The shoot meristem identity gene TFL1 is involved in flower development and trafficking to the protein storage vacuole

Abstract

Plants are unique in their ability to store proteins in specialized protein storage vacuoles (PSVs) within seeds and vegetative tissues. Although plants use PSV proteins during germination, before photosynthesis is fully functional, the roles of PSVs in adult vegetative tissues are not understood. Trafficking pathways to PSVs and lytic vacuoles appear to be distinct. Lytic vacuoles are analogous evolutionarily to yeast and mammalian lysosomes. However, it is unclear whether trafficking to PSVs has any analogy to pathways in yeast or mammals, nor is PSV ultrastructure known in Arabidopsis vegetative tissue. Therefore, alternative approaches are required to identify components of this pathway. Here, we show that an Arabidopsis thaliana mutant that disrupts PSV trafficking identified TERMINAL FLOWER 1 (TFL1), a shoot meristem identity gene. The tfl1-19/mtv5 (for "modified traffic to the vacuole") mutant is specifically defective in trafficking of proteins to the PSV. TFL1 localizes to endomembrane compartments and colocalizes with the putative delta-subunit of the AP-3 adapter complex. Our results suggest a developmental role for the PSV in vegetative tissues.

Fig. 1.

Floral meristems from wild-type Landsberg erecta, clavata3-2, Vac2, mtv5, and mtv5 complemented with TFL1 driven by its native promoter. (Scale bars, 2 mm.)

Fig. 2.

Trafficking defects in mtv5 mutants. (A) CLV3:T7:CTPP_BL relocalizes in mtv5. Electron micrographs show that the chimeric protein is trafficked to the vacuole in the Vac2 parental line but trafficked to the apoplasm in mtv5. Arrows designate the locations of gold particles. V, vacuole; CW, cell wall. (Scale bars, 200 nm.) (B) Col-0, Vac2, and mtv5 protoplasts. Protoplasts were incubated for 24 or 48 hours as indicated. Cells and media were separated, and proteins were analyzed in a dot-blot by using an α-T7 antibody. (C) Trafficking of a transiently expressed Arabidopsis aleurain-like protein (AtALP:GFP) is not affected by the mtv5 mutation. The 70-kDa protein is the AALP:GFP fusion, and the 27-kDa protein is GFP that was released from the 70-kDa chimeric protein upon correct delivery to the vacuole. The experiment was carried out as in B, except that protein extracts were resolved using SDS/PAGE and visualized using an α-GFP antibody.

Fig. 3.

Multiple alleles of TFL1 have trafficking defects. (A) Protoplasts were isolated from Ler, tfl1-2, tfl1-2 complemented with pASM4 (TFL1::TFL1*), tfl1-19, or tfl1-19 complemented with pNVR3068 (TFL1::TFL1). Cells were transformed with GFP:CTPP_BL and collected after 24 and 48 hours; the proteins were analyzed by SDS/PAGE and Western blotting by using α-GFP antibodies. (B) (Upper) Dot-blots of media collected after a 48-hour incubation of protoplasts from Vac2, mtv5, and tfl1-1 and tfl1-2 crossed into the Vac2 line. (Lower) Shown is a Coomassie-stained SDS/PAGE gel of the corresponding cell extracts as a loading control. (C) Vacuolar peroxidases are partially relocalized in mtv5. Immunogold-labeling experiments show that Arabidopsis vacuolar peroxidases containing CTPPs are trafficked to the vacuole and cytoplasm in the Vac2 parental line but trafficked to the cell wall and apoplasm in mtv5. Arrows designate the locations of gold particles. V, vacuole; CW, cell wall. (Scale bars, 200 nm.)

Fig. 4.

Subcellular localization of TFL1. (A) Immunoelectron microscopy of root tips and shoot meristems demonstrated that TFL1 was localized to the plasma membrane, vacuole, and dense vesicles ≈100 nm in diameter. Localization was identical in mtv5 (data not shown). V, vacuole; CW, cell wall; ve, vesicle; PM, plasma membrane. (Scale bars, 100 nm.) (B) Subcellular distribution of TFL1 protein. Proteins were extracted from protoplasts and centrifuged; the soluble and pellet fractions were analyzed by Western blotting using α-TFL1 antibodies. Anti-VSR1 and anti-aleurain antibodies were used as controls for the membrane and soluble fractions, respectively. Total protein extracts from 2-week-old 35S::TFL1 seedlings and purified recombinant 8XHis:TFL1 from Escherichia coli were used as positive controls for the α-TFL1 antibodies. (C) Protoplasts were transformed with either HA:TFL1 or AtδR:GFP constructs. The protoplasts were fixed and visualized with a TRITC-conjugated secondary antibody for HA:TFL1. GFP signals were captured directly from fixed protoplasts. (Scale bar, 20 μm.)