The SOC1-like gene BoMADS50 is associated with the flowering of Bambusa oldhamii

Abstract

Bamboo is known for its edible shoots and beautiful texture and has considerable economic and ornamental value. Unique among traditional flowering plants, many bamboo plants undergo extensive synchronized flowering followed by large-scale death, seriously affecting the productivity and application of bamboo forests. To date, the molecular mechanism of bamboo flowering characteristics has remained unknown. In this study, a SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1)-like gene, BoMADS50, was identified from Bambusa oldhamii. BoMADS50 was highly expressed in mature leaves and the floral primordium formation period during B. oldhamii flowering and overexpression of BoMADS50 caused early flowering in transgenic rice. Moreover, BoMADS50 could interact with APETALA1/FRUITFULL (AP1/FUL)-like proteins (BoMADS14-1/2, BoMADS15-1/2) in vivo, and the expression of BoMADS50 was significantly promoted by BoMADS14-1, further indicating a synergistic effect between BoMADS50 and BoAP1/FUL-like proteins in regulating B. oldhamii flowering. We also identified four additional transcripts of BoMADS50 (BoMADS50-1/2/3/4) with different nucleotide variations. Although the protein-CDS were polymorphic, they had flowering activation functions similar to those of BoMADS50. Yeast one-hybrid and transient expression assays subsequently showed that both BoMADS50 and BoMADS50-1 bind to the promoter fragment of itself and the SHORT VEGETATIVE PHASE (SVP)-like gene BoSVP, but only BoMADS50-1 can positively induce their transcription. Therefore, nucleotide variations likely endow BoMADS50-1 with strong regulatory activity. Thus, BoMADS50 and BoMADS50-1/2/3/4 are probably important positive flowering regulators in B. oldhamii. Moreover, the functional conservatism and specificity of BoMADS50 and BoMADS50-1 might be related to the synchronized and sporadic flowering characteristics of B. oldhamii.

Fig. 1. Sequence analysis of SOC1-like proteins in different plant species.

a The amino acid sequences of SOC1-like proteins were aligned using the ClustalX 2.0 program. Identical and conserved residues are highlighted (black), and the different amino acid site mutations are marked by red stars. b A phylogenetic tree of BoMADS50 and SOC1-like proteins from other species was constructed with MEGA 7 using the maximum likelihood (ML) method with 1000 bootstrap replicates based on a multiple sequence alignment result. Bootstrap values higher than 50% are shown on the nodes. Twenty-seven SOC1-like proteins were used: six from A. thaliana (AGL14, AGL19, AGL42, AGL71, AGL72, and AtSOC1), three from rice (OsMADS50, OsMADS56, and OsMADS60), three from moso bamboo (PH02Gene29534.t1, PH02Gene23951.t1, and PH02Gene36562.t1), two from P. violascens (PvSOC1 and PvMADS56), three from R. guianensis (Rgu002887, Rgu019710, and Rgu010631), two from O. latifolia (Ola021181.1 and Ola031068.1), four from G. angustifolia (Gan021744, Gan002088, Gan031241, and Gan008725) and four from B. amplexicaulis (Bam011868.1, Bam045607.1, Bam012817.1, and Bam004823.1). BoMADS50 and BoMADS50-1/2/3/4 are marked by black triangles. c The subcellular localization of BoMADS50. Agrobacteria carrying BoMADS50 or control vector GFP were infiltrated into leaves of N. benthamiana with the nuclear maker OsART1-RFP, and the fluorescence images were taken in a dark field for green and red fluorescence, in the white field for the morphology of the cell, and in combination. Bright: bright field; GFP: GFP fluorescence; RFP: RFP fluorescence; Merged: GFP/bright/RFP field overlay. Bar = 50 μm

Fig. 2. Detection of flowering in B. oldhamii and the BoMADS50 expression pattern.

