Time-Course Transcriptomic Profiling of Floral Induction in Cultivated Strawberry

Abstract

The initiation and quality of flowering directly affect the time to market and economic benefit of cultivated strawberries, but the underlying mechanisms of these processes are largely unknown. To investigate the gene activity during the key period of floral induction in strawberries, time-course transcriptome analysis was performed on the shoot apex of the strawberry cultivar 'Benihoppe.' A total of 7177 differentially expressed genes (DEGs) were identified through pairwise comparisons. These DEGs were grouped into four clusters with dynamic expression patterns. By analyzing the key genes in the potential flowering pathways and the development of the leaf and flower, at least 73 DEGs that may be involved in the regulatory network of floral induction in strawberries were identified, some of which belong to the NAC, MYB, MADS, and SEB families. A variety of eight hormone signaling pathway genes that might play important roles in floral induction were analyzed. In particular, the gene encoding DELLA, a key inhibitor of the gibberellin signaling pathway, was found to be significantly differentially expressed during the floral induction. Furthermore, the differential expression of some important candidate genes, such as TFL1, SOC1, and GAI-like, was further verified by qRT-PCR. Therefore, we used this time-course transcriptome data for a preliminary exploration of the regulatory network of floral induction and to provide potential candidate genes for future studies of flowering in strawberries.

Keywords: RNA-seq; floral induction; hormone; strawberry; transcription factor.

Figure 1

Overview of floral induction in strawberry ‘Benihoppe.’ (A–E) Morphological observation of the shoot apices at different time points during the induction of flower in strawberry. The observation results of 9 w, 13 w, 14 w, 15 w, and 16 w are shown (A–E). SAM, shoot apical meristem; S, Stipule; YF, young leaf; FM, flower meristem. Scale bar = 1 mm. (F) QRT-PCR analysis of the expression of the flowering marker gene FaAP1 from 8 w to 16 w. Data are displayed as averages ± SD of three biological repeats.

Figure 2

Differential gene expression and GO enrichment analysis during flowering in strawberry. (A) Number of up-regulated and down-regulated DEGs of 13 w and 15 w. (B) Venn diagram of DEGs in the 13 w and 15 w RNA-seq libraries. (C–F) Expression of the DEGs at 9 w, 13 w, and 15 w in clusters 1−4, based on the overlaps in the Venn diagram in panel B. (G–J) GO enrichment analysis of DEGs in cluster 1 (G), cluster 2 (H), cluster 3 (I), and cluster 4 (J).

Figure 3

FPKM values of key genes belonging to the different flowering time pathways. (A) Heat map showing the expression of genes involved in the vernalization pathway. (B) Heat maps showing the expression of genes involved in the autonomous, GA, age-related, carbohydrate, and other pathways. (+) indicates the positive regulators of flower bud differentiation in Arabidopsis, and (−) indicates negative regulator. 9 w: vegetative stage, 13 w: shifts to reproductive stage at gene level, 15 w: reproductive stage. The column with the black boxes represents the student’s t test analysis of DEGs at 9 w vs. 13 w; The column with the pink boxes represents the student’s t test analysis of DEGs at 13 w vs. 15 w; The column with the blue boxes represents the student’s t test analysis of DEGs at 9 w vs. 15 w. (* |Log2FC| ≥ 1, p < 0.05. ** |Log2FC| ≥ 2, p < 0.01).

Figure 4

FPKM values of DEGs related to leaf and flower development. (A) Heat map showing the DEGs that may be involved in leaf development. (B) Heat map showing the DEGs that may be involved in flower development. The purple boxes indicate the genes in which expression was significantly changed at 13 w. 9 w: vegetative stage, 13 w: shifts to reproductive stage at gene level, 15 w: reproductive stage. The column with the black boxes represents the student’s t test analysis of DEGs at 9 w vs. 13 w; The column with the pink boxes represents the student’s t test analysis of DEGs at 13 w vs. 15 w; The column with the blue boxes represents the student’s t test analysis of DEGs at 9 w vs. 15 w. (* |Log2FC| ≥ 1, p < 0.05. ** |Log2FC| ≥ 2, p < 0.01).

