Regulation of flowering time by TGA7 in Arabidopsis
To reveal the function of TGA7 in controlling flowering time, we analyzed the phenotype of TGA7 using a tga7 mutant that contain a point mutation in the seventh exon (Fig. 1a). The C to T mutation led to the loss of an EcoRV site in the TGA7 gene, and resulted in an amino acid change from Ser to Leu in the TGA7 protein (Fig. 1a, b, Additional file 2). All tga7 mutant plants delayed flowering compared to WT seedlings under both LD and SD conditions (Fig. 1c, d, e), suggesting that TGA7 promoters flowering independently of the daylength conditions. We then also examined TGA7 expression in different tissues of WT plants by qRT-PCR, and found that the highest expression of TGA7 was in adult rosette leaves, and almost didn’t expression in siliques (Fig. 1f).
Autonomous Pathway and thermosensory pathway Regulated TGA7 Expression
Since TGA7 is involved in floral transition, we then examined which flowering genetic pathways may relate to TGA7 during flowering time control. The expression of TGA7 remained steady in the photoperiod pathway mutants (Fig. 2a), and the phenotype of tga7 mutant was delay flowering in LD and SD condition (Fig. 1c, d, e), suggesting that TGA7 may not be involved in the photoperiod pathway. In addition, there were almost no effects on TGA7 expression in gibberellin (GA) treatment (Fig. 2b). In both WT and FRI-Col plants, treatment of vernalization did not alter TGA7 expression (Fig. 2c). These observations suggest that the GA and vernalization pathways also did not influence TGA7. By contrast, in the autonomous pathway mutants, the TGA7 expression was increased in fca-2 and fve-4, decreased in fld-3and flk-1 (Fig. 2d), suggesting that the autonomous pathway may affect TGA7 expression.
SVP played crucial roles in the thermosensory pathway, svp-41 mutant displayed steady flowering phenotype under different temperature conditions [19]. We then also analyzed TGA7 expression in different temperature settings. TGA7 expression increased with increasing temperatures (Fig. 2e). Furthermore, TGA7 expression was steady in WT, svp-41 and 35S:SVP plants at 16℃, whilst TGA7 expression was higher in 35S:SVP but lower in svp-41 at 23℃ (Fig. 2f). These findings demonstrate that thermosensory pathway may also regulate TGA7 expression at ambient temperatures.
Transcriptomes of WT and tga7 mutant seedlings
To understand how TGA7 affects flowering time, we identified downstream genes of TGA7 that might be involved in its role in promoting flowering. To obtain a reference transcriptome for the WT and tga7 mutant seedlings, three biological replicates were used for extraction of mRNA from WT and tga7 mutant seedlings at 9 DAG, respectively. In total six RNA-seq libraries were constructed for transcriptome sequencing.
The raw data were qualified and filtered, yielding about 6.67 Gb of sequence data from each library (Additional file 3). By taking Pair-wise Pearson’s correlation coefficients analysis, three replicates of each samples indicated that the sequencing data is highly repeatable (Fig. 3a). In order to gain an overview of the variations among these sequencing data, the principal components analysis (PCA) was performed, and the values of PC1 and PC2 were 97.58 and 2.21%, respectively (Fig. 3b). The PCA clearly separated the six RNA-seq libraries into two groups, WT and tga7 mutant. The size distributions of mRNA are shown in Fig. 3c. The majority of mRNAs (84.55%) were between 500 bp and 3000 bp in length, only 1.60% of the mRNAs were > 5000 bp in length.
Identification of DEGs between WT and tga7 mutant seedlings
RPKM values were calculated to determine to the DEGs between WT and tga7 mutant seedlings at 9 DAG. Totally, 325 DEGs were identified, among them, expression of 133 genes was induced and expression of 192 genes repressed (Fig. 4a). Among the 325 DEGs, AT3G55970, AT5G45570, AT5G44590, AT5G44440, AT4G12480 were the most up-regulated genes, while AT3G01345, AT4G36700, AT3G56980, AT5G28520, AT4G36700 were the most down-regulated genes. The heatmap in Fig. 4b showed the expression profiles of the DEGs between WT and tga7 mutant seedlings. GO term enrichment analysis of these DEGs was performed and the top five largest GO terms in biological process were “photosynthesis, light harvesting in photosystem I”, “photosynthesis, light harvesting”, “protein-chromophore linkage”, “photosynthesis” and “photosynthesis, light harvesting in photosystem II”; in molecular function, “chlorophyll binding”, “protein domain specific binding”, “RNA polymerase II regulatory region sequence-specific DNA binding”, “hydrolase activity, acting on glycosyl bonds” and “carbohydrate kinase activity” were the five largest GO terms; and in cellular component, the top five largest GO terms were “photosystem I”, “photosystem II”, “plastoglobule”, “chloroplast thylakoid membrane” and “chloroplast” (Fig. 4c).
Identification Of Key Flowering Time-related Degs
A large number of genes are flowering time-related, and play vital roles in floral transition, an important turning point from vegetative growth to reproductive growth [20–22]. Among 325 DEGs which were identified between WT and tga7 mutant seedlings (Fig. 4), 4 DEGs were involved in flowering time pathways. The expression level of FLC, MAF5 and SMZ were up-regulated, while NF-YC2 was down-regulated in tga7 mutant seedlings, compare to WT seedlings (Additional file 4).
Validation Of The Expression Of Flowering Time-related Degs
To validate the expression of the 4 flowering time-related DEGs (FLC, MAF5, SMZ and NF-YC2) identified by RNA-seq (Additional file 4), three independent biological duplicates of WT and tga7 mutant seedlings collected at 9 DAG were analyzed by qRT-PCR assay. The expression levels and tendency of the four flowering-related DEGs were consistent with RNA-seq results (Fig. 5). This result suggests that the data gained by RNA-seq are reliable.