A SEPALLATA MADS-Box Transcription Factor, SlMBP21, Functions as a Negative Regulator of Flower Number and Fruit Yields in Tomato

Abstract

MADS-box transcription factors act as the crucial regulators in plant organ differentiation. Crop yields are highly influenced by the flower number and fruit growth. However, flower identification is a very complex biological process, which involves many cascade regulations. The molecular mechanisms underlying the genetic regulation of flower identification in cultivated plants, such as tomato, are intricate and require further exploration. In this study, we investigated the vital function of a SEPALLATA (SEP) MADS-box gene, SlMBP21, in tomato sympodial inflorescence meristem (SIM) development for the conversion from SIMs to floral meristems (FMs). SlMBP21 transcripts were primarily accumulated in young inflorescence meristem, flowers, sepals, and abscission zones. The Ailsa Craig (AC⁺⁺) tomato plants with suppressed SlMBP21 mRNA levels using RNAi exhibited a large increase in flower number and fruit yields in addition to enlarged sepals and inhibited abscission zone development. Scanning electron microscopy (SEM) revealed that the maturation of inflorescence meristems (IMs) was repressed in SlMBP21-RNAi lines. RNA-seq and qRT-PCR analyses showed that numerous genes related to the flower development, plant hormone signal transduction, cell cycle, and cell proliferation et al. were dramatically changed in SlMBP21-RNAi lines. Yeast two-hybrid assay exhibited that SlMBP21 can respectively interact with SlCMB1, SFT, JOINTLESS, and MC, which play key roles in inflorescence meristems or FM development. In summary, our data demonstrate that SlMBP21 functions as a key regulator in SIM development and the conversion from SIMs to FMs, through interacting with other regulatory proteins to control the expression of related genes.

1. Introduction

The generation of flowers and inflorescences always have great influences on the reproduction of plant progeny and crop yields. Agricultural productivity is strongly influenced by the number of flowering branches, flower number in each inflorescence, and the abscission of flowers or fruits during development, both before and at maturity. The generation of flowers in higher plants is a complex genetic and biochemical process which is divided into at least three steps: the flowering transition, the individual flower initiation, and the floral patterning [1]. During this program, the shoot apical meristems (SAMs), a dome of self-renewing cells located at the shoot apex, first generate leaves before transitioning to inflorescence meristems (IMs), which give rise to lateral (axillary) meristems that either directly differentiate into flowers or transition into flower-bearing shoots [2,3]. In monopodial plants, such as rice and Arabidopsis, the SAM is indeterminate and the IM continuously generates floral meristem (FM) laterally, bringing about a finite range of simple inflorescence architectures [4,5]. Whereas, in sympodial plants, such as in tomato, the SAM is determinate and terminated by an inflorescence after 6–12 leaves have been produced and the growth of plants will continue from the bud of axillae at the top leaf [6,7]. Tomato plants continue growth from the generation of a specialized axillary meristem. The axillary meristem grows in the axillary of the last leaf and terminates after generating three leaves. Then, a new axillary meristem is produced in the axil of the last leaf which is generated by the previous axillary meristem. This process is repeated and finally all subsequent axillary meristems generate three leaves, an inflorescence, and the next axillary meristem. These inflorescences are produced by inflorescence meristems (namely, sympodial inflorescence meristems (SIMs) in tomato), which generate a new SIM and the founder cells that generate FM. Each new SIM grows perpendicularly to the previous one, leading to the zigzag pattern of inflorescences in tomato. Finally, each FM forms the floral organs, i.e., sepals, petals, stamens, and pistils [2,8].

In wild species of tomato, inflorescences have multiply branched architectures and produce dozens of flowers, while in most cultivated tomatoes, the inflorescences produce several flowers arranged in a zigzag branching pattern [8]. Several mutants with compound numerous or single flowers have been reported in tomato. The uf (uniflora) mutant, for example, generates fertile, normal, and solitary flowers with a delayed flowering time [9], and plants with SFT (SINGLE FLOWER TRUSS) loss-of-function alleles only generate a few flowers prior to reverting to vegetative branch architectures and flower later than normal [10]. The mutation of TMF (TERMINATING FLOWER) in tomato results in an earlier flowering time and the generation of single-flower primary inflorescence with enlarged leaf-like sepals [11]. In contrast, the s mutant bears hundreds of flowers due to the loss of function of a homeobox protein encoded by the S (COMPOUND INFLORESCENCE, the homolog of WUSCHEL-RELATED HOMEOBOX 9) gene [8,12], while the shoots of the an mutant terminate in cauliflower-like tissues as a result of losing the function of AN (an F-box gene, the homolog of UNUSUAL FLORAL ORGANS) [13]. Mutation of an APETALA2 (AP2) family member TARGET OF EAT1 (SlTOE1) in tomato results in defective floral organs and plenty of flowers [14]. Although many regulators have been reported to participate in tomato IM and FM development, there are still so many related regulators with unknown functions that need to be investigated. This will not only contribute to enriching the regulatory networks of IM and FM development, but also to further revealing the molecular mechanisms of IM and FM development regulation.

