Overexpression of SlMBP22 in Tomato Affects Flower Morphology, Fruit Set and Development

doi:10.21203/rs.3.rs-519249/v1

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Overexpression of SlMBP22 in Tomato Affects Flower Morphology, Fruit Set and Development

https://doi.org/10.21203/rs.3.rs-519249/v1

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published 28 Feb, 2022

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MADS-domain transcription factors have been clarified as key regulators involved in proper flower and fruit development in angiosperms. B_s genes, as members of the MADS-box subfamily, have been suggested to play an important role during the evolution of the reproductive organs in seed plants. Our knowledge about their effects on reproductive development in fruit crops like tomato (Solanum lycopersicum), however, is still unclear. Here, we found that the overexpression of SlMBP22 (SlMBP22-OE) resulted in considerable alterations regarding floral morphology, and affected the expression levels of several floral homeotic genes. Further analysis by yeast-two-hybrid assays demonstrated that SlMBP22 could form dimers with class A protein MACROCALYX (MC) and with SEPALLATA (SEP) floral homeotic proteins TM5 and TM29, respectively. In addition, pollen viability and cross-fertilization assays suggested that the defect in female reproductive development was responsible for infertility phenotype observed in the strong overexpression transgenic plants. The mild overexpression transgenic fruits were reduced in size, as a result of reduced cell expansion, rather than impaired cell division. Additionally, overexpression of SlMBP22 in tomato not only affected proanthocyanidin (PA) accumulation but also altered seed dormancy. Taken together, these findings may provide new insights into the knowledge of B_s MADS-box genes in flower and fruit development in tomato.

Molecular Biology

SlMBP22

flower

fruit

development

tomato

Overexpression of B_sister (B_s) MADS-box gene SlMBP22 impacts tomato reproductive development, including flower morphology, fruit set, fruit size and seed pigmentation.

Reproductive development of higher plants entails a series of biological processes, including floral meristem determination, floral bud generation, fruit development and ripening, all aimed at promoting seed formation and dispersal to ensure progeny survival. MADS-box proteins, have played prominent roles in the transcriptional modulation of the floral organ speciation and reproductive development, and are one of the most thoroughly studied gene family members in plants (Gramzow and Theissen 2010; Ng and Yanofsky 2000).

Floral development is a complex biological process and highly regulated by both the genetic background of plants and environmental factors (Fornara, et al. 2010). Previous studies have revealed that floral homeotic genes determining reproductive floral organ identities can be well understood via ABCDE model. (Coen and Meyerowitz 1991; Colombo, et al. 1995; Theissen 2001; Theissen and Saedler 2001). Interestingly, all genes thus far identified in this model, except for APETALA2 (AP2), encode MIKC ^C-type proteins and belong to MADS-box transcription factor family (Parenicova, et al. 2003). With the deepening of research, more and more MADS-box genes have been identified and characterized as key regulators of tomato flower development. For instance, tomato class A gene MC is involved in the inflorescence determinacy and the sepal development (Vrebalov, et al. 2002). Overexpression of FYFL in tomato presents longer sepals than wild-type (Xie, et al. 2014). Tomato class C gene TOMATO AGAMOUS 1 (TAG1), as the cognate homologs of Arabidopsis AGAMOUS (AG), participants in the regulation of stamen and carpel development as well as floral meristem determinacy (Pan, et al. 2010; Pnueli, et al. 1994). TOMATO AGAMOUS-LIKE1 (TAGL1), is the tomato ortholog of duplicated SHATTERPROOF of Arabidopsis, and also is the most closely related gene to TAG1, playing a key role in regulating carpels development (Vrebalov, et al. 2009). Tomato plants with up-regulated mRNA level of D-class MADS-box gene, Sl-AGL11, display carpel-like sepals (Huang, et al. 2017). SlMBP3 is the most closely related paralog of Sl-AGL11, and is notably expressed in the pistils. Silencing of SlMBP3 affects the development of seeds/placenta, suggesting that this gene specifies carpel/ovule identity (Zhang, et al. 2019). Two E-class MADS-box genes, TM5 and TM29, have predominant functions in the development of floral organs and the determination of floral meristem identity in tomato.

