Arabinogalactan Proteins Are Involved in Salt-Adaptation and Vesicle Trafficking in Tobacco by-2 Cell Cultures

Abstract

Arabinogalactan proteins (AGPs) are a highly diverse family of glycoproteins that are commonly found in most plant species. However, little is known about the physiological and molecular mechanisms of their function. AGPs are involved in different biological processes such as cell differentiation, cell expansion, tissue development and somatic embryogenesis. AGPs are also involved in abiotic stress response such as salinity modulating cell wall expansion. In this study, we describe how salt-adaptation in tobacco BY-2 cell cultures induces important changes in arabinogalactan proteins distribution and contents. Using the immuno-dot blot technique with different anti-AGP antibodies (JIM13, JIM15, and others), we observed that AGPs were highly accumulated in the culture medium of salt-adapted tobacco cells, probably due to the action of phospholipases. We located these AGP epitopes using immunogold labeling in the cytoplasm associated to the endoplasmic reticulum, the golgi apparatus, and vesicles, plasma membrane and tonoplast. Our results show that salt-adaptation induced a significant reduction of the cytoplasm, plasma membrane and tonoplast content of these epitopes. Yariv reagent was added to the control and salt-adapted tobacco cell cultures, leading to cell death induction in control cells but not in salt-adapted cells. Ultrastructural and immunogold labeling revealed that cell death induced by Yariv reagent in control cells was due to the interaction of Yariv reagent with the AGPs linked to the plasma membranes. Finally, we propose a new function of AGPs as a possible sodium carrier through the mechanism of vesicle trafficking from the apoplast to the vacuoles in salt-adapted tobacco BY-2 cells. This mechanism may contribute to sodium homeostasis during salt-adaptation to high saline concentrations.

Keywords: arabinogalactan proteins; phospholipase C; salinity adaptation; vesicle trafficking; yariv reagent.

Figure 1

(A) Immuno-dot blot analysis with JIM13 and LM2 antibodies on samples prepared from culture medium of control and tobacco BY-2 cells adapted to growth in 0, 5, 10, and 15 gL⁻¹ of NaCl. Samples were collected from culture medium of cells growing at Lag, Exponential (Exp) and Stationary (Stat) phases. (B) Immuno-dot blot analysis with JIM15, JIM4, and LM2 antibodies on samples prepared from culture medium of control and 15 gL⁻¹ salt-adapted tobacco BY-2 cells. Samples were collected from culture medium of cells growing at Lag, Exponential (Exp) and Stationary (Stat) phases. (C) Immuno-dot blot analysis of arabinogalactan protein release from control and tobacco BY-2 cells adapted to growth in 15 gL⁻¹ of NaCl. The same weight of washed cells from control and adapted cells were cultured in fresh medium. After 24 h, cells were centrifuged and culture media were assayed with JIM13 and LM2 antibodies.

Figure 2

Immunogold labeling of JIM13 AGP epitope on samples of control (A,B) and salt-adapted (C–F) tobacco BY-2cells. (A) Control cells showing abundant labeling in the cytoplasm. (B) Detail of cell wall showing a high labeling at the plasma membrane and tonoplast. (C) Salt-adapted cells showing labeling in the cytoplasm and the extracellular matrix (arrow). (D) Salt-adapted cells showing labeling in the cytoplasm. (E) Detail of vesicles showing a high labeling close to the plasma membrane (arrows). (F) High labeling of JIM13 epitope in the endomembrane system. Cyt, Cytoplasm; Cw, Cell Wall; G, Golgi, ER, endoplasmic reticulum; M, Mitochondria; V, Vacuole.

Figure 3

Immunogold labeling of JIM15 AGP epitope on samples of control (A,B) and salt-adapted (C,D) tobacco BY-2 cells. (A) Detail of cell wall showing a high labeling at the plasma membrane. (B) General view of control cells showing abundant labeling in the cytoplasm. (C) Details of plasma membrane with low labeling but showing abundant vesicles with high labeling. (D) JIM15 epitope secreted to the apoplast in three-way cell-cell junction. Cyt, Cytoplasm; Cw, Cell Wall; Is, Intercellular space; M, Mitochondria; V, Vacuole.

Figure 4

Immuno-dot blot analysis with JIM13 on samples of control (C) and salt-adapted tobacco BY-2 cells (A) treated with the inhibitor of phospholipase C, U73122 and its inactive analog, U73343. Five samples per each treatment were collected from culture medium of cells growing during 24 h.

Figure 5

Cellular localization by fluorescence microscopy of Yariv precipitates on samples of control (A,B,E,F,I,J) and salt-adapted (C,D,G,H,K,L) tobacco BY-2 cells treated and non-treated with 100 μM Yariv reagent for 24 h. (A–D) DIC images; (E–H) FM4-64 fluorescence images; (I–L) Overlapping of DIC and fluorescence images. Arrows in (B,F,J) indicate the location of the precipitates of Yariv reagent.

Figure 6

Cellular localization by fluorescence microscopy of Yariv precipitates on samples of control tobacco BY-2 cells treated with 100 μM Yariv reagent for 24 h. (A) FM4-64 fluorescence images (Yariv precipitates, white squares). (B) DIC image. (C) Overlapping of DIC and fluorescence images. (D,E) A high magnification from image (A) of Yariv precipitates showing a coil structure.

Figure 7

Subcellular location of Yariv reagent precipitation by transmission electron microscopy on samples of control (A–C) and salt-adapted (D,E) tobacco BY-2 cells. (A) Ultrastructure of control tobacco cells treated with 100 μM Yariv reagent for 24 h. Yariv precipitate is located between the plasma membrane and cell wall (see square). (B) Detail from image (A) showing the coil structure of Yariv precipitate showing a coil structure formed by dense filaments. (C) A high magnification showing the detachment of plasma membrane from the cell walls (arrows) in control tobacco cells treated with 100 μM Yariv reagent for 24 h. (D,E) Ultrastructure of salt-adapted tobacco cells treated with 100 μM Yariv reagent for 24 h. Yariv reagent precipitates were not observed at the plasma membrane. Co, Coil structure of Yariv precipitates; Cyt, Cytoplasm; Cw, Cell Wall; V, Vacuoles.

Figure 8

Immunogold labeling of JIM13 AGP epitope associated with Yariv precipitates on samples of control (A–C) and salt-adapted (D) tobacco BY-2 cells. (A) JIM13 epitope was abundantly located on Yariv precipitates of control tobacco cells treated with 100 μM Yariv reagent for 24 h. (B) Detail from image (A) showing that JIM13 labeling was specifically located in the dense filament (arrow). (C) JIM13 labeling was abundantly located between the plasma membrane and cell wall (arrow). (D) salt-adapted tobacco cells treated with 100 μM Yariv reagent for 24 h showed a similar distribution of JIM13 epitope to untreated cells (see Figure 2).

Figure 9

Proposed model of AGPs as sodium carrier from the apoplast to the vacuoles in salt-adapted tobacco cells. AGPs bind calcium at the plasma membrane but it was partially removed by high sodium concentration in the apoplast. Plasma membrane linked AGPs are internalized by endocytosis transporting sodium by vesicle trafficking to the tonoplast and then liberating sodium in the vacuole. Sodium was located using the fluorochrome Asante Natrium-Green and vesicles were labeled using FM4-64. Cw, Cell Wall; Pm, Plasma membrane.