Isolation and molecular characterization of marigold AGAMOUS subfamily genes
To study the functions of AG subfamily genes in marigold, we amplified TeAG1 (991 bp, MT452648), TeAG2 (837 bp, MT452649), TeAGL11-1 (735 bp, MT394168), and TeAGL11-2 (831 bp, MT394169). Their sequences included the open reading fragment, partial 5’ untranslated region and partial 3’ untranslated region. Multiple alignment with other typical C/D proteins from model plants and Asteraceae species showed that these four proteins were typical MADS-box proteins containing MADS-domain, I-domain, K-domain with conservative amino acid residues, and AG motif I and AG motif II in C-terminal end (Fig. 1). Phylogenetic analysis using neighbor-joining (NJ) method showed that these proteins were divided into two main branches of AG and AGL11 lineages corresponding to the MADS-box class C and class D genes, respectively (Fig. 2). The first WGDs was found to have occurred during evolutionary history of marigold. Two C class proteins TeAG1 and TeAG2 were clustered to core eudicot euAG lineage, and two D class proteins TeAGL11-1 and TeAGL11-2 were clustered into AGL11 lineage (Fig. 2). TeAG1 and TeAG2 proteins are putative orthologs of Sunflower HAM45 and HAM59 proteins, respectively, both of which shared amino acid identity as high as over 85% (Table S2). TeAGL11-1 and TeAGL11-2 proteins shared high similarity with their orthologs Sunflowers HaAGL11-1 and HaAGL11-2 proteins with amino acid identity of 70.76% and 65.38%, respectively (Table S3).
Expression patterns of TeAG and TeAGL11 genes
Here, qRT-PCR was conducted to investigate the expression patterns of the four genes in marigold. In order to determine whether genes’ transcripts were stage-dependent or not, we preliminarily detected their expression levels at four stages of floral buds (FB1-FB4). The qRT-PCR analysis showed that the transcript levels of TeAG1, TeAG2, and TeAGL11-2 showed an increase tendency during floral bud development, while the expression level of TeAGL11-1 was very weak and exhibited no significant changes in four floral bud development stages (Fig. 3a, Fig. S1).
We further analyzed the expression levels of the four genes in vegetative tissues, and anthesis stage of flower organs (Fig. 3a, b; Fig. S1). The results showed that these genes were highly expressed in floral organs. The TeAG1 and TeAG2 were more preferentially expressed in reproductive organs (stamens, pistils and ovaries) than in sepals and petals. Interestingly, the transcript level of TeAG1 was significantly higher in stamens than in pistils and ovaries, while that of TeAG2 was higher in stamens and pistils than in ovaries. The expression patterns of the two AGL11 genes varied in floral organs. TeAGL11-1 had a wide expression region in disk florets, including sepals, petals, stamens, and pistils, whereas this gene was detected only in sepals and pistils of ray flowers, as well as in ovaries. Remarkably, the high expression level of TeAGL11-1 was detected in stamens. In contrast, the TeAGL11-2 was higher expressed in pistils, and ovaries than in stamens, sepals, and petals.
Subcellular localization of TeAG and TeAGL11 proteins
To gain an insight to the subcellular localization of these four genes, four fusion vectors 35S:YFP-TeAG1, 35S:YFP-TeAG2, 35S:YFP-TeAGL11-1, and 35S:YFP-TeAGL11-2 were transiently co-transformed with 35S:RFP-N7 vector into the leaf of tobacco, respectively. The fluorescence signals of these four fusion vectors were mainly observed in the nucleus outside the nucleolus (Fig. 4).
Protein interactions of TeAG and TeAGL11
To confirm the interaction among the four proteins, the yeast two-hybrid experiment was performed. Self-activation of BD constructs was assessed. The results indicated that no autoactivation was observed (Fig. S2a). Although TeAG1 and TeAG2 proteins shared a high similarity in sequences, their interaction manner with other AGAMOUS subfamily proteins were different. As shown in Table 1 and Fig. S2b, the TeAG2 formed heterodimers with TeAG1, TeAGL11-1, and TeAGL11-2, and formed homodimer with itself. However, the TeAG1 only interacted with TeAG2 and TeAGL11-1, but it formed no homodimer. The TeAGL11-1 and TeAGL11-2 showed a limited interactive ability with other AGAMOUS subfamily proteins. Neither homodimer nor heterodimer were formed through the interaction between AGL11-1 and AGL11-2. TeAGL11-1 and TeAGL11-2 interacted with TeAG2 unidirectionally. In addition, TeAGL11-1 strongly interacted with TeAG1, while TeAGL11-2 had no ability to interact with TeAG1.