Generally, early fruit development undergoes three major phases, namely, fruit set, cell division and cell expansion. Fruit development involves complex spatial and temporal regulation by the interplay of numerous biotic and abiotic factors, such as plant hormones, transcription factors, elongation factors, microRNA, RNA-binding proteins, ubiquitin-proteasome and so on (Hussain, et al. 2020). In addition to researches describing the influence of MADS-box proteins on flower development, there have also been numerous studies highlighting the roles of MADS-box transcription factors in mediating various fruit morphologies, ripening and seed dispersal. Besides the role of tomato SHATTERPROOF 1 and 2 (SHP1, 2) ortholog TAGL1 in floral organ identities, this gene also functions in the fruit expansion and ripening process. TAG1 and TAGL1 are paralogous genes, the TAG1 silenced plants display smaller fruits than wild-type, which may be related to the reduction of pericarp thickness (Gimenez, et al. 2016; Vrebalov, et al. 2009). Transgenic plants with reduced tomato SEP1, 2 ortholog TM29 expression levels show phenotype with parthenocarpic fruits (Ampomah-Dwamena, et al. 2002). Class D gene Sl-AGL11, a close paralog of SlMBP3, is involved in the regulation of fruit quality and productivity (Huang, et al. 2017).

The phylogenetic sister clade of the class B genes has been termed B_sister (B_s) (Becker, et al. 2002). Relatively few members of this subfamily involved in the plant vegetative and reproductive growth have so far been characterized in different angiosperm species. It has been considered that B_s genes makeavaluablecontribution to the regulation of reproductive development in seed plants. For instance, the B_s MADS-box transcription factor, GORDITA (GOA), controls fruit size largely by modulating cell expansion in Arabidopsis (Prasad, et al. 2010). ABS/TT16/AGL32, the closest relative of GOA, is involved in the regulation of seed coat pigmentation and proanthocyanidin (PA) accumulation in the inner endothelial cell of the developing seeds in Arabidopsis (de Folter, et al. 2006; Nesi, et al. 2002; Xu, et al. 2017). It has been shown that ABS acts redundantly in the formation of endothelium with the D-class MADS-box protein SEEDSTICK (STK). The very few seeds observed in the Arabidopsis abs stk double mutant caused by the reduction of the number of fertilized ovules and the seed abortions (Mizzotti, et al. 2012). Similarly, FLORAL BINDING PROTEIN 24 (FBP24) is necessary for proper endothelium development in petunia (Petunia hybrida). Nevertheless, a mutant complementation experiment demonstrates that FBP24 fails to replace ABS/TT16, suggesting that there are functional conservation and divergence of the supposed orthologous genes in different angiosperm species (Becker, et al. 2002). OsMADS30 T-DNA insertion plants display the alterations of plant size and architecture in rice, indicating that OsMADS30 may have evolved a new function and therefore is not a canonical B_sgene (Schilling, et al. 2015).

Although we previously identified a tomato B_s gene SlMBP22, homologous to Arabidopsis ABS/TT16 and petunia FBP24, participated in regulating tomato growth and tolerance to drought stress (Li, et al. 2020), its role in reproductive development has not been fully explored. In this study, we found that transgenic tomato plants overexpressing SlMBP22 exhibited phenotypes related to defects in floral architecture, fruit set and development. Moreover, the underlying causes for these phenotypes were respectively analyzed at the morphological, statistical and molecular levels. Our data further expand the understanding of the functions of B_s MADS-box proteins in the regulation of plant reproductive development.