MADS-box proteins are a group of important transcription factors which have been duplicated and diversified extensively during angiosperms evolution, playing vital functions in diverse developmental processes of plants [15], including abiotic stress response, floral organ identity, flowering time, and the regulation of fruit ripening [16,17,18]. For example, OsMADS26 has multiple functions in regulating various stress responses in rice [19]. Tomato SlMBP8 acts as a negative regulator in the salt and drought stress [20]. The Arabidopsis AtAGL21 positively regulates the lateral root development and AtAGL21 overexpression in plants generates longer and more lateral roots [21]. The MADS-box gene TAGL1 plays a significant role in the regulation of cuticle development in tomato [22,23]. In Arabidopsis, five MADS-box genes, SVP (SHORT VEGETATIVE PHASE), AP1 (APETALA1), AGL24 (AGAMOUS-LIKE GENE 24), SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1), and FLC (FLOWERING LOCUS C) participate in regulating flowering time [24,25,26,27,28,29]. Numerous MADS-box genes have been reported to function as the key regulator in the floral organ identity in flowering plants, such as Arabidopsis AP1/2/3 (APETALA1/2/3), AG (AGAMOUS), PI (PISTILLATA) [30,31,32,33,34,35] and TM6, TAP3, TOMATO AGAMOUS 1 (TAG1) in tomato [36,37]. TAP3 and TM6 play vital roles in the regulation of stamen and petal development [36], and TAG1 (TOMATO AGAMOUS 1) functions as an important regulator in the determination of carpels and stamens [37]. In addition, multiple MADS-box genes are also proven to have vital functions in the regulation of fruit development or ripening in tomato, such as RIN, SlMADS1, SlMBP3, FUL1 (FRUITFULL1) and FUL2 (SlMBP7), and so on [38,39,40,41].

In addition, some MADS-box genes are reported to regulate the determination of flower number and inflorescence development. For instance, LFY is a key regulator for the establishment of the FM regulatory network [42]. Two Arabidopsis genes, APETALA1 and CAULIFLOWER, have redundant roles in the formation of FM [43]. The AGL6-like MADS-box gene in wheat is a target of spikelet meristem development regulation and is a key regulator of floral organ identity [44]. Overexpression of two SHORT VEGETATIVE PHASE (SVP) MADS-box genes OsMADS22 and OsMADS55 in rice results in increased flower numbers via the repression of meristem phase transition [45]. In tomato, four MADS-box genes, TM3, STM3, FUL2, and SlMBP20, are reported to regulate tomato inflorescence development; the ful2mbp20 mutants display the increased inflorescence branches with a large number of flowers [46,47]. STM3 is also reported to regulate flower number by regulating the inflorescence meristem via activating FRUITFULL1, and overexpression of STM3 results in highly branched inflorescences with a large flower number [48]. The j mutant displays the determinacy loss of meristem which generates a transformation to vegetative growth after two or three flowers were produced [49]. The MACROCALYX (MC) gene is proven to regulate meristem determinacy and sepal development in tomato; the vegetative inflorescence (mc-vin) mutant which is generated by a T-DNA insertion into MC gene promoter region exhibits a transition to the vegetative growth after generating a few flowers [40,50].

