The Function and Photoregulatory Mechanisms of Cryptochromes From Moso Bamboo (Phyllostachys edulis)

Abstract

Light is one of the most important environmental factors affecting growth and geographic distribution of forestry plants. Moso bamboo is the largest temperate bamboo on earth and an important non-woody forestry species that serves not only important functions in the economy of rural areas but also carbon sequestration in the world. Due to its decades-long reproductive timing, the germplasm of moso bamboo cannot be readily improved by conventional breeding methods, arguing for a greater need to study the gene function and regulatory mechanisms of this species. We systematically studied the photoregulatory mechanisms of the moso bamboo (Phyllostachys edulis) cryptochrome 1, PheCRY1. Our results show that, similar to its Arabidopsis counterpart, the bamboo PheCRY1s are functionally restricted to the blue light inhibition of cell elongation without an apparent activity in promoting floral initiation. We demonstrate that PheCRY1s undergo light-dependent oligomerization that is inhibited by PheBIC1s, and light-dependent phosphorylation that is catalyzed by PhePPKs. We hypothesize that light-induced phosphorylation of PheCRY1s facilitate their degradation, which control availability of the PheCRY1 proteins and photosensitivity of bamboo plants. Our results demonstrate the evolutionary conservation of not only the function but also photoregulatory mechanism of PheCRY1 in this monocot forestry species.

Keywords: PheBIC; PhePPK; Phyllostachys edulis; cryptochrome; light signaling.

FIGURE 1

Bamboo PheCRY1s mediate blue light inhibition of hypocotyl elongation. (A,E) Images of 5-day-old Arabidopsis seedings grown under continuous blue light (10 μmolm^–2s^–1) or darkness. Scale bar, 1 mm. (B,F) Immunoblots showing the expression of FGFP-PheCRY1c (B) or FGFP-PheCRY1d (F) of seedlings shown in (A) or (E). PheCRY1 and HSP90 were detected with anti-GFP antibody or anti-HSP90 antibody, respectively. HSP90 is used as a loading control. (C,D) Measurements of hypocotyl length of seedlings shown in (A), (mean ± SD, n ≥ 20). (G,H) Measurements of hypocotyl length of seedlings shown in (E), (mean ± SD, n ≥ 20).

FIGURE 2

Bamboo PheCRY1 doesn’t function in the flowering time regulation in Arabidopsis. (A,B) Images of the flowering phenotypes of indicated plants grown in LD (16 h light/8 h dark) were shown. Rosette leaf numbers and days to flowering were recorded when flowering. Data were shown as mean ± SD, n ≥ 20.

FIGURE 3

Bamboo PheCRY1s undergo blue light-enhanced homo-oligomerization. (A,B) Co-immunoprecipitation (co-IP) assays showing the blue light-enhanced homo-oligomerization of PheCRY1 in HEK293T cells. Cells co-expressing Flag-PheCRY1 and Myc-PheCRY1 were kept in the dark (Blue –) or exposed to 100 μmol^–2s^–1 of blue light for 2 h (Blue +). Immunoprecipitations (IP) were performed with Flag-conjugated beads. The IP and co-IP products were detected with anti-Flag and anti-Myc antibodies, respectively. (C) Confocal images showing the photobody formation of PheCRY1 in response to blue light in HEK293T cells. Cells were kept in the dark or exposed to blue light (100 μmol m^–2 s^–1) for 1 h. Before imaging, cells were fixed in 4% formaldehyde. Hoechst staining shows the nuclei. BF, bright field; scale bar, 5 μm. (D) Quantitative analysis of PheCRY1-YFP photobody formation in (C). Photobody (%) is the percent of nuclei with photobodies among counted nuclei. The number of nuclei with photobodies and total counted nuclei was indicated above the columns.

