Expression analysis of four pseudo-response regulator (PRR) genes in Chrysanthemum morifolium under different photoperiods

Abstract

Genes encoding pseudo-response regulator (PRR) proteins play significant roles in plant circadian clocks. In this study, four genes related to flowering time were isolated from Chrysanthemum morifolium. Phylogenetic analysis showed that they are highly homologous to the counterparts of PRRs of Helianthus annuus and named as CmPRR2, CmPRR7, CmPRR37, and CmPRR73. Conserved motifs prediction indicated that most of the closely related members in the phylogenetic tree share common protein sequence motifs, suggesting functional similarities among the PRR proteins within the same subtree. In order to explore functions of the genes, we selected two Chrysanthemum varieties for comparison; that is, a short-day sensitive Zijiao and a short-day insensitive Aoyunbaixue. Compared to Aoyunbaixue, Zijiao needs 13 more days to complete the flower bud differentiation. Evidence from spatio-temporal gene expression patterns demonstrated that the CmPRRs are highly expressed in flower and stem tissues, with a growing trend across the Chrysanthemum developmental process. In addition, we also characterized the CmPRRs expression patterns and found that CmPRRs can maintain their circadian oscillation features to some extent under different photoperiod treatment conditions. These lines of evidence indicated that the four CmPRRs undergo circadian oscillation and possibly play roles in regulating the flowering time of C. morifolium.

Keywords: Chrysanthemum; Circadian clock; Flower bud differentiation; Gene expression; PRR.

Figure 1. Phylogenetic relationships and motif composition of PRRs.

(A) Multiple alignment of full-length amino acids of PRRs. (B) Schematic representation of conserved motifs in the PRR proteins.

Figure 2. Flower bud differentiation observation using paraffin section.

(A) Vegetative growth stage; (B) inflorescence primordial differentiation stage; (C) initial stage of involucres primordial differentiation stage; (D) final stage of involucres differentiation; (E) initial stage of ligulae flower differentiation; (F) final stage of ligulae flower differentiation; (G) initial stage of corolla formation; (H) secondary stage of corolla formation; (I) finish stage of corolla formation; Br, Bract; BP, Bract primordium; DFP, Disk primordium; FP, Floret primordium; GRP, Growth point; RFP, Ray floret primordium. For paraffin section operation to observe the flower bud differentiation process under the fluorescence microscope. The paraffin section operation was performed with about 10 buds per samples.

Figure 3. Expression pattern of circadian clock related genes across Zijiao and Aoyunbaixue different flower bud differentiation stages.

A-Vegetative growth stage; B-Inflorescence primordial differentiation stage; C1-Initial stage of involucres primordial differentiation stage; C2-Final stage of involucres differentiation; D1-Initial stage of ligulae flower differentiation; D2-Final stage of ligulae flower differentiation; E1-Initial stage of corolla formation; E2-Secondary stage of corolla formation; E3-Finish stage of corolla formation. Capital letters (A–P) indicate whether the samples are significantly different from each other. Data represents the mean ± SE of three technical and three biological repeats per sample.

Figure 4. Expression pattern of circadian clock genes in Zijiao different development stages.

(A) Relative expression level of CmGI1; (B) Relative expression level of CmGI2; (C) Relative expression level of CmGI3; (D) Relative expression level of CmCOL5; (E) Relative expression level of CmCOL9; (F) Relative expression level of CmCOL13; (G) Relative expression level of CmCOL14; (H) Relative expression level of CmCOL15; (I) Relative expression level of CmCOL16; (J) Relative expression level of CmCOL20; (K) Relative expression level of CmPRR2; (L) Relative expression level of CmPRR7; (M) Relative expression level of CmPRR37; (N) Relative expression level of CmPRR73. AFU, Flower bud undifferentiated stage; SQ, squaring stage; VB, visible bud color stage; IF, initial flowering stage; FB, full blossom stage; SF, senescing flower stage. Letters in (A–N) indicate whether the samples are significantly different from each other. Data represents the average ± SE of three technical and three biological repeats per sample.

Figure 5. Expression pattern of CmPRRs in different tissues of Zijiao.

(A) Relative expression level of CmPRR2; (B) Relative expression level of CmPRR7; (C) Relative expression level of CmPRR37; (D) Relative expression level of CmPRR73. Letters a–d indicate whether the samples are significantly different from each other. Data represents the average ± SE of three technical and three biological repeats per sample.

Figure 6. Expression pattern of CmPRRs of Zijiao in long-day and short-day photoperiod conditions.

(A) Relative expression level of CmPRR2; (B) Relative expression level of CmPRR7; (C) Relative expression level of CmPRR37; (D) Relative expression level of CmPRR73. LD, Long-day condition; SD, short-day condition. Data represents the average ± SE of three technical and three biological repeats per sample.

Figure 7. Expression pattern of CmPRRs of Zijiao in night-break condition.

(A) Relative expression level of CmPRR2; (B) Relative expression level of CmPRR7; (C) Relative expression level of CmPRR37; (D) Relative expression level of CmPRR73. The light break occurred at 15 h, went off at 17 h, and lasted for 2 h. NB, Night-break condition; SD, short-day condition. Data represents the average ± SE of three technical and three biological repeats per sample.

Figure 8. Expression pattern of CmPRRs of Zijiao in continuous light condition.

(A) Relative expression level of CmPRR2; (B) Relative expression level of CmPRR7; (C) Relative expression level of CmPRR37; (D) Relative expression level of CmPRR73. LD, Long-day condition; SD, short-day condition; LL, continuous light condition. Data represents the average ± SE of three technical and three biological repeats per sample.

Figure 9. Expression pattern of CmPRRs of Zijiao in continuous dark condition.

(A) Relative expression level of CmPRR2; (B) Relative expression level of CmPRR7; (C) Relative expression level of CmPRR37; (D) Relative expression level of CmPRR73. LD, Long-day condition; SD, short-day condition; DD, continuous dark condition. Data represents the average ± SE of three technical and three biological repeats per sample.