The flower number which is controlled by the determination of cell fate in inflorescence meristems (IM) is a yield-related trait. In angiosperms, the IM produces the founder cells to generate FMs, which form floral organs, including sepals, petals, stamens, and pistils. Therefore, the developmental progression of flowers is vital for crop yield production. Investigations of the molecular mechanisms underlying the development of flowers are essential for the further understanding of reproductive organ formation and improving yield traits. As a sympodial plant, tomato displays the highly structural plasticity of inflorescences. However, the mechanisms underlying the genetic regulation of SIM development and the conversion from SIMs to FMs remain to be further investigated in tomato. Previous studies have reported that SlMBP21 participates in the regulation of flower abscission zone formation, sepal size, and reproductive development [51,52,53]. In this study, we investigated the vital functions of an SEP MADS-box gene, SlMBP21, in SIM development and the conversion from SIMs to FMs. Its transcripts were primarily accumulated in inflorescences, flowers, sepals, and abscission zone. Suppression of SlMBP21 in the Ailsa Craig (AC⁺⁺) tomato plants using the RNAi method resulted in large increases in flower number and fruit yields, and continuous production of SIM, compared with the wild type. This implies the potential functions of SlMBP21 in the regulation of flower number and fruit yield through regulating SIM development and the conversion from SIMs to FMs. Then, we carried out further functional investigations of SlMBP21 via the phenotypic and molecular characterizations of SlMBP21-RNAi lines to deeply investigate its functions and molecular mechanisms in the regulation of tomato SIM development and the conversion from SIMs to FMs.

2. Methods

2.1. Plant Materials and Growth Conditions

In this study, a near-isogenic tomato line, Solanum lycopersicon Mill. cv. Ailsa Craig (AC⁺⁺), was used as the wild type plants. The transgenic and WT plants were grown in a standard greenhouse (16 h-day/8 h-night cycle, 25/18 °C day/night temperature, 80% humidity). All the harvested samples were immediately frozen in liquid nitrogen and stored at −80 °C until further use.

2.2. SlMBP21 Isolation and Sequence Analysis

Total RNA of tomato inflorescences was obtained using plant RNA kit (OMEGA, Norwalk, CT, USA) following the manufacturer’s instructions. Then, the first strand cDNA was synthesized using 1 µg total RNA with the M-MLV reverse transcriptase (Promega, Madison, WI, USA) and Oligo d(T)₂₀ primer. The full length of SlMBP21 was cloned using primers of SlMBP21-full-F/R (Table S1) with 1–2 µg cDNA as the template. After being tailed, the amplified products were inserted into the pMD18-T vector (TaKaRa, Beijing, China). The PCR was performed to pick out the positive clones, and then sequencing was carried out to further confirm the obtained positive clones.

2.3. Phylogenetic Analysis

Multiple alignment of SlMBP21 protein with other MADS-box proteins was carried out by DNAMAN (V 6.0). A phylogenetic tree was obtained by MEGA 11 using the neighbor-joining method with the following parameters: Poisson model, pairwise deletion, and bootstrap analysis of 1000 replicates.

2.4. Construction of SlMBP21-RNAi Vector and Plant Transformation

To suppress the expression of the SlMBP21 gene, RNA interference (RNAi) vectors were constructed. A 381-bp C-terminal specific fragment of SlMBP21 was obtained using the primers SlMBP21-i-F/R (Table S1). Then, the amplified fragments were digested using the restriction enzyme Kpn I/Xho I and Hind III/Xba I (TaKaRa, Beijing, China). After being purified, the digested SlMBP21 fragments were cloned into the pHANNIBAL vector at Kpn I/Xho I restriction site and at Hind III/Xba I restriction site, respectively. Finally, the needed expression unit including the CaMV (cauliflower mosaic virus) 35S promoter, the sense-orientated SlMBP21 fragment, PDK intron, the antisense-orientated SlMBP21 and OCS terminator, was digested by Sac I/Spe I and inserted into pBIN 19 at the Sac I and Xba I site to produce the SlMBP21-RNAi vector.

The SlMBP21-RNAi binary plasmids were transferred into the Agrobacterium LBA4404 strain using the Agrobacterium-mediated transformation following the protocols reported by Chen et al. [54]. After the transgenic plants were obtained. The PCR was performed using the primers NPTII-F/R (Table S1) to select the positive transgenic plants which will be used for further investigation.

2.5. Morphological Observations and Statistic Analysis

The number of flowers and fruits, the average weight of each fruit, and the total weight of all fruits in each inflorescence were statistically analyzed, respectively. At least 10 plants of each line were used to perform each statistical analysis. For scanning electron microscope (SEM), the second inflorescences of wild type and SlMBP21-RNAi lines were fixed and prepared. SEM was performed according to Molinero-Rosales et al. [10]. Paraffin sectioning was used to perform the morphological observations of abscission zone of breaker stage fruits by light microscopy following our previous method [41].

2.6. RNA-Seq and Data Analysis

The inflorescence meristems (IMs, it is called SIMs in tomato) and floral meristems (FMs) from the second inflorescences of WT and SlMBP21-RNAi plants were harvested from at least 10 plants using the dissecting microscope to carry out RNA-seq analysis. Then, the RNA-seq was performed following the same method in our previous study [41]. The sequencing data can be found in the NCBI Sequence Read Archive (SRA, http://www.ncbi.nlm.nih.gov/Traces/sra accessed on 1 April 2024) with accession numbers SAMN40557986–SAMN40557989.

