Tools to Study Plant Organelle Biogenesis. Point Mutation Lines with Disrupted Vacuoles and High-Speed Confocal Screening of Green Fluorescent Protein-Tagged Organelles

We have focused our studies in the past several years on understanding protein trafficking from the secretory system to the vacuole—an organelle present in all plant cells. Here, we report an approach for generating and screening plants with defects in vacuolar biogenesis. Plant vacuoles are commonly known to be multifunctional organelles, and recent findings have even demonstrated a variety of new roles for vacuoles and the vesicles that deliver cargo to them. Although it has always been assumed that vacuoles are essential for plant survival, the recent isolation of a T-DNA-tagged mutant called vcl1 (vacuoleless1) has unequivocally demonstrated that vacuoles are vital organelles (Rojo et al., 2001). Mutations in the yeast (Saccharomyces cerevisiae) ortholog of VCL1, VPS16, also block vacuole biogenesis and affect all known vacuolar protein transport pathways in yeast; however, in contrast to VCL1, the VPS16 gene product is not essential (Horazdovsky and Emr, 1993). A major effect of VCL1 inactivation is that it blocks the formation of vacuoles, leading to embryonic lethality. Thus, although the isolation of vcl1 served to emphasize the importance of plant vacuoles to plant growth and development, it is difficult to gain additional information about vacuole biogenesis from an embryo lethal mutant. Some important proteins that likely mediate trafficking to the vacuole are represented by single genes in the Arabidopsis genome. For example, each of the six members of the AtC-VPS complex for which VACUOLELESS1 is a member is encoded by a single gene (Rojo et al., 2003); thus, null mutations in these genes would also most likely be lethal.