Isolation and molecular characterization of AGMOUS subfamily genes
To study the function 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), with their sequences covering the open reading fragment, partial 5’ untranslated region and partial 3’ untranslated region. Multiple alignment with other typical C/D proteins 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 C class and D class genes, respectively (Fig. 2). The independent duplication events were also found during marigold evolutionary process. Two C class proteins of TeAG1 and TeAG2 were grouped to core eudicot euAG lineage, and two D class proteins named TeAGL11-1 and TeAGL11-2 were classified into AGL11 lineage (Fig. 2). TeAG1 and TeAG2 proteins are putative ortholog of H. annuus HAM45 and HAM59 proteins respectively, as all of them shared high amino acid identity over 85% (Table S2). TeAGL11-1 and TeAGL11-2 proteins shared higher similarity with H. annuus HaAGL11-1 and HaAGL11-2 proteins, reaching to 70.76% and 65.38% identity of amino acid, respectively (Table S3).
Expression patterns of TeAG and TeAGL11 genes
The expression pattern of genes may be related to the potential specific biological functions. Here, qRT-PCR was conducted to investigate the expression pattern of the four genes of marigold. In order to study whether genes’ transcripts were stage-dependent, we preliminarily detected their expression level among four size of floral buds (FB1-FB4). The qRT-PCR analysis showed that the transcript of TeAG1, TeAG2 and TeAGL11-2 showed increased tendency during floral bud development, while the expression level of TeAGL11-1 was very weak and had no significant changes in four floral bud development stages (Fig. 3a, Fig. S1).
We further analyzed the expression levels of the four genes in vegetable tissues and flower organs in blooming stage (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 ovules) than those in sepals and petals. Interestingly, the transcript of TeAG1 in stamens was significantly higher than that in pistils and ovules, while the TeAG2 was higher in stamens and pistils than ovules. The expression pattern of the two AGL11 genes varied in floral organs. The TeAGL11-1 has a wide expression region in disk florets, including sepals, petals, stamens and pistils, and was only detected in sepals and pistils of ray flowers, as well as in ovules. Remarkably, the high expression level of TeAGL11-1 was detected in stamens. In contrast, the TeAGL11-2 was higher expressed in pistils of ray and disk flowers and ovules than those 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 constructed fusion named 35S:YFP-TeAG1, 35S:YFP-TeAG2, 35S:YFP-TeAGL11-1, 35S:YFP-TeAGL11-2 were transiently transformed into the leaf of Nicotiana benthamiana. For the four constructs, the fluorescence signals were mainly observed in the nucleus (Fig. 4).
Protein-protein Interaction Of Teag And Teagl11 Proteins
The different protein complexes control the floral organ initiation and development based on the ABCDE-model. To confirm the interaction among the four proteins, the yeast two-hybrid experiment was performed. Self-activation of BD constructs was assessed, and no autoactivation were observed (Fig. S2a). Although TeAG1 and TeAG2 proteins shared a high similarity in sequences, they showed different interaction manner with other AGMOUS subfamily proteins. As shown in Table 1 and Fig. S2b, the TeAG2 can form heterodimers with TeAG1, TeAGL11-1 and TeAGL11-2, and homodimer with itself. While the TeAG1 only interacted with TeAG2 and TeAGL11-1, and no homodimer. The TeAGL11-1 and TeAGL11-2 showed a limited interactive ability with other AG subfamily proteins. There is neither homodimer formation nor heterodimer formation in AGL11-1 and AGL11-2 protein interaction. TeAGL11-1 and TeAGL11-2 interacted with TeAG2 in one direction. In addition, TeAGL11-1 strongly interacted with TeAG1, while TeAGL11-2 had no ability to interact with TeAG1.
Table 1
Interactions of Marigold TeAG and TeAGL11 proteins detected by yeast two-hybrid assays.
| AD-TeAG1 | TeAG2 | TeAGL11-1 | TeAGL11-2 | AD-empty | AD-T7 |
BD-TeAG1 | - | ++ | ++ | - | - | / |
BD-TeAG2 | ++ | ++ | + | ++ | - | / |
TeAGL11-1 | ++ | - | - | - | - | / |
TeAGL11-2 | - | - | - | - | - | / |
BD-empty | - | - | - | - | - | / |
BD-53 | / | / | / | / | / | / |
Note: ++, strong interaction; +, weak interaction; -, no interaction, / no done. |
Overexpression of TeAG1 in Arabidopsis has dramatic effect on sepal and petal identity
To further study the functions of TeAG1 and TeAG2, functional analyses were performed using ectopic expression in Arabidopsis. Eighteen independents of 35S:TeAG1 and twenty-three independents of 35S:TeAG2 transgenic lines were obtained. Transcript levels of TeAG1 and TeAG2 were further analyzed by semi-quantitative RT-PCR using the cDNA of flowers as templets (Fig. S3a, b). The transgenic lines harboring 35S:TeAG2 did not show any evident morphological changes compared with the wild type. Among the 35S:TeAG1 transgenic lines, five 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, smaller plant with the early flowering and curled leaves traits were observed both in Sl-TeAG1 and Wl-TeAG1 transgenic lines (Fig. 5b, c, l, Table 2). Furthermore, only in Sl-TeAG1 transgenic lines, normal sepal and petal formation were disrupted (Fig. 5e, g, Table 2). Homeotic conversion of sepal to pistil-like structure were detected at the top margin of sepals. So did the conversion between petal to stamenoid structure (Fig. 5e, g, Table 2). The sepals, petals and stamens persisted 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. 5j, k, l, Table 2).