Dramatic effect of overexpression of TeAG1 in Arabidopsis on sepal and petal identity
To further study the functions of TeAG1 and TeAG2, functional analyses were performed using ectopic expression in Arabidopsis. Eighteen 35S:TeAG1 transgenic lines and twenty-three 35S:TeAG2 transgenic lines were obtained. Transcript levels of TeAG1 and TeAG2 were further analyzed by semi-quantitative RT-PCR with the flower cDNA as templates (Fig. S3a, b). The 35S:TeAG2 transgenic lines did not show any evident morphological changes, compared with the wild type. However, five of the 35S:TeAG1 transgenic lines displayed severe phenotypes (named Sl-TeAG1), seven showed weak phenotypes (named Wl-TeAG1), and six had no remarkable phenotypic changes. Compared with the wild type, Sl-TeAG1 and Wl-TeAG1 transgenic lines displayed early flowering, rosette leaf curling, and small plant size (Fig. 5a, b, c, h, l, Table 2). Furthermore, only in Sl-TeAG1 transgenic lines, normal sepal and petal formations were disrupted (Fig. 5d, e, f, g, Table 2). Homeotic conversion of sepal to pistil-like structure was detected at the top margin of sepals. Similar conversion of petal to stamenoid structure was observed (Fig. 5e, g, Table 2). The sepals, petals, and stamens retained at base of siliques (Fig. 5j, k). The siliques were more bumpy and smaller, and seed setting rate was lower than those of normal siliques in wild-type lines (Fig. 5i, j, k, l, Table 2, S4).
The four whorls of floral organs from Sl-TeAG1 lines and wild-type lines were observed by SEM (Fig. 6). Compared with the sepals structure of wild type(Fig. 6a, b), a cluster of papilla-like cells occurred at the top of carpelloid sepals in transgenic lines (Fig. 6c), and the rough cells with stomata in normal sepals (Fig. 6a) were replaced by the smooth rectangle epidermis cells in adaxial surface of carpelloid sepals (Fig. 6d). In addition, the abaxial epidermis cells were converted from the normal rough types with stomata (Fig. 6b) into irregular smooth convex structure (Fig. 6e), which was similar to the epidermal cell structure of style (Fig. 6p). The second whorl of floral organs in the transgenic plants were converted into stamen-like petals with an anther-like structure (Fig. 6h) consisting of squamous cells (Fig. 6i). Furthermore, the epidermal cells in the lower region of the stamen-like petals were changed from a rough spindle structure (Fig. 6g) to a smooth filament-like structure (Fig. 6k, n). No obvious change was found in stamens and carpels in transgenic lines (Fig. 6l-u). In general, the overexpression of TeAG1 in Arabidopsis resulted in homeotic mutation of flower organs such as carpelloid sepals and stamen-like petals.
Since the phenotypes of ectopic expression of TeAG1 were visually focused on sepal and petal identity, the AP1, AP3, PI, AG, and STK genes in Arabidopsis were selected to detect whether their transcriptional levels were changed, based on the ABCDE model. The results (Fig. 6v) showed that the transcript levels of PI, AG and STK were significantly up-regulated in transgenic line Sl-TeAG1, while that of AP1 was remarkably down-regulated, suggesting that ectopic expression of TeAG1 (class C gene) might suppress the expression levels of AP1 (class A gene) in Arabidopsis. No significant difference in the transcript level of AP3 (B class gene) was observed between wild type and Sl-TeAG1 lines (Fig. 6v). Compared with the results observed in Sl-AG1 line, similar change tendency and mild expression level changes of AP1, PI, AP3, AG and STK were detected in Wl-AG1 lines (Fig. 6v).
