicles, and the proliferation of autophagosome-like structures. These results suggest that GFS9 is required for vacuolar development through membrane fusion at vacuoles. Our findings introduce a concept that plants use GFS9-mediated membrane trafficking machinery for delivery of not only proteins but also phytochemicals, such as flavonoids, to vacuoles. [ABSTRACT FROM AUTHOR]

Proteins in the extracellular space (apoplast) play a crucial role at the interface between plant cells and their proximal environment. Consequently, it is not surprising that plants actively control the apoplastic proteomic profile in response to biotic and abiotic cues. Comparative quantitative proteomics of plant apoplastic fluids is therefore of general interest in plant physiology. We here describe an efficient method to isolate apoplastic fluids from Arabidopsis thaliana leaves inoculated with a nonadapted powdery mildew pathogen.

Spatiotemporal coordination of protein trafficking among organelles is essential for eukaryotic cells. The post-Golgi interface, including the trans-Golgi network (TGN), is a pivotal hub for multiple trafficking pathways. The Golgi-released independent TGN (GI-TGN) is a compartment described only in plant cells, and its cellular and physiological roles remain elusive. In Arabidopsis ( Arabidopsis thaliana ), the SYNTAXIN OF PLANTS (SYP) 4 group Qa-SNARE (soluble N -ethylmaleimide) membrane fusion proteins are shared components of TGN and GI-TGN and regulate secretory and vacuolar transport. Here we reveal that GI-TGNs mediate the transport of the R-SNARE VESICLE-ASSOCIATED MEMBRANE PROTEIN (VAMP) 721 to the plasma membrane. In interactions with a nonadapted powdery mildew pathogen, the SYP4 group of SNAREs is required for the dynamic relocation of VAMP721 to plant-fungus contact sites via GI-TGNs, thereby facilitating complex formation with its cognate SNARE partner PENETRATION1 to restrict pathogen entry. Furthermore, quantitative proteomic analysis of leaf apoplastic fluid revealed constitutive and pathogen-inducible secretion of cell wall-modification enzymes in a SYP4- and VAMP721-dependent manner. Hence, the GI-TGN acts as a transit compartment between the Golgi apparatus and the plasma membrane. We propose a model in which the GA-TGN matures into the GI-TGN and then into secretory vesicles by increasing the abundance of VAMP721-dependent secretory pathway components., (© 2019 American Society of Plant Biologists. All Rights Reserved.)