Regeneration of a Lytic Central Vacuole and of Neutral Peripheral Vacuoles Can Be Visualized by Green Fluorescent Proteins Targeted to Either Type of Vacuoles

Abstract

Protein trafficking to two different types of vacuoles was investigated in tobacco (Nicotiana tabacum cv SR1) mesophyll protoplasts using two different vacuolar green fluorescent proteins (GFPs). One GFP is targeted to a pH-neutral vacuole by the C-terminal vacuolar sorting determinant of tobacco chitinase A, whereas the other GFP is targeted to an acidic lytic vacuole by the N-terminal propeptide of barley aleurain, which contains a sequence-specific vacuolar sorting determinant. The trafficking and final accumulation in the central vacuole (CV) or in smaller peripheral vacuoles differed for the two reporter proteins, depending on the cell type. Within 2 d, evacuolated (mini-) protoplasts regenerate a large CV. Expression of the two vacuolar GFPs in miniprotoplasts indicated that the newly formed CV was a lytic vacuole, whereas neutral vacuoles always remained peripheral. Only later, once the regeneration of the CV was completed, the content of peripheral storage vacuoles could be seen to appear in the CV of a third of the cells, apparently by heterotypic fusion.

A Trafficking Pathway for Anthocyanins Overlaps with the Endoplasmic Reticulum-to-Vacuole Protein-Sorting Route in Arabidopsis and Contributes to the Formation of Vacuolar Inclusions

“…Anthocyanins Share a Golgi-Independent, Vesicular Trafficking Pathway with Proteins Targeted to the PSV The plant secretory system involves multiple pathways for the transport of proteins to the vacuole (Carter et al, 2004), and GFP fusion markers (Chalfie et al, 1994) permit distinguishing between them (Neuhaus, 2000;Di Sansebastiano et al, 2001). To establish whether the ER or ER bodies are a possible initial site of anthocyanin accumulation, as previously suggested for maize (Grotewold et al, 1998) and recently described for flavonols in Brassica and Arabidopsis tapetum cells (Hsieh and Huang, 2007), Arabidopsis seedlings transformed with GFP-HDEL (Haseloff et al, 1997), where HDEL corresponds to an ER-retention signal sequence, were grown under anthocyanin inductive conditions, with (1N) or without (2N) naringenin (Fig.…”

“…4, E-H) of transgenic Arabidopsis lines expressing vacuolar-sorting signals fused to GFP. The GFP-Chi marker, corresponding to a fusion of the GFP to the C-terminal vacuolar-sorting determinant from the barley (Hordeum vulgare) chitinase A protein, is targeted to pH-neutral PSVs directly from the ER in a Golgiindependent manner (Di Sansebastiano et al, 1998Sansebastiano et al, , 2001Fluckiger et al, 2003). Arabidopsis seedlings transgenic for 35STGFP-Chi grown in anthocyanin inductive conditions for 3 d show green fluorescence provided by GFP-Chi in discrete structures that could correspond to the ER and to small peripheral vacuoles (Fig.…”

“…To explore whether anthocyanins would also colocalize with components of the secretory pathway that utilize the TGN for transport from the ER to the vacuole, we utilized Arabidopsis lines expressing an N-terminal vacuolar-sorting determinant from the barley aleurain fused to GFP (Ale-GFP; Di Sansebastiano et al, 2001). Epidermal cells of 35STAle-GFP-expressing seedlings grown under anthocyanin inductive conditions (Fig.…”

“…GFP-HDEL (Haseloff, 1999), GFP-Chi (Di Sansebastiano et al, 1998), and Ale-GFP (Di Sansebastiano et al, 2001) were used as GFP-expressing lines. Arabidopsis (Arabidopsis thaliana) CHI (tt5-1), flavanone 3-hydroxylase (tt6), dihydroflavonol reductase (tt3), and PAP-1D seeds were obtained from the Arabidopsis Biological Resource Center.…”

Demonstration in Yeast of the Function of BP-80, a Putative Plant Vacuolar Sorting Receptor

“…Nevertheless, we had to show that this petunia aleurain sequence was sufficient for vacuolar sorting in plants by using GFP as a reporter. In our laboratory, we had already used the whole propeptide (119 amino acids) from barley aleurain fused to GFP and found that the reporter accumulated in an acidic vacuole (Di Sansebastiano et al, 2001). This necessitated the use of a more stable and brighter variant of GFP5, called GFP6, which differs from GFP5 by the mutations F64L and S65T described previously (Cormack et al, 1996).…”