Effect of ectopic expression of TeAGL11-1 in Arabidopsis on petals and seed development
In order to investigate the function of TeAGL11-1 and TeAGL11-2, the two genes were also ectopically expressed in Arabidopsis. We obtained twenty-one 35S:TeAGL11-1 transgenic lines with seven severe phenotype lines (Sl-TeAGL11-1), ten weak phenotype lines (Wl-TeAGL11-1), and four lines without phenotypic changes. We obtained forty-six 35S:TeAGL11-2 transgenic lines without any evident phenotypic alteration, compared with the wild-type lines. Transcript levels of TeAGL11-1 and TeAGL11-2 were further analyzed by semi-quantitative RT-PCR with flower cDNA as templates (Fig. S3c, d). The overexpression of TeAGL11-1 in Arabidopsis resulted in upward and inward curling of rosette leaves, obvious petal curling, early flowering, and small plant size (Fig. 7a, b, c, d, e, f, g, h, i, l, m, n, p, Table 2). In Sl-TeAGL11-1 lines, the siliques were almost seedless and smaller than those in wild-type lines, and the sepals were not detached from siliques (Fig. 7j, k, p, Table 2, S5). However, in Wl-TeAGL11-1 lines, only bumpy and small siliques were observed (Fig. 7j, k, o, p, Table 2).
To explore whether the phenotype was affected by the expression of the endogenous gene AP1, PI, AP3, AG, and STK regulating the floral organs and ovule development, the qRT-PCR analysis was performed in the two severe phenotype lines, two weak phenotype lines, and two wild-type lines. As shown in Fig. 7q, the transcript levels of AP1 and AP3 exhibited no significant difference among the six samples. The expression level of PI was obviously down-regulated in both Sl-TeAGL11-1and Wl-TeAGl11-1 lines, but the expression level of STK was lower in Sl-TeAGL11-1 lines than in Wl-TeAGl11-1 lines, suggesting that the seedless phenotype in Sl-TeAGL11-1 lines might be related to the downregulation of STK .
Expression profile analysis of endogenous genes related to early flowering and curled leaves
We also detected the expression level of endogenous genes related to flowering time (AP1, FT, LFY, SOC1, AG and SEP3) and curled leaves (GRF1, GRF2, GRF5, TCP3, TCP18, TCP20, and ARF2), when the transgenic and wild-type seedlings were 10 days old. As shown in Fig. 8, the expression levels of AP1, FT, SOC1, AG and SEP3 were significantly higher in all the 35S:TeAG1 transgenic seedlings than in wild-type seedings. However, the expression level of the LFY was remarkably increased in Sl-AG1 lines, and slightly increased in Wl-AG1 lines (Fig. 8a). Transcripts analysis of leaf development-related genes in 35S:TeAG1 transgenic seedlings indicated that expression levels of ARF2, GRF1, GRF5, TCP20 and TCP3 had no significant difference among the six samples (Fig. 8a), whereas the expression levels of GRF2 and TCP18 were obviously higher than those in wild-type lines, suggesting that high expression of GRF2 and TCP18 might have caused the leaf curling.
In Wl-TeAGL11-1 lines and Sl-TeAGL11-1 lines, AP1, AG, FT and SEP3 were strongly up-regulated. The expression levels of SOC1 were increased in Sl-TeAGL11-1 lines, and no significant difference in the expression level of SOC1 was observed between Wl-TeAGL11-1 lines and wild-type lines (Fig. 8b). The results suggested that AP1, AG, FT and SEP3 might contribute to the early flowering in 35S:TeAG11-1 transgenic lines. Expression levels of leaf development-related genes showed a complex expression pattern in 35S:TeAGL11-1 transgenic lines. The transcript level of TCP18 in Sl-TeAGL11-1 lines was up-regulated and obviously higher than that in wild-type lines, and slightly increased in Wl-AG11-1 lines (Fig. 8b). ARF2 and TCP3 were down-regulated in Sl-TeAGL11-1 lines, and TCP3 was significantly decreased in Wl-TeAGL11-1 lines. Expression levels of GRF1, GRF2, GRF5 and TCP20 had no significant changes, compared with those in wild type (Fig. 8b).