RcSRR1 interferes with the RcCSN5B-mediated deneddylation of RcCRL4 to modulate RcCO proteolysis and prevent rose flowering under red light

Abstract

Light is essential for rose (Rosa spp.) growth and development. Different light qualities play differing roles in the rose floral transition, but the molecular mechanisms underlying their effects are not fully understood. Here, we observed that red light suppresses rose flowering and increases the expression of sensitivity to red light reduced 1 (RcSRR1) compared with white light. Virus-induced gene silencing (VIGS) of RcSRR1 led to early flowering under white light and especially under red light, suggesting that this gene is a flowering repressor with a predominant function under red light. We determined that RcSRR1 interacts with the COP9 signalosome subunit 5B (RcCSN5B), while RcCSN5B, RcCOP1, and RcCO physically interact with each other. Furthermore, the RcCSN5B-induced deneddylation of Cullin4-RING E3 ubiquitin ligase (RcCRL4) in rose was reduced by the addition of RcSRR1, suggesting that the interaction between RcSRR1 and RcCSN5B relieves the deneddylation of the RcCRL4-COP1/SPA complex to enhance RcCO proteolysis, which subsequently suppresses the transcriptional activation of RcFT and ultimately flowering. Far-red light-related sequence like 1 (RcFRSL3) was shown to specifically bind to the G-box motif of the RcSRR1 promoter to repress its transcription, removing its inhibition of RcFT expression and inducing flowering. Red light inhibited RcFRSL3 expression, thereby promoting the expression of RcSRR1 to inhibit flowering. Taken together, these results provide a previously uncharacterized mechanism by which the RcFRSL3-RcSRR1-RcCSN5B module targets RcCO stability to regulate flowering under different light conditions in rose plants.

Figure 1

Red light delays flowering of R. chinensis. A. Spectral photon distributions and PPFD in WL, BL, and RL treatment. B. Flowering phenotypes and C. flowering time of rose cuttings under WL, BL, and RL. D. The relative expression levels of RcFT and E. RcCO in rose cuttings under WL, BL, and RL. The relative expression levels were determined by RT-qPCR with RcGAPDH as a reference gene. The representative result was shown from three-time repetitions. Mean value ± standard deviation was shown from at least 10 plants for flowering time, and from 3 biological replications each with three technical replications for gene expression. The different letters meant significant differences at P < 0.05 conducted with one-way ANOVA followed by Tukey’s multiple range test. WL: White light; BL: Blue light; RL: Red light

Figure 2

Silencing of RcSRR1 leads to rose early flowering under both WL and RL. A and B. Flowering phenotypes of RcSRR1-silencing rose cuttings (TRV: RcSRR1) compared with control (CK) under WL and RL. The representative result was shown from three-time repetitions. C. Flowering time of rose cuttings. D. Relative expressions of RcSRR1 and E. RcFT in CK and TRV: RcSRR1 rose cuttings under WL and RL. The relative transcriptions were determined by RT-qPCR with RcGAPDH as a reference gene. Mean value ± standard deviation was shown from at least 10 plants for flowering time, and from three biological replications each with three technical replications for gene expression. The different letters meant significant differences at P < 0.05 conducted with one-way ANOVA followed by Tukey’s multiple range test. WL: White light; RL: Red light

Figure 3

RcSRR1 physically interacts with RcCSN5B. A. Interaction assay between RcSRR1 and RcCSN5B by yeast two-hybrid. The coding sequences of RcCSN5B was inserted into pGADT7 vector containing activating domain and the coding sequence of RcSRR1 was inserted into pGBKT7 vector containing DNA-binding domain, the empty pGADT7 vector was used as negative control. The binding of pGBKT7-RcSRR1 to pGADT7-RcCSN5B was determined by yeast cell growth on synthetic dropout nutrient medium lacking Trp, Leu, His and Ade containing 20 μg/ml X-α-gal (SD/-Trp/-Leu/-His/-Ade + X-α-gal), while that growth on SD/-Leu/-Trp was used as positive control. B. Interaction of RcSRR1 and RcCSN5B in BiFC assay. The combination of nYFP-RcSRR1 + cYFP-RcCSN5B was infiltrated into leaves of genetically modified N. benthamiana carrying nucleus-localized red florescent protein (mcherry) and imaged under a confocal microscopy. The empty nYFP or cYFP co-infiltrated with corresponding construct were used as controls. YFP, yellow florescent protein. Scale bar corresponds to 20 μm. C. Interaction assay between RcSRR1 and RcCSN5B by split LUC complementation in rose seedlings. The combinations of nLUC + cLUC, nLUC + cLUC-RcCSN5B, nLUC-RcSRR1 + cLUC, nLUC-RcSRR1 + cLUC-RcCSN5B were co-infiltrated into rose seedlings and imaged by a CCD camera. All the representative results above were shown from three-time repetitions

Figure 4

RcCSN5B shows deneddylation activity on RcCUL4. A. Schematic representation of 35S: RcCUL4-GFP, 35S: RcCSN5B-GFP and 35S: RcSRR1-GFP constructs used for gene over-expression in rose seedlings. B. Neddylated and deneddylated RcCUL4 levels detected by Western Blotting in rose calli over-expressing 35S: empty-GFP or 35S: RcCSN5B-GFP. C. Ratios of neddylated/deneddylated RcCUL4 in rose calli shown in B. The asterisks represented statistically signification differences determined by Student’s t-test with *P < 0.05 as the threshold of significance. D. Neddylated and deneddylated RcCUL4 levels detected by Western Blotting in rose seedlings over-expressing 35S: RcCUL4-GFP alone, or together with 35S: RcCSN5B-GFP and 35S: RcSRR1-GFP in different combinations. Band intensities in B and D were quantified by ImageJ ver 1.53c. The representative results were shown from three-time repetitions