2.7. Quantitative Real-Time PCR (qRT-PCR) Analysis

The harvested SIMs and FMs from the second inflorescences of WT and SlMBP21-RNAi plants that were used for RNA-seq were also used for the qRT-PCR analysis. Total RNA of each sample of WT and SlMBP21-RNAi lines was obtained using plant RNA kit (OMEGA, Norcross, GA, USA). The qRT-PCR analysis was performed using the same method as our previous study [55]. The primers q-SlMBP21-F/R (Table S2) were used to examine the expression levels of SlMBP21 in WT and SlMBP21-RNAi lines. Furthermore, the expression levels of numerous genes were determined simultaneously, including inflorescence meristem fate-related genes SP (NM_001247045), STM3 (XM_010317148), JOINTLESS (NM_001319841), and S (NM_001247143), cell cycle-related genes cycA2 (NM_001246839), cycD3 (NM_001246865), cdkB2 (NM_001246976), and E2FE (XM_004236104), cell wall modification-related genes cel2 (NM_001247938), XTH1 (NM_001246929), EXPA18 (NM_001247832), and MAN1 (NM_001247646), cytokinin synthesis and degradation-related genes LOG3 (XM_004246608), LOG6 (NM_001257982), CKX2 (NM_001257980), and CKX7 (NM_001257979), auxin synthesis and response-related genes IAA2 (XM_010324706), IAA5 (NM_001279133), ARF5 (NM_001247616), and ARF9 (NM_001247605), and ethylene synthesis and response-related genes ACS1A (NM_001246993.1), ACS6 (NM_001247235.1), ERF-H1 (NM_001247919), and ERF5 (NM_001330445). Primers are shown in Supplementary Table S2. Three independent biological repeats were performed.

2.8. Yeast Two-Hybrid Assay

The ORFs of SlMBP21, SFT, SlCMB1, JOINTLESS, and MC were amplified by PCR using the primers SlMBP21 (Y2H)-F/R, SFT (Y2H)-F/R, SlCMB1 (Y2H)-F/R, JOINTLESS (Y2H)-F/R, and MC (Y2H)-F/R (Table S1), respectively. The PCR products of each gene were respectively digested with restriction enzymes EcoR I and BamH I (TaKaRa, China). After that, SlMBP21 was linked into the EcoR I/BamH I site of the pGBKT7 to obtain the pGBKT7-SlMBP21 vector. Simultaneously, the ORFs of SFT, SlCMB1, JOINTLESS, and MC were digested with restriction enzymes EcoR I and BamH I and inserted into the EcoR I/BamH I site of the pGADT7 to obtain the pGADT7-SFT, pGADT7-SlCMB1, pGADT7-JOINTLESS, and pGADT7-MC vector, respectively. Then, pGBKT7-SlMBP21 vector was transferred into Y2HGold. The pGADT7-SFT, pGADT7-SlCMB1, pGADT7-JOINTLESS, and pGADT7-MC vectors were transferred into Y187, respectively. The yeast two-hybrid assays were performed using the same method as described in our previous study [55].

3. Results

3.1. Molecular Characterization of SlMBP21

To study the genetic regulation of inflorescence architecture in tomato (Solanum lycopersicum), we concentrated our functional investigations on the SEP MADS-box member SlMBP21. Alignment analysis of MADS-box protein sequences showed that SlMBP21 had the conserved domains (namely MADS domain, I domain, and K domain) and its C-terminal region was highly different than other sequences of MADS-box proteins (Figure 1A). Moreover, phylogenetic analysis exhibited that SlMBP21 belonged to the SEPALLATA (SEP) clade (Figure 1B).

3.2. Expression Patterns of SlMBP21 in Different Tomato Tissues

To understand the potential functions of SlMBP21 in tomato development, the expression levels of SlMBP21 in roots, stems, leaves, sepals, and a series of developmental stages of flowers inflorescences and fruits were analyzed. Low-level expression of SlMBP21 was observed in vegetative organs such as roots, stems, leaves, and B + 4 (4 days after breaker fruits) to B + 14 fruits (14 days after breaker fruits) (Figure 2A). In all detected tissues, SlMBP21 was transcribed more abundantly in sepals, flowers, inflorescences, and 7d to B (breaker) fruits (Figure 2A). In the different developmental stages of flowers and inflorescences, SlMBP21 transcripts were preferentially accumulated at the onset of inflorescence production and young flowers, and decreased with the development of flowers and inflorescences (Figure 2B,C). Furthermore, SlMBP21 transcript accumulation was detected at low levels in the early stage of pedicel abscission zone formation, and increased dramatically in the pedicel abscission zones of B + 7 (7 days after breaker fruits) stage fruits (Figure 2D). These results indicate that SlMBP21 may have vital functions in the development of inflorescences, flowers, sepals, and pedicel abscission zone.

