Expression patterns of SlMBP22
Bs MADS-box transcription factors have important regulatory functions during the evolution of the reproductive organs in seed plants (Becker, et al. 2002). Our previous report indicated that SlMBP22 may play essential roles in tomato flowers, fruits and roots development based on its expression pattern analysis (Supplementary Fig. S1) (Li, et al. 2020). To further explore the potential functions of SlMBP22 in tomato, we further evaluated its relative expression levels in flowers at different development stages and in four-whorl floral organs at the anthesis stage by qRT-PCR analysis. The results showed that the transcript abundances of SlMBP22 were higher in four days ahead of anthesis and anthesis flowers than that in two days before and after anthesis flowers (Fig. 1a). In addition, SlMBP22 transcripts were mainly abundant in the pistils of floral organs, consistent with other plant Bs MADS-box genes (Chen, et al. 2012) (Fig. 1b). These results hinted that SlMBP22 may participate in the regulation of floral organ development, especially female reproductive organ development in tomato.
Overexpression of SlMBP22 alters tomato flower morphology and affects floral organ identity genes
We successfully generated five independent transgenic OE lines that were used for further study (Supplementary Fig. S2) (Li, et al. 2020), and observed that all of these lines displayed aberrant characteristics related to reproductive parts. The most evident phenotype was that all the SlMBP22 overexpression plants displayed smaller flowers, especially the strong overexpression transgenic lines OE18 and OE2 (Fig. 1c, d). The measurements of the lengths of the four types of floral organs (sepals, petals, stamens and pistils) indicated significant reductions in the strong overexpression transgenic plants than those in the wild-type plants (Fig. 1e). Of particular note, the strong overexpression transgenic sepals were extremely abnormal in development, their color was a lighter green, the size was reduced by approximately 61% to 63% and then could not wrap the petals, when compared to the equivalent organs in the wild-type plants. Also, the petals of the SlMBP22-OE plants were more yellow than those of the WT, and had curly edges (Fig. 1c, d). To determine whether the light green sepal phenotype represented a change in total chlorophyll content, we extracted chlorophyll from sepals of fully opened flowers and observed that the WT plants possessed higher chlorophyll levels compared with the strong SlMBP22-OE lines (Fig. 1f). Furthermore, the expression levels of genes related to chlorophyll biosynthesis and degradation, CHLH, CHLM, CAO1 and SGR1 (Hu, et al. 2011), were examined in sepals of both the wild-type and SlMBP22-OE transgenic plants by qRT-PCR analysis. The results showed that these genes were dramatically down-regulated in the transgenic plants (Supplementary Fig. S3).
MADS-box family genes, the major members of plant floral organ identity genes, have a central role in the regulation of flower development. Based on the functions of MADS-box genes in floral organs, these genes are subdivided into five classes according to the ABCDE model (Deng, et al. 2012; Smaczniak, et al. 2012; Theissen 2001). Considering that overexpressing SlMBP22 caused defects in flower morphology, the expression levels of floral organ identity genes were assessed by qRT-PCR. As shown in Fig. 2a, the mRNA level of MC, one of the A-class genes and plays an essential role in the sepal development and inflorescence determinacy (Vrebalov, et al. 2002), was sharply downregulated in both sepals and pistils in the SlMBP22 overexpression plants compared to the WT. TAG1 and TAGL1, two tomato C-class floral organ identity genes, are orthologs of AGAMOUS (AG) and SHATTERPROOF1/2 (SHP1/SHP2) genes of Arabidopsis, respectively (Gimenez, et al. 2010; Pnueli, et al. 1994; Vrebalov, et al. 2009). In the overexpression transgenic plants, TAG1 expression was dramatically upregulated in the pistils, but the TAGL1 expression was greatly downregulated in both the stamens and pistils when compared with the WT (Fig. 2b, c). The transcript level of SlMBP3, one member of the class D MADS-box genes and specifies carpel/ovule identity, was evidently increased in the transgenic pistils compared to the wild-type (Fig. 2d). Two E-class genes, TM5 and TM29, participate in the maintenance of floral meristem identity and the regulation of floral organ development (Ampomah-Dwamena, et al. 2002; Pnueli, et al. 1994). Relative to WT, the TM5 showed increased expressions in the transgenic sepals, stamens and pistils (Fig. 2e). Fig. 2f showed that the transcript for the TM29 was much lower in the transgenic pistils than that in the wild-type. Our results suggested that overexpression of SlMBP22 leading to the morphological alterations of flowers might be attributed to the changes in the expressions of the floral organ identity genes.