Plant materials and growth conditions

Tomato (Solanum lycopersicum Mill. cv. Ailsa Craig) wild-type (WT) and transgenic plants were grown under normal greenhouse conditions (16-h-day/8-h-night cycle, 25°C/18°C day/night temperature, and 250 μmol m ^-2 s ^-1 light intensity). For gene expression analysis, flowers were harvested at different developmental stages: -4D (4 d ahead of anthesis), -2D (2 d ahead of anthesis), anthesis and 2D (2 d after anthesis), and four-whorl mature floral organs (sepals, petals, stamens and pistils) were harvested at the anthesis stage. All samples were collected and promptly frozen in liquid nitrogen and then stored at –80°C until required.

Vector construction and plant transformation

For the construction of the SlMBP22-overexpressing (SlMBP22-OE) vector, the SlMBP22 full-length coding region was amplified by PCR with primers SlMBP22-F and SlMBP22-R, adding the Xba I and Sac I site to the 5´end and 3´end, respectively (Supplementary Table S1). The amplified SlMBP22 products were digested with Xba I and Sac I and then ligated into the plant binary vector pBI121 placed under the control of CaMV 35S promoter. The resulting binary vectors were transferred into S. lycopersicum variety AC⁺⁺ cotyledons, according to the transformation and regeneration methods as previously reported (Chen, et al. 2004).

Gene expression analysis

Total RNA was isolated using RNAiso Plus (Takara). The cDNA was synthesized using M-MLV Reverse Transcriptase Kit (Promega). Gene expression levels in different organs were evaluated by quantitative real-time PCR (qRT-PCR) using a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad). SlCAC (Solyc08g006960), a tomato housekeeping gene, was used as an internal control (Exposito-Rodriguez, et al. 2008). The analysis of relative expression levels was conducted using the 2 ^−ΔΔCTmethod (Livak and Schmittgen 2001). All primers for qRT-PCR are presented in Supplementary Table S1.

Yeast two-hybrid assays

The ORFs of SlMBP22, MC, TAGL1, SlMBP3, TM5 and TM29 were amplified by PCR using primers (Supplementary Table S1). The PCR fragments of SlMBP22 were cloned into the pGBKT7 vector to generate a bait construct BD-MBP22, and MC, TAGL1, SlMBP3, TM5 and TM29 were linked into pGADT7 vector to obtain prey constructs, namely, AD-MC, AD-TAGL1, AD-SlMBP3, AD-TM5 and AD-TM29, respectively. Different combinations of bait and prey vectors were co-transformed into Y2Hgold. The yeast two-hybrid assays were conducted as described previously (Tang, et al. 2020). The experiments were repeated three times.

Pollen viability assay

Pollen viability assay was tested by TTC staining method described previously (Huang, et al. 2017). Briefly, soaked pollen grains of the fully opened flower from the wild-type and transgenic plants in a 0.1% 2,3,5-triphenyl-2 h-tetrazolium chloride (TTC) solution at room temperature for 15 min, and then, stained pollen grains were observed and photographed under the microscope (Nikon E100). The experiments were repeated three times.

Cross assay

A cross assay was conducted according to the method described in a previous report (Shen, et al. 2019). The anthers of the WT and three OE plants (lines 18, 2 and 17) were carefully removed at 2 days ahead of anthesis, and the emasculated flowers were labeled and bagged with small plastic bags to prevent natural pollination. Two days after emasculation, mature pollen grains of newly opened flowers from the two OE lines, OE18 and OE2, were collected and brushed to the styles of the emasculated flowers from the WT plants. Meanwhile, we brushed mature pollen grains from the newly opened WT flowers to the styles of the emasculated flowers of the transgenic lines: OE 18, 2 and 17, respectively.

Histological analysis

Histological analyses of sepal at the anthesis stage and fruit pericarp at the breaker stage from the WT and transgenic plants were processed following the method as described in our previous report (Li, et al. 2020). The morphological observations of the paraffin sectioning were performed under a light microscope (Nikon E100). The number of cell layers and the mean mesocarp cell size were estimated according to our previous report (Zhu, et al. 2019).