3.3. SlMBP21-RNAi Lines Exhibited Abundant Flower Production and Increased Fruit Yield

To further study SlMBP21 functions in tomato development, we constructed the SlMBP21-RNAi vector (Figure S1) and transformed it into wild-type tomato plants by Agrobacterium tumefaciens-mediated T-DNA transfer. Finally, twelve independent RNAi lines in which the transcripts of SlMBP21 were dramatically decreased were obtained, and three lines (RNAi02, RNAi03, RNAi07) which displayed the SlMBP21 lowest expression levels were selected for the further investigations (Figure 3A). Then, the expression of SlMBP21 was also detected in sepals and the abscission zone of the RNAi02, RNAi03, and RNAi07 lines, respectively. The results exhibited that SlMBP21 transcripts were markedly reduced in sepals and the abscission zone of these three lines, respectively (Figure S2A,B).

After being suppressed, we found that the RNAi lines generated abundant flowers and more fruits. It seems that the SIMs in inflorescences of SlMBP21-RNAi lines were always in the state of continuous and vigorous differentiation, while the SIMs of wild-type plants stopped differentiation after producing several flowers. As a result, the RNAi lines generated abundant flowers and more fruits apart from enlarged sepals and inhibited abscission zone formation, while the WT plants generated a normal number of flowers, sepal size, and abscission zone (Figure 3B,C and Figure S2C–H). Then, the flower numbers of the first, second, and third inflorescences of SlMBP21-RNAi plants and wild-type plants were counted. The results showed that there were about 7–10 flowers in one inflorescence of WT plants, while the SlMBP21-RNAi plants can possess over 100 flowers in each inflorescence (Figure 4A). Finally, we found that although the SlMBP21-RNAi lines can produce hundreds of flowers, not every flower can successfully complete fruit setting and produce fruits. This may be due to the fact that too many flowers needed more nutrition, which eventually resulted in insufficient nutrition to supply so many flowers to complete the fruit setting. However, the average fruit number of each inflorescence in SlMBP21-RNAi lines was still significantly higher than that in WT plants (Figure 4B). Furthermore, another statistic analysis exhibited that although the average weight per fruit in each inflorescence (Figure 4C) was reduced, the total weight of all fruits in each inflorescence was dramatically higher than that in WT plants (Figure 4D). These data together demonstrate that SlMBP21 has vital functions in controlling floral meristem development, flower production and fruit yield in tomato.

3.4. Maturation of SIMs Was Repressed in SlMBP21-RNAi Lines

Tomato inflorescences are compound shoots, resulting from sympodial inflorescence meristems (SIMs, i.e., IMs), each of which gives rise to another SIM before failure of the meristems emerging from the SIM to specify floral identity [8]. The rate of SIM maturation drives inflorescence complexity and flower production, and a delay in SIM maturation can increase the complexity of inflorescence [56,57]. Finally, the SIMs terminate in the last FMs and no new SIM is produced after 6–10 flowers are produced. However, in our study, the scanning electron microscopy (SEM) analysis showed that the inflorescences in SlMBP21-RNAi lines can simultaneously give rise to multiple SIMs before terminating in one FM (Figure 5). These SIMs can further generate more FMs and finally produce a very large number of flowers (Figure 5). These results reveal that the conversion from SIMs to FMs is delayed in SlMBP21-RNAi lines, which is regarded as the immediate reason of the extreme branched inflorescences. These data suggest that SlMBP21 is a crucial regulator in the differentiation and maturation of SIMs.

