The wheat grain contains pectic domains exhibiting specific spatial and development-associated distribution

Abstract

Cell walls are complex structures surrounding plant cells with a composition that varies among species and even within a species between organs, cell types and development stages. For years, cell walls in wheat grains were described as simple walls consisting mostly of arabinoxylans and mixed-linked beta glucans. Proteomic and transcriptomic studies identified enzyme families involved in the synthesis of many more cell wall polysaccharides in the wheat grains. Here we describe the discovery of pectic domains in wheat grain using monoclonal antibodies and enzymatic treatment to degrade the major cell wall polymers. Distinct spatial distributions were observed for rhamnogalacturonan I present in the endosperm and mostly in the aleurone layer and homogalacturonan especially found in the outer layers, and tight developmental regulations were unveiled. We also uncovered a massive deposition of homogalacturonan via large vesicular bodies in the seed coat (testa) beneath a thick cuticle during development. Our findings raise questions about the function of pectin in wheat grain.

Figure 1. Homogalacturonan epitopes in wheat mature grain: effect of different treatments on LM20 labeling.

A. Half wheat grain harvested at 750°D and stained with toluidine blue showing the tissues of the grain and the region where the immunofluorescence acquisitions were taken (black square). B. Differential interference contrast (DIC) showing the tissue structures. C, D, E, F, G, H, I. Immunofluorescence images localizing pectic LM20 epitope. C. The sections non treated (NT) to degrade arabinoxylans and beta-glucans exhibit labeling in the outer layers but no labeling in the endosperm. D, E. Similar results are obtained when the sections are treated only with lichenase (L) or only with xylanase (X). F,G. Incubation with lichenase and xylanase (LX) revealed LM20 labeling in the endosperm and outer layers (F) and removal of HG methylesters by Na₂CO₃ decreased LM20 labeling (G). H, I. Pectin lyase treatment which degrades HG decreased LM20 signal. al: aleurone layer, cc: cross cells, ne: nucellar epidermis, op: outer pericarp, se: starchy endosperm, t: testa,. Bars represent 250 µm for A, 50 µm for I (the same scale was applied for B, C, D, E, F, G and H).

Figure 2. Pectic epitopes in wheat mature grain are heterogeneously distributed.

A. Half wheat grain harvested at 750°D and stained with toluidine blue showing the tissues of the grain and the regions 1, 2 and 3 where the immunofluorescence acquisitions were taken. B, C, D, E, F, G, H, I, J, K L. Immunofluorescence images localizing low (or non) methylesterified HG epitope using LM19 antibody (B, C, D and E), methylesterified HG epitope using LM20 antibody (F, G, H, I), rhamnogalacturonan I backbone epitope (J) using RUI antibody or galactan and arabinan side chains epitopes using LM5 and LM6 antibodies (K and L). In C and H, immunofluorescence and DIC images are merged to identify the labeled layers, the testa (t) and outer pericarp (op) for LM19 in C, and the outer wall of the cross cells (cc) and outer pericarp for LM20 in H. The sections were all treated with lichenase and xylanase prior to immunolabeling. An heterogeneity of labeling is noticed in the starchy endosperm (se) for both antibodies, in the dorsal region 1 the starchy endosperm is not labeled and in the peripheral region 2 and central region 3, it is heterogeneously labeled. al: aleurone layer, ne: nucellar epidermis. Bars represent 250 µm for A, 50 µm for K (the same scale was applied for B, D, E, F, G, I, J and L), and 20 µm for H and C.

Figure 3. Homogalacturonan (LM20 and LM19 epitopes) deposition in developing wheat grain.

A, B, and C. Half wheat grain harvested at 45°D, 150°D and 250°D stained with toluidine blue showing the evolution of the grain tissues and the regions 1, 2 and 3 where the immunofluorescence acquisitions were taken. D, E, F, G, H, I, J, K and L. Immunofluorescence images localizing methylesterified HG epitope using LM20 and low (or non) methylesterified HG epitope using LM19 antibody. The sections were all treated with lichenase and xylanase prior to immunolabeling. D. LM20 labeling is observed from 45°D in the pericarp and especially in the epiderm of the pericarp (ep). F, G. LM20 labeling is observed only at 250°D in the testa (t), and from 250°D in the endosperm (G, H, I). J. LM19 labeling is observed from 250°D especially in the testa, the pericarp is labeled from 250°D and the starchy endosperm (se) from 350°D (K). al: aleurone layer, cc: cross cells, e: endosperm, esw: embryo sac wall, mp: mesocarp, ne: nucellar epidermis, op: outer pericarp, p: pericarp, se: starchy endosperm. Bars represent 250 µm for A, B and C and 50 µm for the remaining images (all at the same scale).

Figure 4. Rhamnogalacturonan I-related epitopes detection in developing wheat grain.

A, B, and C. Half wheat grain harvested at 45°D, 150°D and 250°D stained with toluidine blue showing the evolution of the grain tissues and the regions 1 and 2 where the immunofluorescence acquisitions were taken. D, E, F, G, H, I, J, K, L. Immunofluorescence images localizing rhamnogalacturonan I backbone epitope using RUI antibody or galactan and arabinan side chains epitopes using LM5 and LM6 antibodies. The sections were all treated with lichenase and xylanase prior to immunolabeling. D, E, F. RU1 labels weakly most of the cell layers at 250°D, labeling is later confined to the aleurone layer (al) and the starchy endosperm (se). G, H, I. LM5 epitopes are already detected in the nucellus (n) at 45°D. At 150°D, the labeling is localized in the walls of the nucellar epidermis (ne), and later in the development the aleurone layer and starchy endosperm become labeled. J, K, L. LM6 labeling is detected in the nucellar epidermis at 150°D, then in the nucellar epidermis and the the endosperm (aleurone and starchy endosperm) at 250°D and later in the development only in the endosperm. e: endosperm, mp: mesocarp, p: pericarp. Bars represent 250 µm for A, B and C and 50 µm for the remaining images (all at the same scale).

Figure 5. Transmission electron micrograph showing immunogold LM20 detection in the pericarp cell walls.

TEM of the outermost cell layer (A) and of a cell junction in the outer pericarp (B) of the wheat grain at 250°D showing labeling in cell wall and in the middle lamella of a cell junction. c: cuticle, cw: cell wall.

Figure 6. The testa at 250°D secretes homogalacturonans beneath the cuticle with spatial variations in methylesterification.

A. Lipophilic Sudan red staining of wheat grain tissues showing the reactive cuticles: a thick cuticle (c) outside the testa (t) and a thinner cuticle outside the nucellar epidermis (ne). The staining is confined to the cuticle and does not react to structures beneath the cell wall of the testa. B. Polysaccharide staining (PATAg) of the outer layer of the testa, the cuticle and the cross cells (cc) showing a thick layer beneath the cell wall (cw) reactive to PATAg. This layer is referred as sub-cell wall layer (scw). C and D. TEM of the outer layer of the testa showing immunogold labeling with LM20 in the sub-cell wall layer, and in the cell wall between the two cell layers of the testa. Large bodies (b) and smaller vesicles (v) not labeled with LM20 are seen. E, F, G. TEM of the outer layer of the testa showing immunogold labeling with LM19 in the sub-cell wall layer, and in the large bodies and vesicles. Bars represent 20 µm in A, 2 µm in B, and 1 µm in C, D, and G (E, F are at the same scale than G).