The trans-Golgi sorting and the exocytosis of xylogalacturonan from the root border/border-like cell are conserved among monocot and dicot plant species

ABSTRACT

Root border cells lie at the interface between the root cap and the soil, secreting mucilage containing polysaccharides and molecules influencing microbial growth around the root. Border cells are sloughed off from the root surface, and the detachment is associated with secretion of xylogalacturonan (XGA). Recently, we showed that in alfalfa XGA secretion is mediated by large vesicles arising from the trans-Golgi in root cap cells. These vesicles are detected in precursor cells of border cells, but their fusion with the plasma membrane is observed only in border cells. We have now examined XGA secretion from maize border cells and Arabidopsis border-like cells using transmission electron microscopy and immunolabeling. In the root caps of both species, XGA is packaged into vesicles derived from the trans-Golgi, not in the vesicles from the trans-Golgi network as in the alfalfa root cap. Border cell-specific exocytosis of XGA was observed in the maize root suggesting that sorting and secretion of XGA in the root cap are conserved in monocot plants.

The root cap covers the growing tip of the root to protect the meristem from biotic and abiotic stresses.¹ The root cap secrets massive amounts of mucilage that facilitates penetration of the root into the soil. The mucilage also contains substances that control the microbiome in the vicinity of root surfaces.^1,2 Epidermal cells of the root cap are loosely attached, and they are released from the root cap when the root tip comes into contact with water. These cells are called border cells, and these are the type of root caps cells primarily responsible for mucilage secretion.

Polysaccharides are the main constituents of the mucilage produced in the lumen of the Golgi. Characteristics of polysaccharide synthesis are increased numbers of Golgi cisternae and swollen margins of the cisternae in the border cell. In transmission electron microscopy (TEM) images of Golgi stacks in plant cells preserved by high-pressure freezing/freeze substitution, cis, medial, and trans cisternae, and associated vesicles are clearly differentiated by their morphological features.^3–6 When we examined alfalfa root border cells and their precursor cells (i.e., root cap peripheral cells), their Golgi stacks had two more trans-type cisternae than Golgi stacks of other plant cells, and the margins of these trans cisternae were hypertrophied.^7–9

Interestingly, staining patterns of the enlarged Golgi peripheries were not uniform. Swellings of trans cisternae closer to the medial Golgi were more darkly stained than those of the trans-most cisterna, suggesting differential compositions of their luminal contents. The darkly stained swellings were observed to separate from trans cisternae, giving rise to spheroid or ovoid vesicles. These vesicles were readily distinguished from other types of vesicles by their darker staining and larger sizes.⁷

Immunogold labeling experiments with antibodies against cell wall polysaccharides revealed that the trans-Golgi derived vesicles of the alfalfa root border and peripheral cells contain epitopes of LM8, a monoclonal antibody raised against xylogalacturonan (XGA). The vesicles, however, do not contain xyloglucan (XG) or pectic polysaccharides observed in the primary cell walls of dicot plants. Instead, the polysaccharides were present in the trans-Golgi network (TGN) compartment and vesicles derived from the TGN.⁷ These results indicate that proliferation of trans cisternae and their peripheral swellings are related to the production of the XGA-carrying vesicles in the alfalfa root cap cells.

The Arabidopsis root cap sloughs off its epidermal cells, but they are shed in cell layers, unlike the root cap cells of pea or alfalfa that release individual border cells. These cells are called border-like cells,^10,11 and it was observed that some Brassicaceae family plants and flax generate border-like cells. Both border cells and border-like cells have been as shown to play roles in plant defense against pathogens.^12–14

Maize is one of the most widely cultivated monocot species. Its root cap produces large numbers of border cells, and these border cells and the mucilage that they secret constitute a protective barrier between the maize root tip and environment.¹⁵ Previous studies have suggested that maize root border cells are also involved in defense reactions.^16,17

To examine how XGA sorting and secretion happen in Golgi stacks of Arabidopsis border-like cells and maize border cells, we carried out immunogold labeling of Arabidopsis and maize root tip samples processed by high-pressure freezing/freeze substitution with the LM8 antibody. LM8 epitopes are detected in the apoplasts of border-like cells and in the border cells of Arabidopsis and maize root tips, respectively.¹⁸ Golgi stacks in border-like cells of Arabidopsis and border cells of maize exhibited morphological features very similar to those that we observed in Golgi stacks of alfalfa border cells. These cells of Arabidopsis and maize have trans cisternae with swollen and darkly stained peripheries. The staining is stronger in the trans cisternae than in the TGN (Figure 1A–C). We determined the localization of XGA in the two cell types and observed that the polysaccharide was concentrated at the swollen margins of the trans-Golgi (Figure 1D–F).