Vanillin Assay and PA extraction

Vanillin staining of mature tomato seeds was carried out using vanillin reagent as previously described (Chen, et al. 2013; Debeaujon, et al. 2000). Briefly, fresh seeds of the three independent mild transgenic OE lines and the WT plants at the 42d post anthesis (DPA) stage were harvested and soaked in a 1% vanillin solution at room temperature for 30 min. PAs of tomato seeds were determined by using the Vanillin-HCl method according to a previous report (Gao, et al. 2018; Mitsunaga, et al. 1998).

Seed germination assays

The seed germination assays were performed as previously described (Zhou, et al. 2019). In brief, the WT and T2 homozygous transgenic seeds were sown onto MS medium after surface sterilization, and then germinated in the dark at 25°C for 7 days. Seed germination rates were recorded on days 3, 5 and 7 respectively. The experiments were repeated three times.

Statistical analysis

Data presented in this report are means ± standard deviation from three independent repeats. The significant difference was assessed using Student’s t-test (P < 0.05).

Expression patterns of SlMBP22

B_s 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 B_s 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.

TT16/ABS/AGL32 gene has been identified as a member of the B_sister subfamily of MADS-domain proteins, which is well described in Arabidopsis thaliana (Kaufmann, et al. 2005; Mizzotti, et al. 2012; Nesi, et al. 2002; Xu, et al. 2017), Petunia hybrida (de Folter, et al. 2006; Tonaco, et al. 2006) and Brassica napus (Chen, et al. 2013; Deng, et al. 2012), respectively. In Arabidopsis, TT16 is involved in the regulation of endothelium development and PA accumulation of developing seeds (Nesi, et al. 2002). The FBP24, is closely related to TT16 in petunia, functions in endothelium layer development similar to previously described TT16 in Arabidopsis (de Folter, et al. 2006). However, canola TT16 ortholog BnTT16 controls multiple physiological functions, especially seed oil synthesis and embryo development, which are beyond endothelial cell specification and flavonoid biosynthesis (Deng, et al. 2012). More recently, we have uncovered important biological roles of tomato TT16 ortholog SlMBP22 in plant vegetative development and drought stress responses (Li, et al. 2020). Furthermore, we found that the overexpressing SlMBP22 transgenic tomato plants exhibited defects in reproductivegrowth and development, including morphological alterations of flowers, reduced fruit set and growth, and abnormal seed color. These data indicate that the functional conservation and diversity of TT16 genes in different plant species.

B_sistergenes are predominantly expressed in female reproductive organs, suggesting that this subfamily is involved in the evolution of the reproductive organs in seed plants (Becker, et al. 2002). Here, flowers of the SlMBP22 overexpression plants showed considerable changes regarding floral organs size, sepal and petalcolor as compared with the wild-type, suggesting that the overexpression of SlMBP22 in tomato may affect the development of flower, following what would be expected for a typical B_sister gene. To obtain further insights into the potential molecular regulation mechanism explaining the phenotypes associated with floral organ development, several MADS-box genes related to flower development were tested by qRT-PCR analysis in mature floral organs of both the WT and overexpression plants. These results revealed that overexpression of SlMBP22 caused alterations in the expression levels of these genes such as the close tomato of SHP1/SHP2 (TAGL1), SEP1/2 (TM29), AG (TAG1) and SEP3 (TM5)，AP1 (MC) and STK (SlMBP3). These results suggest that the impacts of SlMBP22 on flower development may be associated with other transcription factors. It has been proposed that MADS-box transcription factors carry out their functions in floral organ formation and identity or other developmental processes by a complex network of protein-protein and protein-DNA interactions (Tonaco, et al. 2006). In the case of Arabidopsis, ABS/TT16 can form dimers with SEPALLATA (SEP) floral homeotic proteins and form higher-order complexes that also include the SEEDSTICK (STK) or SHATTERPROOF1/2 (SHP1, SHP2), which are verified by yeast-two-hybrid and three-hybrid assays, respectively (Kaufmann, et al. 2005). Hence, the formations of dimers and higher-order complexes may have a key role in regulating plant growth and development among MADS-box genes. In our study, a yeast two-hybrid experiment showed that SlMBP22 could physically interact with a MADS-box transcription factor, MC, which is known as a regulator of sepal size (Vrebalov, et al. 2002), indicating that SlMBP22 and MC can potentially form a dimer and then may explain the sepals with extremely reduced size observed in the SlMBP22-OE lines compared with WT. Meanwhile, protein-protein interactions were also observed in the yeast two-hybrid assay between SlMBP22 and another two SEP MADS-box proteins, TM5 and TM29, which are previously verified to mediate organ differentiation of the inner three whorls of tomato flowers (Ampomah-Dwamena, et al. 2002; Pnueli, et al. 1994). It is possible that overexpressing SlMBP22 can affect flower morphology at least partly be interpreted as the consequence of forming dimers, trimers, or even tetramers with other floral homeotic proteins in tomato plants.

