F-actin-dependent endocytosis of cell wall pectins in meristematic root cells. Insights from brefeldin A-induced compartments

Abstract

Brefeldin A (BFA) inhibits exocytosis but allows endocytosis, making it a valuable agent to identify molecules that recycle at cell peripheries. In plants, formation of large intracellular compartments in response to BFA treatment is a unique feature of some, but not all, cells. Here, we have analyzed assembly and distribution of BFA compartments in development- and tissue-specific contexts of growing maize (Zea mays) root apices. Surprisingly, these unique compartments formed only in meristematic cells of the root body. On the other hand, BFA compartments were absent from secretory cells of root cap periphery, metaxylem cells, and most elongating cells, all of which are active in exocytosis. We report that cell wall pectin epitopes counting rhamnogalacturonan II dimers cross-linked by borate diol diester, partially esterified (up to 40%) homogalacturonan pectins, and (1-->4)-beta-D-galactan side chains of rhamnogalacturonan I were internalized into BFA compartments. In contrast, Golgi-derived secretory (esterified up to 80%) homogalacturonan pectins localized to the cytoplasm in control cells and did not accumulate within characteristic BFA compartments. Latrunculin B-mediated depolymerization of F-actin inhibited internalization and accumulation of cell wall pectins within intracellular BFA compartments. Importantly, cold treatment and protoplasting prevented internalization of wall pectins into root cells upon BFA treatment. These observations suggest that cell wall pectins of meristematic maize root cells undergo rapid endocytosis in an F-actin-dependent manner.

Figure 1

Development- and tissue-specific distributions of RGII-borate pectins in cells of control (A) and BFA-treated (B–F) maize root apices. A, RGII-borate pectins localize preferentially to cell walls of all cells of the root apical meristem. B, In BFA-treated roots, all meristematic cells accumulate RGII-borate pectins within BFA compartments. The only exceptions to this feature are metaxylem elements (asterisk in C) and secretory cells of the root cap periphery (asterisk in D). Prominent BFA compartments are found in epidermis cells in the meristem (E) and in the apical part of the elongation region (F). Note the switch in positioning of BFA compartments in post-mitotic epidermis cells in E and F. In contrast, all other elongating root cells are devoid of BFA compartments; for cortical cells see G. The basical-apical root axis of each cell (in this and all other figures) runs from the top to bottom of the page. Stars indicate nuclei. Bar = 10 μm in A and G; 46 μm in B through D; 12 μm in E; and 15 μm in F.

Figure 2

Distributions of JIM5- (A–C), LM5- (D and E), and LM7-reactive (F and G) pectins in control (A, D, and F) and BFA-treated (B, C, E, and G) root apices. B and C, In BFA-treated root apices, JIM5-reactive pectins accumulate within BFA compartments in all meristematic cells (B) but not in elongating cells (C). D and E, LM5-reactive pectins redistribute almost completely from cell walls (D) into BFA-induced compartments (E). F and G, In contrast, LM7-reactive pectins do not accumulate within BFA compartments and remain in cell walls also in BFA-treated cells. Bar = 20 μm in A and B; 40 μm in C; and 11 μm in D through G.

Figure 3

Effects of latrunculin B (A and B) and oryzalin (D) on accumulation of JIM5-reactive pectins within BFA compartments. A and B, Cells devoid of F-actin in latrunculin B pretreated root apices do not internalize JIM5-reactive pectins both in cortex (A) and stele (B). C and D, Cells of oryzalin pretreated roots (D) form even slightly larger JIM5-positive BFA compartments than cells treated only with BFA (C). Bar = 25 μm.

Figure 4

Distribution of JIM7-reactive pectins (A and B), GA-derived AGPs (C and D), ER-based HDEL proteins (E), and PM-associated recycling proteins (F through H) in control (A) and BFA-treated (B through H) cells. In control cells, JIM7 antibody recognizes numerous spots distributed throughout the cytoplasm (A), and this pattern does not change dramatically in BFA-treated cells (B). C and D, Secretory AGPs reactive to MAC207 (C) and LM2 (D) antibodies and ER-based HDEL proteins (E) do not accumulate within BFA compartments of BFA-treated root cells. F through H, In contrast, both PM-H⁺-ATPase (F and G) and PIN1 auxin efflux carrier (H) accumulate abundantly within BFA compartments. Stars indicate nuclei. Bar = 20 μm in A through E; 11 μm in F and H; and 23 μm in G.

Figure 5

Distribution of ARF1 (A and B) and 58K (C and D) GA proteins in control (A and C) and BFA-treated (B and D) cells. Although ARF1 accumulates prominently within BFA compartments (B), 58K antibody labels spots distributed throughout the cytoplasm that correspond to GA (C), and this pattern does not change dramatically in BFA-treated cells (D). Stars indicate nuclei. Bar = 8 μm in A; 12 μm in B; and 17 μm in C and D.

Figure 6

Protoplasts of maize root apex cells do not accumulate cell wall pectins in BFA compartments. JIM5-reactive pectins (A and B), LM5-reactive pectins (C and D), and RGII-borate pectins (E and F) show the same distribution pattern both in control (A, C, and E) and BFA-treated (B, D, and F) protoplasts. Bar = 20 μm in A through E; and 34 μm in F.

Figure 7

Cold treatment (B and D) prevents internalization of cell wall pectins into BFA compartments (for BFA treatment at room temperature, see A and C). A and B, RGII-borate pectins; C and D, JIM5 pectins. Stars indicate nuclei. Bar = 8 μm in A; 10 μm in B; 12 μm in C and D.