Numerousresearches have demonstrated that MADS-box transcription factors carry out their functions in flower development by forming dimers or higher-order complexes (Tonaco, et al. 2006). Subsequently, a yeast two-hybrid assay was performed to assess the self-activation of pGBKT7-SlMBP22 and to confirm the interactions between the SlMBP22 and other floral homeotic MADS-box proteins. As shown in Fig. 2g, no autoactivation activity was detected on SD/-Leu-Trp-Ade-His and SD/-Leu-Trp-Ade-His containing X-α-Gal plates. Besides, SlMBP22 could physically interact with MC, TM5 and TM29 but not with TAGL1, SlMBP3 in yeast. These results suggested that SlMBP22 may carry out its role in flower development by forming dimers with MC, TM5 and TM29, respectively.
Overexpression of SlMBP22 results in reduced fecundity in tomato
The relatively high mRNA accumulation of SlMBP22 in tomato fruit suggested the possibility of additional functions in fruit development (Supplementary Fig. S1) (Li, et al. 2020). Thus, the effects of the overexpression of the SlMBP22 on fruit development were then investigated in the transgenic OE lines. We found another remarkable phenotype was that the strong overexpression lines (OE18 and OE2) could not bear fruit, whereas the mild overexpression transgenic lines (OE17, OE12 and OE14) showed reduced fruit size and produced fewer seeds. Occasionally, the strong overexpression line OE2 produced a much smaller fruit, while could not expand as normally as the wild type (Fig. 3a, b).
To further compare fruit development, some parameters were measured, including fruit weight, fruit volume, fruit diameter, pericarp thickness and the number of seeds in the B4 (4 d after breaker) stage fruits. As shown in Fig. 3c-e, the mild overexpression of SlMBP22 resulted in significant reductions in fruit weight, fruit volume and fruit diameter when compared with those in the control fruits. Moreover, the pericarp thickness of fruits was also measured, and the result showed that the OE lines showed thinner pericarp tissues than WT plants (Fig. 3f). Additionally, compared to WT, the seed number per fruit of the mild overexpression transgenic plants were reduced by approximately 62% to 68% (Fig. 3g). Previous studies indicate that there are close relationships between fruit size and seed number per fruit (Hussain, et al. 2020), and then we speculate that the notable differences in fruit size between the mild SlMBP22-OE lines and WT plants might be attributed to significantly reduced pericarp thickness and seed yield.
Additionally, to further investigate whether the reduced fertility of transgenic flowers is a result of the defects in either the male or the female parts, cross-pollination experiments were conducted between wild-type and transgenic plants. The results showed that failed fertilization occurred by crosses between the wild-type pollen and strong transgenic pistils (data not shown), while seeds were produced successfully when mild overexpression transgenic flowers and wild-type flowers were respectively crossed with wild-type pollen and strong overexpression transgenic pollen (Fig. 3h). Besides, pollen viability was detected by TTC staining, and the result suggested that the strong overexpression transgenic pollen stained similarly to the WT pollen (Fig. 3i), hinting that the pollen viability may not be affected in the transgenic plants. Moreover, less viable pollen grains were observed in the strong overexpression transgenic flowers than in the WT flowers (data not shown). Subsequently, the relative transcript accumulation of pollen development-related genes SlCRK1 (Kim, et al. 2014), SlPRALF (Covey, et al. 2010), LePRK3 (Kim, et al. 2002), SlPMEI (Kim, et al. 2013) were also examined by qRT-PCR assay. Intriguingly, all of these four gene transcripts were consistently reduced in the strong SlMBP22-overexpression lines compared to those in the non-transgenic plants (Fig. 3j-m), which were likely to be associated with the reduction of pollen grains in the transgenic lines with a strong SlMBP22 overexpression. Thus, we propose that the infertility phenotype observed in the SlMBP22-OE transgenic tomato plants is probably attributed to low pollen production and defect in female reproductive development.