The phases of fruit initiation and development have been considered to be the continuation of the floral developmental program, and MADS-box transcription factors play considerable and multiple functions during flower, fruit and seed development (Busi, et al. 2003). For instance, transgenic tomato plants with reduced TAG1 expression levels exhibit defects in stamen and carpel identity, while overexpression lines display alteration in the first whorl, male and female sterility, and parthenocarpic fruit (Pnueli, et al. 1994). Another C class homeotic gene TAGL1 plays an important role in both regulating fleshy fruit expansion and ripening processes. (Vrebalov, et al. 2009). The down-regulation of SEP homolog TM29 leads to infertile stamens and ovaries, parthenocarpic fruit, and green-colored petals and stamens which suggest a partial conversion of these organs into sepals (Ampomah-Dwamena, et al. 2002) Besides modifying the flower morphology, we also found that the artificial enhancement of SlMBP22 affected fruit set and growth, and the strong SlMBP22 overexpression transgenic plants bore no fruit, while the smaller fruits with fewer seeds were observed in the mild overexpression transgenic lines compared to those in the WT. The strong SlMBP22-OE transgenic flowers were manually cross-pollinated with wild-type pollen, which failed to produce fruit, whereas normal seeds could develop when strong overexpression transgenic pollen was used to pollinate WT styles. In addition, TTC staining and qRT-PCR assays were carried out and the results showed that the pollen viability was not affected in SlMBP22-OE flowers, but the strong overexpression of SlMBP22 led to reduced viable pollen grains. These data indicated that the strong overexpression of SlMBP22 caused female sterility and disturbed mature pollen formation, thus resulting in reduced fertility.

The final size of an organ is controlled by two phases of growth, namely, cell proliferation and subsequent cell expansion (Anastasiou and Lenhard 2007; Horiguchi, et al. 2006). SlMBP22 overexpressing plants showed smaller flowers and fruits with thinner pericarp and fewer seeds than those of the WT. qRT-PCR analysis was conducted and the results revealed that the expression of several genes involved in cell expansion, including SlEXP1, LeEXP2 and LeEXP8, was greatly down-regulated in the OE plants, but the transcripts for cell cycle genes such as CDKA1, SlCycA3;1, SlCycB1;1 and SlCycB2;1 displayed no obvious differences, when compared to their respective expression in the WT. These expression data agreed with the findings of the microscopic analysis, implying that the overexpression of SlMBP22 affects flower and fruit size is likely to originate from the reduced cell expansion but not the impaired cell division. There have been reports highlighting the influence of phytohormones especially auxin and GA (de Jong, et al. 2009; de Jong, et al. 2011; Fuentes, et al. 2012; Hu, et al. 2018; Serrani, et al. 2008). Auxin response factor SlARF7 may negatively regulate fruit set and moderates the auxin response during fruit growth (de Jong, et al. 2009). Silencing of Sl-IAA17 in tomato results in increased fruit size as a result of the alteration of endoreduplication-related cell expansion (Su, et al. 2014). Plants overexpressing transcription factor SlGRAS24 affect fruit set and development via coordinating gibberellin and auxin homeostasis (Huang, et al. 2017). Taking into account two previous studies have shown that the transcript levels of FBP24/ SlMADS29 (renamed here as SlMBP22) were greatly reduced after 2,4-D treatment in tomato ovaries by qRT-PCR analysis (Hu, et al. 2018; Tang, et al. 2015), indicate that SlMBP22 may participate in the regulation of fruit development by mediating auxin signalling. Indeed, our qRT-PCR assay showed that the mRNA accumulations of several genes related to auxin signalling were altered in the transgenic ovaries. In addition, auxin signalling is also important to regulate floral organ size. For instance, tomato MADS-box gene SlMBP21 negatively regulates cell expansion to affect sepal size, which was mediated by ethylene and auxin (Li, et al. 2017). Thus, it is likely that the overexpression of SlMBP22 affected tomato reproductive development including ﬂoral organ size, fruit initiation and growth via disturbing auxin signalling, which is consistent with its effect on tomato plant vegetative development (Li, et al. 2020).