Table 2
Mutant morphological traits of the transgenic plants via overexpression TeAG1 and TeAGL11-1.
| Rosette leaf | Flowering time | Sepal | Petal | Stamen | Pistil | Silique |
Sl-TeAG1 | Less and curled rosette leaves | Early flowerin | Carpelloid sepals | Stamen-like petals | - | - | Bumpy and small, low seed setting rate |
Wl-TeAG1 | Less and curled rosette leaves | Early flowerin | - | - | - | - | - |
Sl-TeAGL11-1 | Less and curled rosette leaves | Early flowering | Curled petal | - | - | - | Bumpy and small, almost seedless |
Wl-TeAGL11-1 | Less and curled rosette leaves | Early flowering | Curled petal | - | - | - | Bumpy and small, low seed setting rate |
Note: -, no morphological change compared with wild-type lines. |
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 taken place at the top of carpelloid sepals in transgenic lines (Fig. 6c), and the smooth rectangle epidermis cells in adaxial surface of carpelloid sepals(Fig. 6d) had instead of the rough cells with stomata in normal sepals (Fig. 6a). In addition, the abaxial epidermis cells were converted from the normal rough types with stomata (Fig. 6b) to 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 changed into stamen-like petals which had the anther-like structure (Fig. 6h) consisting of squamous cells (Fig. 6i). Furthermore, the epidermal cells in the lower region of the stamen-like petals changed from a rough spindle structure (Fig. 6g) to a smooth strip filament-like structure (Fig. 6k, n). No obvious change could be found in stamens and carpels between transgenic lines and wild-type 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 due to ectopic expression of TeAG1 were visually focused on sepal and petal identity, the AP1, AP3, PI, AG and STK genes of 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 upregulated in Sl-TeAG1 lines, while that of AP1 was remarkably downregulated, suggesting that ectopic expression of C class gene (TeAG1) may suppress the expression levels of A class gene (AP1) in Arabidopsis. The transcript level of AP3 (B class gene) displayed no significantly changes between the control and Sl-TeAG1 lines (Fig. 6v). Compared with the results observed in Sl-AG1 lines, similar but mild pattern of expression level changes of AP1, PI, AP3, AG and STK were detected in Wl-AG1 lines (Fig. 6v).
Ectopic expression of TeAGL11-1 in Arabidopsis affects 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 generated twenty-one 35S:TeAGL11-1 independent transgenic lines with seven severe phenotype lines, named Sl-TeAGL11-1, ten weak phenotype lines, named Wl-TeAGL11-1, and four lines without phenotypic changes. We obtained forty-six 35S:TeAGL11-2 independent transgenic lines without evident phenotypic alterations compared with the wild-type lines. Transcript levels of TeAGL11-1 and TeAGL11-2 were further analyzed by semi-quantitive RT-PCR using cDNA of flowers as templets (Fig. S3c, d). The overexpression of TeAGL11-1 in Arabidopsis resulted in upwardly curled rosette leaves, virtually curled petals, early flowering and small body plant (Fig. 7b, c, e, f, h, i, l, m, n, p, Table 2). In Sl-TeAGL11-1 lines, the siliques were almost seedless and smaller than that in wild-type lines, and the sepals were not detached from siliques (Fig. 7j, p, Table 2). While in Wl-TeAGL11-1 lines, only bumpy and smaller siliques were observed (Fig. 7o, p, Table 2).
To explore whether the phenotypic changes were affected by the expression of the endogenous gene AP1, PI, AP3, AG and STK, which regulated the floral organs and ovules development, the qRT-PCR analysis was carried out among the two transgenic severe phenotype lines, two weak phenotype lines and two wild-type lines. As shown in Fig. 7q, the transcript levels of AP1 and AP3 had no significantly difference among the six samples. The expression level of PI was obviously down regulated both in Sl-TeAGL11-1and Wl-TeAGl11-1 lines. Whereas, the expression level of STK in Sl-TeAGL11-1 was lower than in Wl-TeAGl11-1 line, suggesting that the seedless in Sl-TeAGL11-1 lines might related to down regulation of the STK.
Expression profile analysis of endogenous genes related to early flowering and curled leaves
We also detected the expression level of endogenous genes involved in 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 seedling were 10 days old. As shown in Fig. 8, the expression level of AP1, FT, SOC1, AG and SEP3 were significantly higher in all of 35S:TeAG1 transgenic seedlings than those in wild-type lines. However, the LFY was remarkably induced 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 level of ARF2, GRF1, GRF5, TCP20 and TCP3 had no significant changes 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 seemed to cause the curled leaves.
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 shown no visual changes in Wl-TeAGL11-1 lines compared with those in wild-type lines (Fig. 8b). The results suggested that AP1, AG, FT and SEP3 may regulated the early flowering in 35S:TeAG11-1 transgenic lines. Expression levels of leaf development-related genes shown a complex expression pattern in 35S:TeAGL11-1 transgenic lines. The transcript level of TCP18 in Sl-TeAGL11-1 lines were up-regulated and obviously higher than those in wild-type lines, and slightly increased in Wl-AG11-1 lines (Fig. 8b). The ARF2 and TCP3 were down-regulated, in which TCP3 is obviously decreased in Wl-TeAGL11-1 lines. Expression level of GRF1, GRF2, GRF5 and TCP20 had no significant changes (Fig. 8b).