Figure 5

Phylogenetic analyses and relative expression of the DEGs in the MADS and SEB families. (A) Phylogenetic analysis of DEGs in the MADS family. (B) Heatmap showing the relative expression of DEGs in the MADS family. (C) Phylogenetic analysis of DEGs in the SEB family. (D) Heatmap showing the relative expression of DEGs in the SEB family. The purple boxes indicate the genes in which expression was significantly changed at 13 w. 9 w: vegetative stage; 13 w: shifts to reproductive stage at gene level; 15 w: reproductive stage. The column with the black boxes represents the student’s t test analysis of DEGs at 9 w vs. 13 w; The column with the pink boxes represents the student’s t test analysis of DEGs at 13 w vs. 15 w; The column with the blue boxes represents the student’s t test analysis of DEGs at 9 w vs. 15 w. (* |Log2FC| ≥ 1, p < 0.05).

Figure 6

Kyoto Encyclopedia of Genes analysis of DEGs. KEGG pathway analysis of the DEGs showing the top 20 enriched pathways. The most enriched pathway—Plant hormone signal transduction is indicated by the red box.

Figure 7

DEGs involved in auxin signaling pathways. (A) Auxin signal transduction pathways by referring to the KO04075 pathway in KEGG enrichment analysis. (B) Heatmaps showing the relative expression of DEGs involved in the auxin. The purple boxes indicate the genes in which expression was significantly changed by 9 w vs. 13 w. 9 w: vegetative stage, 13 w: shifts to reproductive stage at gene level, 15 w: reproductive stage. The column with the black boxes represents the student’s t test analysis of DEGs by 9 w vs. 13 w. The column with the pink boxes represents the student’s t test analysis of DEGs by 13 w vs. 15 w; The column with the blue boxes represents the student’s t test analysis of DEGs by 9 w vs. 15 w. (* |Log2FC| ≥ 1, p < 0.05. ** |Log2FC| ≥ 2, p < 0.01).

Figure 8

DEGs involved in gibberellin, cytokinin, ethylene, and jasmonic acid signaling pathways. (A–D) Gibberellin, cytokinin, ethylene, and jasmonic acid signal transduction pathways by referring to the KO04075 pathway in KEGG enrichment analysis. (E) Heatmaps showing the relative expression of DEGs involved in the above-mentioned hormone signaling pathways. The purple boxes indicate the genes in which expression was significantly changed by 9 w vs. 13 w. 9 w: vegetative stage, 13 w: shifts to reproductive stage at gene level, 15 w: reproductive stage. The column with the black boxes represents the student’s t test analysis of DEGs by 9 w vs. 13 w; The column with the pink boxes represents the student’s t test analysis of DEGs by 13 w vs. 15 w; The column with the blue boxes represents the student’s t test analysis of DEGs by 9 w vs. 15 w. (* |Log2FC| ≥ 1, p < 0.05. ** |Log2FC| ≥ 2, p < 0.01).

Figure 9

DEGs involved in abscisic acid, brassinosteroid, and salicylic acid signaling pathways. (A–C) Abscisic acid, brassinosteroid, and salicylic acid signal transduction pathways by referring to the KO04075 pathway in KEGG enrichment analysis. (D) Heatmaps showing the relative expression of DEGs involved in the above-mentioned hormone signaling pathways. The purple boxes indicate the genes in which expression was significantly changed by 9 w vs. 13 w. 9 w: vegetative stage, 13 w: shifts to the reproductive stage at the gene level, 15 w: reproductive stage. The column with the black boxes represents the student’s t test analysis of DEGs by 9 w vs. 13 w; The column with the pink boxes represents the student’s t test analysis of DEGs by 13 w vs. 15 w; The column with the blue boxes represents the student’s t test analysis of DEGs by 9 w vs. 15 w. (* |Log2FC| ≥ 1, p < 0.05. ** |Log2FC| ≥ 2, p < 0.01).

Figure 10

QRT-PCR analysis of 24 strawberry genes that showed differential expression at 9 w, 13 w, and 15 w to verify the RNA-seq data. (A–T) QRT-PCR identification of DEGs mentioned in the above text. Error bars indicate the standard deviation (SD) for three biological replicates. (* p < 0.05, ** p < 0.01).