Overexpression of SlMBP22 affects auxin signalling-related genes
Auxin plays critical roles in regulating fruit development, including fruit set and growth, ripening and abscission (Pattison, et al. 2014). Recently, we demonstrated that the mild overexpression of SlMBP22 led to reduced plant height by affecting gibberellin (GA) and auxin homeostasis. (Li, et al. 2020). In this study, the artificial enhancement of SlMBP22 resulted in infertility phenotype in the strong overexpression lines, and then we speculated that it was also related to the alteration of auxin signalling. Subsequently, qRT-PCR assay was conducted to further investigate the expression levels of auxin pathway-related genes in the wild-type and strong overexpression transgenic ovaries. It was found that the transcripts of an auxin biosynthesis gene ToFZY5 (Exposito-Rodriguez, et al. 2011), an auxin response gene (ARF3) (Zouine, et al. 2014), three Aux/IAA transcription factor genes (IAA13, IAA14 and IAA29) (Audran-Delalande, et al. 2012), were greatly increased in the SlMBP22 strong overexpression transgenic ovaries at the anthesis stage, compared with the WT ovaries (Fig. 4a-e). By contrast, transcripts of an AUX/LAX gene LAX1, and three PIN genes (PIN1, PIN2 and PIN4) (Pattison and Catala 2012), respectively encoding auxin influx and efflux transport proteins, were sharply decreased in the transgenic ovaries than in the WT (Fig. 4f, g and I). Relative to WT, PIN2 gene expression was upregulated in the transgenic ovaries (Fig. 4h). Based on the results described above, we inferred that the overexpression of SlMBP22 may alter tomato productive development via disturbing auxin signaling.
Overexpression of SlMBP22 affects flowers and fruits size mainly by inhibiting cell expansion
Plant organ growth is controlled by multiple regulatory factors that coordinate cell proliferation and cell expansion (Anastasiou and Lenhard 2007; Horiguchi, et al. 2006). In our work, the SlMBP22-OE tomato plants exhibited reduced flower size, particularly first-whorl sepals, and smaller fruits with thinner fruit pericarp (Fig. 1c-e and Fig. 3b, f). Therefore, anatomical analyses were performed to investigate the cytological differences between the WT and transgenic sepals and fruit pericarps. Obviously, the cells in the sepal and pericarp respectively from the strong and mild SlMBP22 overexpression transgenic lines were much smaller than those in the wild-type plants (Fig. 5a, b). Compared with the WT, the transgenic fruit pericarps contained slightly reduced cell layers, while the reduction in mean cell size of the mesocarp cell of the transgenic fruits reached up to a 60 % difference (Fig.5c, d). Furthermore, we analyzed the transcript levels of genes associated with plant cell division and cell expansion in the inflorescences from the WT as well as the strong SlMBP22-OE plants by qRT-PCR. CDKA1, involved in the progression of the cell cycle (Czerednik, et al. 2015; Czerednik, et al. 2012), showed no significant difference in the mRNA accumulation between the transgenic and non-transgenic plants (Fig. 6a). The transcripts for three cyclin genes, SlCycA3;1, SlCycB1;1 and SlCycB2;1 were not clearly affected (Fig. 6b-d). In contrast, the expression levels of cell expansion-related genes, SlEXP1 (Perini, et al. 2017), LeEXP2 (Caderas, et al. 2000) and LeEXP8 (Chen and Bradford 2000), were distinctly repressed in the SlMBP22-overexpressing plants (Fig. 6e-g). FUL2, as a member of the MADS-box transcription factor family, affects style abscission and cell expansion (Wang, et al. 2014). In the SlMBP22-OE plants, FUL2 was strongly up-regulated when compared with its respective expression in the WT (Fig. 6h). Overall, these findings support the possibility that SlMBP22 up-regulation leads to the alterations in tomato flowers and fruit size are, atleastinpart, due to the reduced cell expansion, rather than impaired cell division.
Mild overexpression of SlMBP22 promotes proanthocyanidin accumulation and affects seed germination
The tt16 seeds are yellow in color and the PA accumulation was restricted to the chalazal bulb and the micropylar end in the mutant seed coat, while the ectopic expression of TT16 produced brown seeds as a result of ectopic PA biosynthesis (Nesi, et al. 2002). According to our observations, the SlMBP22 overexpression transgenic seeds had a dark brown color (Fig. 7a). To further explain the phenotype regarding the pigmentation of the seed coat, a vanillin assay was conducted and indicated that the mild OE transgenic seeds may accumulate more PA than WT, and then we decided to measure the PA content. As expected, the OE plant seeds possessed higher PA levels than wild-type plants (Fig. 7b). The overaccumulation of pigments in the seed coat has a negative effect on seed germination (Debeaujon, et al. 2000). Subsequently, a seed germination assay was performed to try to detect the germination energy of the transgenic tomato seeds. The results exhibited lower seed germination rates in seeds from transgenic lines compared with those from the non-transgenic plants (Fig. 7c, d), implying that mild overexpression of SlMBP22 may inhibit the germination ability of the transgenic tomato seeds.