Low-temperature-induced changes in the transcriptome reveal a major role of CgSVP genes in regulating flowering of Cymbidium goeringii

Abstract

Background: Cymbidium goeringii is one of the most horticulturally important and popular ornamental plants in the orchid family (Orchidaceae). It blooms in winter during January-March and a period of low temperature is necessary for its normal flowering, otherwise there is flower bud abortion, which seriously affects the economic benefits. However, the molecular mechanism underlying winter-blooming behavior in C. goeringii is unclear.

Results: In this research, we firstly study the flowering physiology of C. goeringii by cytobiology observations and physiological experiments. Using comparative transcriptome analysis, we identified 582 differentially expressed unigenes responding to cold treatment that were involved in metabolic process, flowering time, hormone signaling, stress response, and cell cycle, implying their potential roles in regulating winter-blooming of C. goeringii. Twelve MADS-box genes among them were investigated by full-length cDNA sequence analysis and expression validation, which indicated that three genes within the SHORT VEGETATIVE PHASE (SVP) sub-group had the most significant repressed expression after cold treatment. Further analysis revealed that the SVP genes showed population variation in expression that correlated with cold-regulated flowering and responded to low temperature earlier than the flowering pathway integrators CgAP1, CgSOC1, and CgLFY, suggesting a potential role of CgSVP genes in the early stage of low-temperature-induced blooming of C. goeringii. Moreover, a yeast two-hybrid experiment confirmed that CgSVP proteins interacted with CgAP1 and CgSOC1, suggesting that they may synergistically control the process of C. goeringii flowering in winter.

Conclusions: This study represents the first exploration of flowering physiology of C. goeringii and provides gene expression information that could facilitate our understanding of molecular regulation of orchid plant winter-flowering, which could provide new insights and practical guidance for improving their flowering regulation and molecular breeding.

Keywords: Cymbidium goeringii; Flowering; Low-temperature; SVP; Transcriptome.

Fig. 1

Floral development of Cymbidium goeringii. a–e Scanning electron micrograph (SEM) of early floral developmental stages of wild-type C. goeringii. Bar = 100 mm. Im, inflorescence meristem; Fm, floral meristem; Se, sepal primordium; Pe, petal primordium; Li, lip primordium; Co, column primordium. (f-h) developing floral bud, Bar = 1 cm. f potential floral bud initiated in the lateral buds of developing shoots. g semi-endodormant floral bud. h developing bud after cold-treatment in winter. i blooming flower

Fig. 2

Cold pre-treatment and floral development stage of Cymbidium goeringii. a–c 2 representative floral buds of the plants treated with a prolonged cold condition of 0, 20, and 40 days, respectively, and then returned to normal condition. d–f Developing floral organs the plants treated with a prolonged cold condition of 0, 20, and 40 days, respectively, and then returned to normal condition. g blooming flower. Se, sepal; Pe, petal; Li, lip; Co, column, Bar = 1 cm

Fig. 3

GO classification of unigenes differentially expressed after cold treatment

Fig. 4

The quantitative RT-PCR analysis of gene expression before and after cold treatment The y-axis indicates fold change in expression among the samples. Expression levels were normalized using the threshold cycle values obtained for the Ubiquitin and Actin genes. Error bars indicate the standard deviation of the mean (SD) (n = 3). Three replicates were analyzed, with similar results. One way ANOVA with Bonferroni multiple comparison test significant at P < 0.05 between the two samples

Fig. 5

Phylogenetic analysis of the SVP-like proteins from different plant species. Amino acid sequences were aligned by the ClustalW 2.0, and phylogenetic relationships were reconstructed using a maximum-likelihood (ML) method in PHYML software with JTT amino acid substitution model. Bootstrap values for 1000 replicates were used to assess the robustness of the trees. Previously published plant SVP protein sequences were retrieved from GenBank database. Aa: Anthurium amnicola, Ao: Apostasia odorata, Bj: Brassica juncea, Br: Brassica rapa, Cc: Carya cathayensis, Ct: Citrus trifoliate, Eg, Elaeis guineensis, Ep: Erycina pusilla, Gm:Glycine max, Lp: Lolium perenne, Mh: Monotropa hypopitys, Os, Oryza sativa.(AtSVP: BAD43004, BM10: ABM21529, LpMADS10: AAZ17549, ZMM19: NP_001105148, ZMM26: NP_001105154, ZMM21: CAD23411, OsM55: BAD35842, RMD1: BAA81880, INCO: CAG27846, BrSVP: XP_009112514, EgSVP: XP_010942683, AaSVP: JAT54145, GmSVP: NP_001240951, AoSVP: AIZ95422, CtSVP: ACJ09170, EpMADS18: AJB29196, EpMADS19: AJB29197, MhAGL24: AQM52285, CcAGL24: AHI85951, AtAGL24: OAO97218, BjAGL24: AFM77904)

Fig. 6

Expression patterns of SVP genes in different cultivated varieties of Cymbidium genus (a-c).. The y-axis indicates fold change in expression among the floral buds at different developmental stages. Expression levels were normalized using the threshold cycle values obtained for the Ubiquitin and Actin genes. Error bars indicate the standard deviation of the mean (SD) (n = 3). Three replicates were analyzed, with similar results

Fig. 7

Time course cold response of CgSVP2 and floral pathway integrators. The y-axis indicates fold change in expression among the samples. Expression levels were normalized using the threshold cycle values obtained for the Ubiquitin and Actin genes. Error bars indicate the standard deviation of the mean (SD) (n = 3). Three replicates were analyzed, with similar results

Fig. 8

Transient over-expression of CgSVP2 inhibits flower bud development. a Flower bud of wild-type (WT), 35S:eGFP and 35S:CgSVP2. Photos were taken in 3 weeks after injection of Agrobacterium. b Increased expression level of CgSVP2 in the flower bud infiltrated with 35S:CgSVP2, c Appearance of floral organs in the flower bud infiltrated with 35S:CgSVP2 and 35S:eGFP. D-G: GFP signal detected after injection of Agrobacterium for 3 days (d), 5 days (e), 3 weeks (f), and 5 weeks (g) as a control to confirm continuous gene expression

Fig. 9

Protein–protein interaction among CgAP1, CgSOC1 and CgSVP proteins. Protein interaction behavior is indicated by growth on selection medium lacking leucine, tryptophan, histidine, and adenine