Polar localization and degradation of Arabidopsis boron transporters through distinct trafficking pathways

Abstract

Boron (B) is essential for plant growth but is toxic when present in excess. In the roots of Arabidopsis thaliana under B limitation, a boric acid channel, NIP5;1, and a boric acid/borate exporter, BOR1, are required for efficient B uptake and subsequent translocation into the xylem, respectively. However, under high-B conditions, BOR1 activity is repressed through endocytic degradation, presumably to avoid B toxicity. In this study, we investigated the localization of GFP-tagged NIP5;1 and BOR1 expressed under the control of their native promoters. Under B limitation, GFP-NIP5;1 and BOR1-GFP localized preferentially in outer (distal) and inner (proximal) plasma membrane domains, respectively, of various root cells. The polar localization of the boric acid channel and boric acid/borate exporter indicates the radial transport route of B toward the stele. Furthermore, mutational analysis revealed a requirement of tyrosine residues, in a probable cytoplasmic loop region of BOR1, for polar localization in various cells of the meristem and elongation zone. The same tyrosine residues were also required for vacuolar targeting upon high B supply. The present study of BOR1 and NIP5;1 demonstrates the importance of selective endocytic trafficking in polar localization and degradation of plant nutrient transporters for radial transport and homeostasis of plant mineral nutrients.

Fig. 1.

Polar localization of GFP-NIP5;1 and BOR1-GFP in root cells. (A–C) GFP-NIP5;1 in the root tip (A) and elongation zone (B and C) of roots under low-B conditions. (D–F) BOR1-GFP in the root tip (D), elongation zone (E), and root hair zone (F) of roots under low-B conditions. (G) BOR1-GFP and PI-stained cell wall in the root hair zone. (H) BOR1-GFP and FM4-64–stained membranes in the root hair zone. FM4-64 was applied for 3 h. en, endodermis; co, cortex; ep, epidermis. (Scale bars, 50 μm.)

Fig. 2.

B-dependent degradation of BOR1-GFP, but not GFP-NIP5;1, in roots. BOR1-GFP (A–C) or GFP-NIP5;1 (D) in plants grown on low-B medium and then transferred to low-B medium (−B) or high-B medium (+B) for the indicated time periods. (B) Colocalization of BOR1-GFP (green) and mCherry-RabF2a/Rha1 (red) in epidermal cells. (C) GFP fluorescence derived from BOR1-GFP in the vacuole of epidermal cells in the absence of light under high-B conditions but not under low-B conditions. (Scale bars, 100 μm in A and D; 10 μm in B and C).

Fig. 3.

Involvement of Y373, Y398, and Y405 in polar localization of BOR1. (A) A topology model of BOR1. (B–F) BOR1-GFP with Y-to-A mutations in root tips (Left) and elongating epidermal cells (Right) under low-B conditions. (G) BOR1(Y373A/Y398A/Y405A)-GFP in the root hair zone. en, endodermis; co, cortex; ep, epidermis. BOR1-GFP (H) and BOR1(Y373A/Y398A/Y405A)-GFP (I) in elongating epidermal cells of roots incubated with 50 μM CHX for 30 min and then with 50 μM CHX and 50 μM BFA for 2 h. (Scale bars, 50 μm.)

Fig. 4.

Lateral diffusion of BOR1-GFP, BOR1(Y373A/Y398A/Y405A)-GFP, and GFP-NIP5;1. (A, B, and D) FRAP analysis after bleaching of 2-μm regions of interest (ROIs). Recovery of BOR1-GFP (A) and BOR1(Y373A/Y398A/Y405A)-GFP (B) fluorescence at the apical and basal PMs and recovery of GFP-NIP5;1 fluorescence (D) at the outer PM in elongating epidermal cells. (C) Quantitative FRAP analysis. Data points and error bars represent averages and SD from 8 to 12 ROIs in two to three roots. CHX (50 μM) was applied for 60 min before FRAP analysis (C and D).

Fig. 5.

Involvement of Y373, Y398, and Y405 in B-dependent degradation of BOR1. (A–E) BOR1-GFP with Y-to-A mutations in plants grown on low-B medium and then transferred to low-B medium for 5 h (−B) or high-B medium for 2 h (+B2h) or 5 h (+B5h). (Scale bar, 100 μm.) (F) Immunoblot analysis of BOR1-GFP and BOR1(Y373A/Y398A/Y405A)-GFP. The plants were grown on low-B medium (−B) and transferred to high-B medium for 2 h (+B2h) or grown on high-B medium (+B).