Identification of two distinct periods of flower bud differentiations in M. lifiiflora ‘Hongyuanbao’
M. lifiiflora ‘Hongyuanbao’ flowers in both spring and summer. The entire flower bud differentiation process was studied by microscopy observation of paraffin sections of flower buds sampled at regular intervals (Fig. 1A). In spring, ‘Hongyuanbao’ flowers from late March to the middle of April, which is relative later than the once flowering Magnolia plants. In summer, it started to flower from early June to middle August and continued to bloom for a longer period than the flowers in spring (Fig. 1B).
The flower bloomed in spring was borne primarily at the top of the raw branches from the previous year. Unlike other Magnolia, the flowering in ‘Hongyuanbao’ was rather synchronized. When the spring flowers had fallen, the axillary buds sprouted new shoots and formed flower buds at the top. The differentiation of buds occurred with simultaneous elongation of new shoots. In summer, these differentiated flower buds would bloom from early June to middle August, which lasted longer than the spring flowers. It appeared that some variations existed in the development and opening process of flower buds between spring and summer flowers, which could be attributed to the variations in plant nutritional status and environmental conditions.
Untargeted metabolomic analysis of various primary metabolites between the first and second flower bud differentiation
In order to explore the mechanism of twice flowering trait in ‘Hongyuanbao’, the primary metabolites of different flower buds periods during twice flowering were analyzed using gas chromatography-mass spectrometry (GC-MS).
In most metabolomic data analyses, compounding factors orthogonal to the variables of interest may obscure the intended class separation, and an orthogonal projection to latent structures discriminant analysis (OPLS-DA) was used as a common analytical method. In the study, OPLS-DA
was used to filter out the orthogonal variables that are not related to the classification variables and analyze both the nonorthogonal and orthogonal variables in order to investigate the relationship between the metabolites and twice flower bud differentiation(Fig. 2). The coordinates value for to1 and t1 showed a clear separation of the first predicted component between the two groups (Fig. 2D-F), and the value of the Q2 is deemed to represent the prediction ability of OPLS-DA model. In this study, Fig. 2-A was compared with Fig. 2-D, which had an overall cross-validation coefficient, Q2(y), of 58%. In addition, when Fig. 2-B was compared with Fig. 2-E, it had an overall cross-validation coefficient, of 78% and the model had an overall cross-validation coefficient, of 77% when Fig. 2-C was compared with Fig. 2-F. Thus, the OPLS-DA model can be used to reliably identify the categories, and OPLS-DA is more suitable than principal components analysis to identify the source of ‘Hongyuanbao’ samples.
To investigate the impact of twice flowering on metabolism in flower buds, the metabolic profiles of both the first and the secondary flower buds, each with five replications, were used to conduct hierarchical cluster analysis (Fig. 2). A total of 41 differential metabolites were identified (Supplementary Table 1).
For the purpose of identifying the metabolites during various stages of flower bud differentiation, the responses of all the 41 metabolites were analyzed. Based on the Mass Bank and Kyoto Encyclopedia of Genes and Genomes (KEGG) and Human Metabolome Database analyses, the metabolites were divided into six groups: Carboxylic acids and derivatives (including 12 compounds), Organooxygen compounds (13), Fatty Acyls (5), Prenol lipids (3), Indoles and derivatives (2), and other compounds (6). The levels of most sugars tended to increase compared with the twice differentiation in flower buds,while the levels of glutamate and pyroglutamate were lower in the flower buds, and the level of organic acids, such as pyruvate, tended to significantly increase. The level of malate tended to decrease in the second flower buds. Furthermore, the levels of citrate and 2-oxoglutarate were lower in the flower buds. These data suggest that many metabolites, especially sugars, are necessary for the differentiation of secondary flower buds.
The correlation analysis of metabolite-metabolite interaction network in M. lifiiflora ‘Hongyuanbao’
To study the correlation between the metabolites in different stages of flower bud differentiation in M. lifiiflora ‘Hongyuanbao’, Pearson correlation coefficients were used to calculate the data of differential metabolites between the first and secondary flower bud differentiations in three different developmental stages. MetaboAnalyst 4.0 (http://www.metaboanalyst.ca) was used to map the correlation network among metabolites. There were 22 groups of metabolites in S1, 41 groups in S2, and 25 groups in S3 that were measured and the False Discovery Rate (FDR) was 0.05. The correlation network of S2 was found to be much closer than that of S1 and S3 (Fig. 3). These data suggest that various metabolic activities of S2 are active during flower bud differentiation and we would focus on the correlations between the metabolites in S2.
