Immunolocalization and Changes of Hydroxyproline-Rich Glycoproteins During Symbiotic Germination of Dendrobium officinale

Abstract

Hydroxyproline-rich glycoproteins (HRGPs) are abundant cell wall components involved in mycorrhizal symbiosis, but little is known about their function in orchid mycorrhizal association. To gain further insight into the role of HRGPs in orchid symbiosis, the location and function of HRGPs were investigated during symbiotic germination of Dendrobium officinale. The presence of JIM11 epitope in developing protocorms was determined using immunodot blots and immunohistochemical staining procedures. Real-time PCR was also employed to verify the expression patterns of genes coding for extensin-like genes selected from the transcriptomic database. The importance of HRGPs in symbiotic germination was further investigated using 3,4-dehydro-L-proline (3,4-DHP), an inhibitor of HRGP biosynthesis. In symbiotic cultures, immunodot blots of JIM11 signals were moderate in mature seeds, and the signals became stronger in swollen embryos. After germination, signal intensities decreased in developing protocorms. In contrast, in asymbiotic cultures, JIM11 signals were much lower as compared with those stages in symbiotic cultures. Immunofluorescence staining enabled the visualization of JIM11 epitope in mature embryo and protocorm cells. Positive signals were initially localized in the larger cells near the basal (suspensor) end of uninfected embryos, marking the future colonization site of fungal hyphae. After 1 week of inoculation, the basal end of embryos had been colonized, and a strong signal was detected mostly at the mid- and basal regions of the enlarging protocorm. As protocorm development progressed, the signal was concentrated in the colonized cells at the basal end. In colonized cells, signals were present in the walls and intracellularly associated with hyphae and the pelotons. The precise localization of JIM11 epitope is further examined by immunogold labeling. In the colonized cells, gold particles were found mainly in the cell wall and the interfacial matrix near the fungal cell wall. Four extensin-like genes were verified to be highly up-regulated in symbiotically germinated protocorms as compared to asymbiotically germinated ones. The 3,4-DHP treatment inhibited the accumulation of HRGPs and symbiotic seed germination. In these protocorms, fungal hyphae could be found throughout the protocorms. Our results indicate that HRGPs play an important role in symbiotic germination. They can serve as markers for fungal colonization, establishing a symbiotic compartment and constraining fungal colonization inside the basal cells of protocorms.

Keywords: Dendrobium; hydroxyproline-rich glycoproteins; immunolocalization; mycorrhiza; symbiotic germination.

FIGURE 1

The successive developmental stages of D. officinale from seed germination to protocorm formation in symbiotic culture. (A) Stage 0, the embryos enclosed by the intact seed coats in the first week of inoculation. Scale bar = 0.5 mm. (B) Stage 1, swollen seeds after 1 week of inoculation. Scale bar = 0.5 mm. (C) Stage 2, globular protocorm rupturing the seed coat after 2 weeks of inoculation. Scale bar = 1 mm. (D) Stage3, green protocorm with the shoot tip and rhizoids after 3 weeks of inoculation. Scale bar = 1 mm.

FIGURE 2

Light micrographs of sections showing the successive developmental stages of D. officinale from seed germination to protocorm formation in symbiotic culture. (A) Stage 0, the uncolonized embryo covered by a thin seed coat in the first week of inoculation. The embryo cells filled with storage products, such as protein and lipid bodies. Scale bar = 50 μm. (B) Stage 1, the infected embryo after 1 week of inoculation. Fungal hyphae have colonized the embryo and formed the pelotons (arrows) within embryo cells. Larger vacuoles make the embryo cells expand and the storage nutrients disappear from most embryo cells. Scale bar = 50 μm. (C) Stage 2, the enlarged embryo with the rupture of the seed coat after 2 weeks of inoculation. The pelotons (arrows) locate in outer cortical cells and inner cortical cells of the spherical protocorm. Scale bar = 50 μm. (D) Stage3, protocorm with the first crest and rhizoids after 3 weeks of inoculation. At this stage, the pelotons (arrows) are digested in the inner cortical cells. c, crest; r, rhizoid. Scale bar = 100 μm.