Figure 1.

Immunolocalization of XGA in alfalfa, Arabidopsis, and maize root border cells/border-like cells.

A-C. Electron micrographs showing Golgi stacks in root border cells/border-like cells. Hypertrophied margins of trans-Golgi cisternae (arrow) are more darkly stained than the TGN swellings (arrowheads). D. Electron micrograph of a Golgi stack from an alfalfa root border cell double immunogold labeled with LM8 (arrow) and α-XG (arrowhead). E-F. Electron micrographs of Golgi stacks labeled with an anti-XGA antibody, LM8 (arrows), in (E) an Arabidopsis root border-like cell and (F) a maize border cell. G-H. Electron micrographs of free vesicles carrying LM8 epitopes (arrows) in maize peripheral (G) and border (H) cells. I-J. Immunofluorescence micrograph showing XGA detected with LM8 (red) in a maize root cap sample. The boxed area in I is magnified in J. Note that LM8 epitopes are associated with cytosolic puncta. LM8-labeled puncta are detected in the cell wall only in the outer most cell layer (arrowheads in J). Cell walls were stained with Calcofluor White (blue). Scale bars in A-H indicate 200 nm. Scale bar in I indicates 20 μm. G: Golgi.

We were able to discern large spheroid vesicles labeled by LM8 in the cytosols of maize peripheral cells and border cells (Figure 1G–H). When XGA in the maize root cap was visualized by immunofluorescence microscopy, the polysaccharide was associated with cytosolic puncta that correspond to trans-Golgi and to free vesicles (Figure 1I). LM8-specific epitopes were not detected in the cell walls except for the walls of some cells at the root cap surface (Figure 1J) as we previously reported for the alfalfa root cap.⁷

The primary cell walls of Arabidopsis, alfalfa, and most angiosperm plants are classified as type I. In these cell walls, XG crosslinks cellulose microfibrils into a stable network. The primary cell wall of maize is called type II; in maize, glucuronoarabinoxylan, not XG, interlocks cellulose microfibrils.¹⁹ Furthermore, amounts of pectic polysaccharides are lower in the type II cell wall than in the type I cell wall²⁰ and mixed-linkage β-glucan abundant in the grass cell wall is produced in the Golgi.²¹ These indicate that the Golgi organization in maize cells involved in cell wall polysaccharide synthesis differs significantly from that in Arabidopsis and alfalfa cells. However, remodeling of the Golgi stack associated with the synthesis and secretion of XGA in the border/border-like cells of the three plant species seems to be conserved.

The XGA-carrying large vesicles (XGA-LVs) derived from the trans-Golgi accumulate in the cytosol of peripheral cells, but they do not fuse with the plasma membrane until the cells become exposed to the soil (Figure 2). By contrast, XG and other pectic polysaccharides contained in TGN-derived vesicles are constitutively secreted from root cap cells.⁷ Intracellular retention of XGA and its release from the root cap surface were reported in a recent study on the cell wall modification in the pea root border cell.²² Exocytosis of XGA coincides with the detachment of border cells, and this regulated secretion of XGA appears to ensure that XGA is not deposited into the cell wall of peripheral cells that are firmly attached to each other.

Figure 2.

Border cell-specific secretion of XGA.

XGA-carrying large vesicles (pink) arise from the trans-Golgi, but the vesicles are retained in the cytosol of peripheral cells. XGA-LVs fuse with the plasma membrane of the distal side of border cells, releasing XGA to the root-soil interface (red dots). Border cells are detached from the root surface to become free border cells. TGN-derived vesicles (green) contain components of the primary cell wall such as xyloglucan, and they fuse with the plasma membrane of both peripheral cells and border cells.

Our three-dimensional electron microscopy and immunogold labeling analyses uncovered a novel mode of cell wall polysaccharide secretion from the root cap that is conserved among angiosperm species analyzed. The spatial segregation of XGA epitopes in the Golgi and production of XGA-LVs provide suggest that synthesis and sorting of XGA are mediated by machinery distinct from the machinery that produces other cell wall polysaccharides that are constitutively secreted. Given that XGA is released into the extracellular matrix of cells that are separating from the root, we speculate that the functions of XGA are related to modulating the interaction of the plant with the environment rather than to conventional functions of cell wall polysaccharides such as facilitation of cell-cell adhesion.^22–24

Funding Statement

This work was supported by the Research Grants Council of Hong Kong (GRF14126116, AoE/M-05/12, C4011-14R), Direct Grant for Research (Chinese University of Hong Kong), and the Rural Development Administration, Republic of Korea (project 10953092018).