3.5. Transcriptome Analysis in Meristems of the WT and SlMBP21-RNAi Lines

Numerous genes of different families have been proven to function as the vital regulators in regulating plant development. To explore the expression changes of genes during meristem (SIM and FM) development in WT and SlMBP21-RNAi plants, we performed the RNA-seq analyses. In this RNA-seq, four cDNA libraries including the WT and three RNAi lines were produced to be sequenced. More than 23 million sequence reads were obtained from each cDNA library, representing the sequence data of >7 Gb for each sample. Finally, a total number of 23,855 genes were identified from meristems (SIMs and FMs) of WT and SlMBP21-RNAi lines using FPKM > 1 as the standard to determine whether genes were expressed. RNA-seq data of these samples showed good correlations and were appropriate for further study (Figure S3). A summary of RNA-seq, mapping, annotation, and assembly was shown in Table S3. As a consequence, a total number of 2229 genes with 805 genes up-regulated and 1424 genes down-regulated were identified as the differentially expressed genes (DEGs) between WT and SlMBP21-RNAi lines using padj < 0.05 and |log2 (fold-change)| ≥ 1 as the significant cut-off value in DESeq2 (Figure 6A–C, Table S4). Among these DEGs, numerous genes belonging to different significant gene families and phytohormone synthesis were identified, such as AUX/IAA, GRAS, ARF, WRKY, Dof, MADS-box, AP2/ERF, bZIP, EXP, bHLH, synthesis and response of gibberellin, auxin and ethylene, and so on.

Gene ontology (GO) functional enrichment analysis of these DEGs was implemented to classify them according to their functions in molecular function (MF), cellular component (CC), and biological process (BP). The results showed that these DEGs were enriched in 638 GO terms (82 CC, 235 MF, 321 BP; padj < 0.05). For CC, DEGs were mainly enriched in ribosome, organelle, photosystem, ribonucleoprotein complex, and so on. For MF, most of the DEGs had something to do with protein activity, protein binding, and cell structural constituent. For BP, DEGs were primarily enriched in DNA replication, cell cycle, cell wall modification, response to stress and hormone, and so on (Figure 6D, Table S5). KEGG analysis displayed that 79 pathways were identified and a majority of these DEGs were primarily enriched in some pathways in connection with plant hormone signal transduction, metabolism, biosynthesis, DNA replication, and MAPK signaling pathway, and so on (Figure 6E, Table S6 KEGG). These data demonstrate that these DEGs participate in regulating tomato SIM and FM development via controlling the transcripts of genes related to cell cycle, enzyme activity, plant hormone signal transduction, and cell proliferation.

3.6. Expression Profile Analysis of Genes Related to SIM and IM Development

To further verify our transcriptome data and explore the vital functions of SlMBP21 in meristem (SIM and FM) development, some genes which participate in floral meristem development, cell wall metabolism, cell cycle, signal transduction, cell wall modification, and plant hormone metabolism and response were selected from our RNA-seq data to perform the qRT-PCR analysis in the meristems of WT and SlMBP21-RNAi plants. The results exhibited that the transcripts of these genes showed varying degrees in meristems (Figure 7 and Figure 8). The transcriptional accumulations of SP, STM3, and JOINTLESS were dramatically increased, whereas S was markedly reduced in SlMBP21-RNAi lines (Figure 7A). The transcripts of four cell cycle-related genes (cycA2, cycD3, cdkB2, and E2FE) were observably increased in SlMBP21-RNAi lines (Figure 7B). The expression levels of four genes, cel2, XTH1, EXPA18, and MAN1, which were involved in cell wall modification were significantly increased in SlMBP21-RNAi lines (Figure 7C).

Phytohormones, such as cytokinin, auxin, gibberellin, ethylene, and so on play crucial regulatory functions in plant development [58,59,60]. Here, we also detected several genes that are involved in regulating the synthesis, degradation, or responses of three kinds of phytohormones (cytokinin, auxin, ethylene). These phytohormones promote cell development at the early stage of organ development. Our results displayed that the transcripts of two genes (LOG3 and LOG6) related to cytokinin synthesis were significantly up-regulated, while two degradation-related genes (CKX2 and CKX7) were decreased (Figure 8A). The transcripts of auxin synthesis-related genes (IAA2 and IAA5) and auxin response-related genes (ARF5 and ARF9) were obviously increased in SlMBP21-RNAi lines (Figure 8B). Moreover, the transcripts of two known genes (ACS1A and ACS6), which participated in ethylene synthesis, and two ethylene response genes (ERF-H1 and ERF5) were prominently up-regulated in SlMBP21-RNAi lines (Figure 8C). These data indicate that SlMBP21 may regulate SIM development and the switch from SIMs to FMs by either directly or indirectly regulating the transcripts of genes related to meristem development, cell cycle, plant hormone signal transduction, and cell wall modification, and so on.