FIGURE 3

Longitudinal sections of developing protocorms from symbiotic cultures showing the cells with the development and degeneration of fungal hyphae. (A) In the first week of inoculation, the embryo has become slightly swollen, and the fungal hyphae (arrow) have just touched the suspensor end of embryo. Within the embryo cells, the protein bodies are degenerating (double arrowhead), and the starch grains appear (arrowhead). Scale bar = 50 μm. (B) Fungal hyphae have penetrated the embryo through the suspensor end (arrow) and colonized the middle and basal cortical cells of the enlarged embryo at stage 1. Scale bar = 50 μm. (C) In a developing protocorm at stage 2, the peloton hyphae are primarily found in the middle and basal cortical cells. In the infected cortical cells, starch grains were depleted, while in the top portion of the protocorm, chloroplasts had appeared and starch grains (arrows) were noticeable within the cytoplasm. Scale bar = 50 μm. (D) As the protocorm further grows up (stage3), there are a number of collapsing peloton hyphae (arrows) and degenerated hyphal clumps (arrowheads) in the cortical cells. Scale bar = 50 μm.

FIGURE 4

Expression of genes coding for extensin-like genes in the mature seeds (S), symbiotically (SP), and asymbiotically (AP) germinated protocorms measured by real-time PCR. ^∗∗∗Indicates significant difference at P < 0.001 probability.

FIGURE 5

Semi-quantitative analysis by immunodot blots of the relative abundance of JIM11 epitope in extracts prepared from the mature seeds and developing protocorms in the symbiotic and asymbiotic cultures.

FIGURE 6

Immunofluorescence localization of JIM11 epitope during symbiotic seed germination of D. officinale. (A) The uncolonized embryo with strong signals of JIM11 epitope (green color, excitation 488 nm and emission 500–530 nm). The signals (arrows) are present mainly in the walls of the middle and basal regions of the embryo. The orange color indicates the autofluorescence (excitation 488 nm and emission 565–615 nm). Scale bar = 50 μm. (B) After 1 week of inoculation, the infected embryo showing strong signals of JIM11 epitope (arrows) in the walls of the middle and basal regions of the swollen embryo. Scale bar = 50 μm. (C) After 2 weeks of inoculation, the enlarged embryo with the rupture of the seed coat showing prominent signals of JIM 11 epitope in the pelotons (arrowheads) and the walls of colonized cells (arrows) in the basal protocorm. Scale bar = 50 μm. (D) After 3 weeks of inoculation, the signals of JIM 11 epitope (arrows) are mainly observed in the walls of colonized cells of the developing protocorm. Scale bar = 100 μm.

FIGURE 7

Immunogold staining of JIM11 epitope in the symbiotic protocorms of D. officinale. (A) In the apical portion of the protocorm, no immunogold particles were observed. Scale bar = 1 μm. (B) TEM of a basal portion of protocorm cell with fungal hyphae (H). Labeling occurred in the host cell wall (CW) and the interfacial matrix (arrowhead) or fungal cell wall. Scale bar = 1 μm. (C) TEM of a portion of protocorm cell with collapsed fungal hyphae (H). Scale bar = 1 μm. (D) The control staining. Thin sections were incubated with the mouse pre-immune IgG instead of JIM11 antibody. Immunogold particles were absent in these treatments. Scale bar = 1 μm.

FIGURE 8

The effect of 3,4-DHP on the localization of JIM11 epitope during symbiotic seed germination of D. officinale. (A) After 3 weeks of inoculation, most embryos did not enlarge, and weak signals of JIM11 epitope (arrow) are observed in the walls of basal end of the embryo. Scale bar = 50 μm. (B) After 3 weeks of inoculation, the infected embryo showing weak signals of JIM11 epitope (arrow) in the walls of basal end of the swollen embryo. Scale bar = 50 μm. (C) Most seeds in the treatment with 3,4-DHP are unable to germinate, and the severe fungal invasion in the ungerminated seeds could be commonly observed. Scale bar = 50 μm.