a Morphology of B. oldhamii inflorescences of different sizes and b paraffin sections with scales of 25.1 or 62.7 μm. c Morphology of floral organs in a mature flower. d and e Expression patterns of BoMADS50 in different vegetative and reproductive tissues. AM apical meristem, FP floret primordium, St stamen, An anther, Pi pistil, Le lemma, Pa palea, Lo lodicule, MP mature pollen, Ov ovule

Fig. 3. Overexpression of BoMADS50 promotes flowering in rice.

a Morphology of rice plants overexpressing BoMADS50 at flowering. Bar = 10 cm. b Morphology of seeds of BoMADS50-overexpressing rice. c Expression level of BoMADS50 in three independent lines. d Heading days and (e) height of three BoMADS50-overexpressing lines. All data are the mean ± s.d. (n ≥ 10 independent plants for each line). Asterisks indicate significantly different values (**P < 0.01). f Analysis of mRNA abundance of flowering-related genes in transgenic rice lines and WT. Asterisks indicate that the value is significantly different from that of the WT at the same time point (*P < 0.05, **P < 0.01)

Fig. 4. Protein–protein interaction of BoMADS50 and AP/FUL-like proteins by yeast two hybridization (Y2H) and bimolecular fluorescence complementation (BiFC) assays.

(a) and (b) Show that BoMADS50 could form dimers with BoMADS14-1, BoMADS14-2, BoMADS15-1, and BoMADS15-2 in both yeast cells and A. thaliana protoplasts. In (a), pGBKT7-53 and pGADT7-T, pGBKT7-Lam, and pGADT7-T were used as positive controls and negative controls, respectively. In (b), the empty vectors were used as negative controls. The cotransformation results of BoMADS50, BoMADS14-1, BoMADS14-2, BoMADS15-1, and BoMADS15-2 with the YN-P or YC-P vector are shown in Fig. S4. Scale bar = 20 μm. Bright bright field, YFP YFP fluorescence, Chl chlorophyll autofluorescence, Merged YFP/bright/Chl field overlay

Fig. 5. BoMADS14-1 promotes BoMADS50 expression.

a Diagram of the BoMADS50 promoter region containing the putative CArG motif. b BoMADS14-1 bound to the BoMADS50 promoter regions with the putative CArG motif and activated the expression of the nutritional reporter gene HIS2 by yeast one-hybrid assays. c Effector and reporter constructs used in transient dual-luciferase assays. d BoMADS14-1 triggered the expression of BoMAD50pro:LUC with the integration of the 2000-bp BoMADS50 genomic sequence upstream of the ATG before LUC. All data are the mean ± s.d. (n ≥ 3). Asterisks indicate significantly different values (**P < 0.01)

Fig. 6. BoMADS50-1 can interact with BoMADS50 and promote its own expression and BoSVP.

a The subcellular localization of BoMADS50-1. Agrobacteria carrying BoMADS50-1 or control vector GFP were infiltrated into leaves of N. benthamiana with the nuclear maker OsART1-RFP, and the fluorescence images were taken in dark fields for green and red fluorescence, in the white field for the morphology of the cell, and in combination. Scale bar = 50 μm. Bright: bright field, GFP: GFP fluorescence, RFP: RFP fluorescence, Merged: GFP/bright/RFP field overlay. b and c BoMADS50-1 could form dimers with BoMADS50 in both yeast cells and Arabidopsis protoplasts. Scale bar = 20 μm. Bright: bright field, YFP:YFP fluorescence. Chl: chlorophyll autofluorescence, Merged: YFP/bright/Chl field overlay. d Both BoMADS50 and BoMADS50-1 bound to the BoMADS50 and BoSVP promoter regions with putative CArG motifs and activated the expression of the nutritional reporter gene HIS2 in yeast one-hybrid assays. e BoMADS50-1 triggered the expression of BoMAD50pro:LUC and BoSVPpro:LUC by integration of 2000-bp BoMADS50 and BoSVP genomic sequences upstream of the ATG before LUC. The cotransformation of BoMADS50 decreased the binding ability of BoMADS50-1 to itself and BoSVP. All data are the mean ± s.d. (n ≥ 3). Asterisks indicate significantly different values (**P < 0.01)