3.7. SlMBP21 Could Interact with SFT, SlCMB1, JOINTLESS, and MC

Transcription factors usually form homologous or heterologous dimers or polymers with other regulatory proteins to control plant development. To explore the interactions between SlMBP21 and other regulators, SFT, SlCMB1, JOINTLESS, and MC were selected for the yeast two hybrid assay. In tomato, these four proteins have been proven to be the vital regulators during tomato inflorescence development [10,40,61,62]. The open reading frame (ORF) of SlMBP21 was amplified and cloned into pGBKT7 as the bait and the ORFs of SFT, SlCMB1, JOINTLESS, and MC were each inserted into pGADT7 as prey, respectively (Figure S4A,B). Self-activation of pGBKT7-SlMBP21 was tested and a negative result was observed (Figure S5). Moreover, the empty pGBKT7 and pGADT7 vectors were also used as the negative controls, respectively. Figure 9 shows that the yeast can grow on selective media (QDO) and turn blue on X-α-gal indicator plates (QDO/X), demonstrating that SlMBP21 can interact with SFT, SlCMB1, JOINTLESS, and MC, respectively.

4. Discussion

The transition from vegetative to reproductive growth is a significant event for flowering plants. In this process, the noticeable changes are the production of flowers. Previous investigations have proven that MADS-box proteins play crucial functions in reproductive development [63,64,65,66]. SlMBP21 is an SEP MADS-box gene according to the phylogenetic analysis. Its expression patterns in tomato tissues suggest that it may take part in regulating tomato inflorescence development. Then, SlMBP21-RNAi lines produced abundant flowers with the enlarged sepals and lacked pedicel abscission zone, which are consistent with previous studies [52,67,68]. In addition, the repressed maturation of SIM and a delay in the conversion from SIMs to FMs were also observed in SlMBP21-RNAi lines. These results demonstrate that SlMBP21 plays indispensable roles in regulating tomato SIM development and the conversion from SIMs to FMs in addition to its functions in the abscission zone and sepal development.

In tomato, multiple genes which play crucial functions in plant inflorescence development have been identified. This will be conducive to the regulatory network investigations of flower formation and meristem (IM and FM) development. For instance, STM3 regulates inflorescence meristem development by activating FRUITFULL1, overexpression of STM3 results in increased flowers [48]. Overexpression of the SP (SELF-PRUNING) gene in tomato results in replaced flowers by leaves and suppressed transformation from vegetative to reproductive growth [69]. In jointless mutants, after one or two flowers were produced, the inflorescences produce a shoot rather than new flowers [62]. Mutation of the S gene in tomato results in highly branched inflorescences with abundant flowers [13]. In this study, the transcripts of STM3, SP, JOINTLESS were increased, whereas the expression of S was observably suppressed in SlMBP21-RNAi lines based on our RNA-seq and qRT-PCR data. Therefore, increased transcripts of STM3, SP, JOINTLESS, and reduced expression of S may lead to the branched inflorescences with dramatically increased flowers in SlMBP21-RNAi lines. Based on these results, we speculate that decreased SlMBP21 transcripts results in the expression changes of some genes relating to inflorescence or meristem development, and finally the SlMBP21-RNAi lines produce highly branched inflorescences with significantly increased flowers and fruits.

In the differentiation of flowers from SAM to IM, from the IM (it is named SIM in tomato) to FM, and from the FM to flowers, numerous genes play crucial regulatory roles in these processes, such as genes related to biosynthesis and metabolism of plant hormones, cell wall modification, cell cycle, and other biological processes. In eukaryotes, the cell cycle is principally controlled by heterodimeric complexes of cyclins (CYCs) and cyclin-dependent kinases (CDKs) [70,71]. Pectin lyases (PLs), β-xylosidases (XYLs), endo-1,4-β-glucanases (CELs), β-galactosidases (TBGs), pectin methylesterases (PMEs), polygalacturonases (PGs), expansins (EXPs), and xyloglucan endotransglucosylases/endohydrolases (XTHs) play important regulatory roles in the modification of cell walls [72]. The plant hormones (gibberellins (GAs), IAA, ethylene, cytokinin, et al.) can regulate plant development via tissue elongation or expansion, such as roots, stems, leaves, flowers, and fruits [73,74,75]. During gibberellin (GAs) synthesis, GA20oxs and GA3oxs control the bioactive GAs production, whereas GA2oxs catalyze GAs deactivation to produce inactive GAs [76]. GAs can regulate numerous aspects of plant development, such as the stem elongation, flowers development, pollen maturation, and so on [75]. Auxin response factor ARF5 controls fruit set and development through mediating auxin and gibberellin signaling, and ARF9 regulates cell division during early fruit development in tomato [77,78]. Overexpression of IAA1 with domain II mutation results in impaired cell division and elongation in Arabidopsis [79]. Cytokinin and auxin regulate almost every aspect of plant development, such as the cell division and differentiation, meristem function, senescence and stress responses, and so on. Cytokinin can maintain the stem cell population in the SAM, and auxin can specify the meristematic fate in the SAM [80]. In specific developmental conditions, ethylene can positively influence cell division and expansion. Ethylene and its downstream regulators AtERF018 and AtERF109 promote cell division in the process of stem vasculature development in Arabidopsis [81]. In grape berry and Sagittaria pygmaea, ethylene increases the transcripts of XTHs and promotes cell expansion and cell wall loosening [82,83].