KEGG Enrichment Analysis of sugar metabolites and the expression of MlTPS genes
KEGG enrichment analysis showed that sucrose and trehalose in the sucrose and starch metabolic pathways were significantly upregulated. To reveal how the sugars affect continuous flowering, genes in relevant metabolic pathways were mined and five MlTPS genes, which were related to sugar pathways, were obtained from transcriptome data. The expression of MlTPS genes during the second differentiation process was significantly upregulated as revealed by qRT-PCR analysis. Further analysis found that MlTPS1 and MlTPS7 were the most prominent in expression, and MlTPS5 was barely discernible during the second differentiation. The overall trend demonstrates that the level of expression of MlTPS genes increased significantly during the middle stage during the second flower bud differentiation (Fig. 4), suggestive of potential function role of MlTPS genes in the secondary flower bud differentiation.
Trehalose promoted early flowering
In order to confirm the effects of sucrose and trehalose on the twice flowering trait in M. lifiiflora ‘Hongyuanbao’, the plants grown in the nursery of Zhejiang A&F University, were sprayed with a solution of sucrose and trehalose (Fig. 5A). Once the plants stopped flowering, leaf spraying commenced with three concentrations (30 mM, 60 mM, 90 mM) of sucrose or trehalose. The leaves were sprayed once every 5 days and the samples were taken for microscopy observation on the 20th and 35th day following the first spraying.
Following 20 days spaying, the plants that had been sprayed with solutions of 90 mM and 30 mM of sucrose were still in the undifferentiated status, while the plants sprayed with a solution of 30 mM trehalose had reached the sepal differentiation status. Simultaneously, the flower buds of the plants sprayed with a solution of 60 mM trehalose had reached petal differentiation stage. Among the samples taken after 35 days spraying, the control (CK- plants without any treatment) was still in the stage of pistil development, but the plants that had been sprayed with solutions of 60 mM and 90 mM of trehalose had started to show flower bud differentiation. However, the plants treated with a 30 mM solution of sucrose were in the petal developmental stage, while those treated with a 60 mM solution of sucrose were in the pistil developmental stage. The plants treated with a 90 mM solution of sucrose were in the stamen developmental stage. Taken together, these results demonstrate that spraying of ‘Hongyuanbao’ leaves with different concentrations of sugars, either sucrose or trehalose, have variable effects on flower bud differentiation, with trehalose showing prominent promotion effects on the process of flower bud differentiation (Fig. 5B).
Expression of TPS genes and flowering genes under sucrose treatment
To investigate the effect of trehalose and sucrose on the levels of expression of the MlTPS genes in ‘Hongyuanbao’, the expression patterns of the MlTPS genes in ‘Hongyuanbao’ leaves that had been treated with 60 mM of trehalose or sucrose were examined (Fig. 6). It was apparent that the MlTPS genes were widely expressed throughout the flower bud differentiation period. In the treatment with a solution of 60 mM trehalose, relative to CK, the expression of MlTPS1 increased during the middle and later stage, the expression of MlTPS5 continuously increased, the expression of MlTPS6 decreased following an initial increase, while the expression of MlTPS7 and MlTPS9 remained unchanged. Under the treatment with 60 mM sucrose, the expression of MlTPS1 was barely detectable, the expression of MlTPS5 decreased following an initial increase, the expressions of MlTPS6 and MlTPS7 was always higher than that of the CK, and the MlTPS9 expression remained unchanged.
Moreover, the flowering integrators also responded to sugar treatment (Fig. 6). MlFT might have influenced the differentiation of flower buds in the beginning, particularly when the plants were sprayed with a solution of trehalose. The level of expression of MlLFY was higher in the beginning of flower differentiation compared to the later stages, suggestive of its functional role in the development of floral meristem. The expression of MlCO was raised moderately by the sugar treatments, while MlAP1 was not responsive. These results directly demonstrated that MlTPS genes could be responsive to sugar signal to regulate floral differentiation and the acceleration in flowering promotion may depend on the enhanced expression of MlFT and MlLFY.
Analysis of the expression of transcription factor SPL following sugar treatment
SPL genes have been shown to be regulated by diverse flowering signals and to form the molecular output of a pathway that regulates flowering as a function of a plant’s age . In the current studies, the age pathway gene SPL3 have been suggested as a participant in the T6P pathway and affect the process of flowering. Two MlSPL3 genes (MlSPL3-1 and MlSPL3-2) were identified from the ‘Hongyuanbao’ transcriptome. MlSPL3-1 showed significantly higher expression relative to CK under both sugar treatments, with the sucrose treatment showing relatively higher expression than the trehalose treatment. While the expression level of MlSPL3-2 did not differ significantly between the spraying treatments with trehalose and sucrose, the overall expression of MlSPL3-2 was higher following spraying with trehalose (Fig. 7). These results suggest that the T6P pathway can directly affect the expression of important flowering-time and flower-patterning genes MlSPL via the age pathway to promote the floral differentiation process.