Figure 5

RcSRR1 and RcCSN5B antagonistically regulate RcCO protein stability. A. Schematic diagrams of 35S: RcCO-LUC, 35S: empty-GFP, 35S: RcCOP1-GFP, 35S: RcCSN5B-GFP, and 35S: RcSRR1-GFP constructs for LUC assays. B. Representative images of LUC activities representing RcCO protein abundance in rose seedlings by the over-expression of RcCO-LUC with empty-GFP, RcCOP1-GFP, RcCSN5B-GFP or RcSRR1-GFP in different combinations. C. LUC intensities presented in B measured by Andor Solis ver 4.15. Mean value ± standard deviation was shown from three replications. The different letters meant significant differences at P < 0.05 conducted with one-way ANOVA followed by Tukey’s multiple range test. D. Western blotting results showing the RcCO-LUC fusion protein levels of the combinations described above in rose seedlings. Band intensities were quantified by ImageJ ver 1.53c. All the representative results in B and D were shown from three-time repetitions

Figure 6

RcSRR1 suppresses RcFT transcription through interfering with RcCO stability. A. Schematic diagrams of proRcFT-LUC, 35S: empty-GFP, 35S: RcCO-GFP, 35S: RcCOP1-GFP, 35S: RcCSN5B-GFP, and 35S: RcSRR1-GFP constructs for LUC assays. B. Representative images of proFT-LUC activity in rose seedlings infiltrated with empty vector or co-infiltrated with the constructs shown above in different combinations. The representative result was shown from three-time repetitions. C. The LUC intensity of each treatment shown in B. measured by Andor Solis ver 4.15. Mean value ± standard deviation was shown from three replications. The different letters meant significant differences at P < 0.05 conducted with one-way ANOVA followed by Tukey’s multiple range test

Figure 7

RcFRSL3 binds to RcSRR1 promoter to inhibit its transcription. A. RcFRSL3 bound to RcSRR1 promoter in a yeast-one-hybrid assay system. The binding of RcFRSL3-prey to proRcSRR1-bait was determined by yeast cell growth on synthetic dropout nutrient medium lacking Trp, Leu and His containing 50 mM 3-AT (SD/-Trp/-Leu/-His +50 mM 3-AT), while that growth on SD/-Trp/-Leu + 50 mM 3-AT was used as positive control. B. Schematic representation image showing ChIP-qPCR regions P1 (−1848 to −1703 bp), B (−777 to −587 bp) and C (−484 to −248 bp), marked by black bars below the RcSRR1 genomic diagram. C. Relative expression level of RcFRSL3 in RcFRSL3 over-expressing rose seedlings. D. Relative enrichment levels of P1 to P3 fragments in RcFRSL3 over-expressing rose seedlings by ChIP-PCR. IgG was used as a negative control. Mean value ± standard deviation was shown from 3 biological replications (n = 3) of transient transgenic rose seedlings. The asterisks represented statistically signification differences determined by Student’s t-test with ****P < 0.0001 as the threshold of significance. E. Binding assay of RcFRSL3 to the G-box motif within RcSRR1 promoter in EMSA. F. Schematic diagrams of empty-LUC, proRcSRR1-LUC, 35S: empty-GFP, and 35S: RcFRSL3-GFP constructs used for LUC assays. G. Representative images of LUC activity in rose seedlings co-infiltrated proRcSRR1-LUC with 35S: empty-GFP or 35S: RcFRSL3-GFP. H. The LUC intensity of each treatment shown in G. measured by Andor Solis ver 4.15. Mean value ± standard deviation was shown from 3 replications (n = 3). The different letters meant significant differences at P < 0.05 conducted with one-way ANOVA followed by Tukey’s multiple range test. ND: Value not detected. The representative results above were presented from three-time repetitions

Figure 8

The silencing of RcFRSL3 delays rose flowering under both WL and RL. A and B Flowering phenotypes of RcFRSL3-silencing rose cuttings (TRV: RcFRSL3) compared with control (CK) under WL and RL. C. The flowering time and relative expressions of D RcFRSL3, E RcSRR1, and F RcFT in CK and TRV: RcFRSL3 rose cuttings under WL and RL. The relative transcriptions were determined by RT-qPCR with RcGAPDH as a reference gene. The representative result was presented from three-time repetitions. Mean value ± standard deviation was shown from at least 10 plants for flowering time, and from three biological replications each with three technical replications for gene relative expression levels. The different letters meant significant differences at P < 0.05 conducted with one-way ANOVA followed by Tukey’s multiple range test. WL: White light; RL: Red light

Figure 9

Simplified schematic model of rose flowering time regulated by RcSRR1. Under white-light conditions, RcFRSL3 binds to the RcSRR1 promoter to suppress its transcription, and releases its downstream targets to promote flowering. Under red light conditions, the repression of RcFRSL3 to the transcription of RcSRR1 is reduced with the reduction of RcFRSL3. The accumulated RcSRR1 protein associates with and interferes with RcCSN5B to decrease the RcCUL4 deneddylation, thereby enhances the E3 ubiquitin ligase activity of RcCRL4^COP1-SPA complex. This in turn promotes the RcCOP1/RcSPA-mediated RcCO proteolysis, which consequently diminishes the RcFT transcription and delays flowering of R. chinensis