In our RNA-seq analysis, the transcripts of DEGs related to cell cycle, enzyme activity, plant hormone signal transduction, cell proliferation, and cell wall modification were significantly increased or decreased. Furthermore, the expression levels of genes related to inflorescence development (SP, STM3, JOINTLESS, and S), cell cycle (cycA2, cycD3, cdkB2, and E2FE), cell wall modification (cel2, XTH1, EXPA2, and MAN1), the metabolism or responses of cytokinin (LOG3, LOG6, CKX2, and CKX7), auxin (IAA2, IAA5, ARF5, and ARF9), and ethylene (ACS1A, ACS6, ERF-H1, and ERF5) were dramatically up- or down-regulated. In addition, our previous study had shown that three ethylene-responsive elements (ERE motif), one auxin-responsive element (TGA-box), and one gibberellin-responsive element (P-box) were found in promoter sequence of SlMBP21 [68], further suggesting that SlMBP21 may regulate tomato organ development by affecting plant hormone levels. These results demonstrate that SlMBP21 may regulate tomato SIM differentiation and the conversion from SIMs to FMs through controlling the transcripts of genes relating to cell development, plant hormone signal transduction and meristem development, and biological processes.

Up to now, MADS-box proteins have been widely proven to play vital functions in regulating plant reproductive development. Previous studies have reported that MADS-box proteins can interact with other regulators to form the dimers or higher-order protein complexes to control plant growth and development [84,85]. MC encodes an AP1/SQUAMOSA-like MADS-box transcription factor and is an important regulator of inflorescence and sepal development, and also controls tomato fruit abscission zone development by interacting with J [40,86]. The MADS-box protein JOINTLESS functions as an important regulator in inflorescence development and the abscission zone of tomato [49,62]. JOINTLESS has also been proposed to be involved in the timing of floral meristem maturation and to reduce the time window for floral meristem maturation, and the jointless mutants generate two or three flowers followed by leaves in inflorescences [87]. The sft mutant displays the conversion from reproductive to vegetative growth after generating one or several flowers [10,12]. An additional study reports that a target of SFT and JOINTLESS participates in a protein complex to suppress the floral meristem fate [87]. Our previous study showed that the suppression of MADS-box protein SlCMB1 results in enlarged sepals and longer and loss of determinacy of SIM [61]. It has been reported that MC can interact with JOINTLESS to form a dimer [86], and JOINTLESS can interact with itself to form a homodimer, but MC cannot [52]. Furthermore, SlMBP21, MC, and J can form trimer in vivo [52]. In our study, the yeast two-hybrid assay showed that SlMBP21 can interact with SFT, MC, JOINTLESS, and SlCMB1, respectively. Hence, we further speculate that SlMBP21 may form dimer or trimer with SFT, MC, JOINTLESS, and SlCMB1 to control SIM and FM fate via regulating the expression of related genes.

5. Conclusions

The fate of inflorescence meristem and floral meristem is a very complex biological process, which involves many cascade regulations, and still needs to be further explored. In this study, we evaluated the physiological, molecular, and morphological features of SlMBP21-RNAi tomato plants. Although the accurate regulatory mechanisms of SlMBP21 in tomato SIM development and the conversion from SIMs to FMs is still to be investigated, SlMBP21 should be a significant target to explore the regulatory mechanisms exploring tomato SIM development. This will provide a new method to improve the fruit yield of tomato plants and other crops. In summary, our data demonstrate that SlMBP21 functions as the crucial regulator in tomato SIM development and the conversion from SIMs to FMs through interactions with other regulatory proteins to regulate the activities of related regulators or the expression levels of genes relating to numerous biological processes. Identifying the functions of SlMBP21 in tomato SIM development and the conversion from SIMs to FMs not only extends our understanding of the biological roles of MADS-box proteins in plants but also provides new insights into IM development, an important agronomic trait.