FIGURE 4

Bamboo PheBIC1 interacts with PheCRY1s to inhibit the photo-oligomerization of PheCRY1. (A,B) Co-immunoprecipitation (co-IP) assays showing the blue light-dependent interaction of PheCRY1 and PheBIC1 in HEK293T cells. Cells co-expressing Flag-PheCRY1 and PheBIC1-GFP were kept in the dark (Blue –) or exposed to 100 μmol^–2s^–1 of blue light for 2 h (Blue +). IP were performed with Flag-conjugated beads. The IP and co-IP products were detected with anti-Flag and anti-GFP antibodies, respectively. (C) Confocal images showing the inhibition of PheBIC1 on PheCRY1 photobody formation in HEK293T cells. Cells expressing indicated plasmid pairs were kept in the dark or exposed to blue light (100 μmol m^–2 s^–1) for 1 h. Cells were fixed in 4% formaldehyde before imaging. Hoechst staining shows the nuclei. BF, bright field; scale bar, 5 μm. (D) Quantitative analysis of the photobody formation in (C). Photobody (%) is the percent of nuclei with photobodies among counted nuclei. The number of nuclei with photobodies and total counted nuclei was indicated above the columns.

FIGURE 5

Bamboo PheBIC1 promotes hypocotyl elongation in blue light. (A) Images of 5-day-old Arabidopsis seedings grown under continuous blue light (10 μmolm^–2s^–1) or darkness. Scale bar, 1 mm. (B) Immunoblots showing the expression of FGFP-PheBIC1a of seedlings shown in (A). PheBIC1 and HSP90 were detected with anti-GFP antibody or anti-HSP90 antibody, respectively. HSP90 is used as a loading control. (C,D) Measurements of hypocotyl length of seedlings shown in (A), (mean ± SD, n ≥ 20). (E,F) Immunoblots showing the phosphorylation and degradation of endogenous Arabidopsis CRY1 and CRY2 in the wild-type or FGFP-PheBIC1a transgenic Arabidopsis. 7-day-old etiolated seedlings were irradiated with 30 μmolm^–2 s^–1 of blue light for the indicated time. Anti-AtCRY1, anti-AtCRY2 and anti-HSP90 antibodies were used to probe AtCRY1, AtCRY2 or HSP90. HSP90 is used as a loading control.

FIGURE 6

Bamboo PheCRY1s undergo blue light-dependent proteolysis. (A) Immunoblots showing the degradation of PheCRY1 in transgenic Arabidopsis. 6-day-old etiolated seedlings were irradiated with 100 μmolm^–2 s^–1 of blue light for the indicated time. Two independent transgenic lines were used for analysis. Anti-Flag antibody was used to detect AtCRY2 and PheCRY1. Anti-HSP90 antibody was used to detect HSP90. HSP90 is used as a loading control. (B) Quantitative analysis of CRY protein degradation at time 0 and 12 h of blue light treatment from immunoblots shown in (A). CRY (B/D) = [CRY/HSP90]^blue/[CRY/HSP90]^dark. Data are presented as mean ± SD (n = 3 individual immunoblots).

FIGURE 7

Bamboo PhePPKb catalyzes the blue light-dependent phosphorylation of bamboo PheCRY1 in HEK293T cells. (A–C) Cells co-expressing indicated plasmid pairs were exposed to green light (G, 50 μmolm^–2 s^–1), blue light (B, 50 μmolm^–2 s^–1), red light (R, 50 μmolm^–2 s^–1), or far-red light (FR, 5 μmolm^–2 s^–1) for 60 min. Immunoblots were probed with the anti-Myc or anti-Flag antibodies. (D–F) Cells co-expressing indicated plasmids were kept in the dark (Blue –) or treated with 100 μmolm^–2 s^–1 of blue light for 2 h (Blue +). Lysates were treated without (- λ-PPase) or with λ-PPase (+ λ-PPase), and analyzed by immunoblots probed with the anti-Flag or anti-Myc antibodies. Arrowheads indicate the phosphorylated CRY.