Proanthocyanidins (PAs), as one of the main flavonoids in Arabidopsis seeds (Routaboul, et al. 2012), play a crucial role in modulating seed dormancy and longevity during storage (Debeaujon, et al. 2000). Once oxidized, PAs confer brown pigmentation in the mature seed coat (Devic, et al. 1999). Arabidopsis ttg1 mutant seeds have a yellow color, and appear reduced seed dormancy can be ascertainedby a highergermination rate (Debeaujon, et al. 2000). Arabidopsis TT16 gene participates in the control of seed coat pigmentation and proanthocyanidin (PA) accumulation in the endothelium of developing seeds (Deng, et al. 2012; Nesi, et al. 2002; Xu, et al. 2017). In our study, SlMBP22-OE transgenic seeds were dark brown in color, and with more PA accumulation and exhibited lower germination than non-transgenic seeds, indicating that the overexpression of SlMBP22 in tomato not only affected biosynthesis of PAs but also altered seed dormancy. As reported, endogenous plant flavonoids not only affect seed germination and dormancy, but also play an important role in regulating cellular auxin efflux and consequent polar auxin transport. Auxin transport is elevated in tt4 (no flavonoid production) but reduced in tt7 and tt3 mutants (which accumulate excess flavonols) (Peer, et al. 2004). Additionally, previous reports indicate that seeds could produce or deliver auxins to the surrounding fruit tissues and then promote fruit expansion (Ariizumi, et al. 2013; Gillaspy, et al. 1993). The agl62 mutant shows impaired transport of auxin was responsible for seed abortion (Figueiredo, et al. 2016). Our data demonstrated that overexpression of SlMBP22 resulted in altered expression of a series of genes related to auxin transport. Based on the results described above, we infer that SlMBP22 may act as a key regulator that affects flower development, seed development, fruit set and growth via affecting auxin signalling.

In summary, we aimed to investigate the effects of SlMBP22 overexpression on the tomato reproductive development regarding flowers, fruits and seeds in this study. Our data demonstrate that the overexpression of SlMBP22 not only results in morphological alterations of flowers but also affects fruit set, fruit size and seed pigmentation. Morphological, physiological,andmolecularanalyses have been carried out to preliminarily elucidate the causes related to the defects of SlMBP22 overexpressing transgenic plants. These findings further advance our knowledge of physiological effects of B_s MADS-box transcription factors and provide valuable information for modern breeding biotechnologies.

Author contributions Z.H., and G.C. designed research; F.L., Y.J., X.C., S.Z. and Q.X., performed the experiments; F.L. wrote the paper. All authors have read and approved the ﬁnal version of the manuscript.

Acknowledgments This work was supported by the National Natural Science Foundation of China (nos. 31872121, 31801870), and the Natural Science Foundation of Chongqing of China (csts2019jcyj-msxmX0094).

Conflict of interest The authors declare that they have no conflict of interest.

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Overexpression of SlMBP22 in Tomato Affects Flower Morphology, Fruit Set and Development

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