Next Article in Journal
Development and Application of a Cleaved Amplified Polymorphic Sequence Marker (Phyto) Linked to the Pc5.1 Locus Conferring Resistance to Phytophthora capsici in Pepper (Capsicum annuum L.)
Next Article in Special Issue
Physiological Changes and Transcriptomic Analysis throughout On-Tree Fruit Ripening Process in Persimmon (Diospyros kaki L.)
Previous Article in Journal
New Finds and Ecology of the Rare Liverworts Scapania apiculata, Scapania carinthiaca, and Scapania scapanioides in Austria
Previous Article in Special Issue
Transcriptional Regulation of Female and Male Flower Bud Initiation and Development in Pecan (Carya illinoensis)
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 12
Issue 15
10.3390/plants12152754
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

TM3 and STM3 Promote Flowering Together with FUL2 and MBP20, but Act Antagonistically in Inflorescence Branching in Tomato

by
Iris E. Zahn
1,
Chris Roelofsen
1,
Gerco C. Angenent
1,2 and
Marian Bemer
2,*
1
Laboratory of Molecular Biology, Wageningen University & Research, 6708 PB Wageningen, The Netherlands
2
Business Unit Bioscience, Wageningen University & Research, 6708 PB Wageningen, The Netherlands
*
Author to whom correspondence should be addressed.
Plants 2023, 12(15), 2754; https://doi.org/10.3390/plants12152754
Submission received: 23 June 2023 / Revised: 17 July 2023 / Accepted: 19 July 2023 / Published: 25 July 2023
(This article belongs to the Special Issue Gene Regulatory Mechanisms of Flower and Fruit Development in Plants)
Browse Figures
Versions Notes

Abstract

:
The moment at which a plant transitions to reproductive development is paramount to its life cycle and is strictly controlled by many genes. The transcription factor SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) plays a central role in this process in Arabidopsis. However, the role of SOC1 in tomato (Solanum lycopersicum) has been sparsely studied. Here, we investigated the function of four tomato SOC1 homologs in the floral transition and inflorescence development. We thoroughly characterized the SOC1-like clade throughout the Solanaceae and selected four tomato homologs that are dynamically expressed upon the floral transition. We show that of these homologs, TOMATO MADS 3 (TM3) and SISTER OF TM3 (STM3) promote the primary and sympodial transition to flowering, while MADS-BOX PROTEIN 23 (MBP23) and MBP18 hardly contribute to flowering initiation in the indeterminate cultivar Moneyberg. Protein–protein interaction assays and whole-transcriptome analysis during reproductive meristem development revealed that TM3 and STM3 interact and share many targets with FRUITFULL (FUL) homologs, including cytokinin regulators. Furthermore, we observed that mutating TM3/STM3 affects inflorescence development, but counteracts the inflorescence-branching phenotype of ful2 mbp20. Collectively, this indicates that TM3/STM3 promote the floral transition together with FUL2/MBP20, while these transcription factors have opposite functions in inflorescence development.

1. Introduction

The developmental switch to flowering requires an identity change in the shoot apical meristem (SAM) [1]. In tomato (Solanum lycopersicum), this switch underlies important agricultural traits such as flowering time and inflorescence architecture. When progressing from the vegetative to the reproductive stage, the flat and vegetative tomato SAM forms a dome-like structure and gradually changes into a transition meristem (TM) and subsequently floral meristem (FM). The FM terminates the shoot by producing a flower, and gives rise to a new inflorescence meristem (IM) on its flank. The IM will develop into an FM and form a new IM, and so forth. This iterative process results in a zigzagged inflorescence. Inflorescence branching depends on the development of the different reproductive meristems (TM, FM, IM). When FM maturation is delayed, multiple IMs are formed on the flank of a single FM, resulting in a (sometimes highly) branched inflorescence [2,3]. Thus, the development of the apical meristem determines both flowering time and inflorescence architecture.
A tomato shoot repeatedly makes the transition to flowering due to its sympodial architecture. After the primary apex terminates in an inflorescence, a new vegetative shoot is formed from the sympodial meristem (SYM), derived from the axillary meristem of the youngest leaf. The vegetative SYM again has to transition to flowering, thereby terminating the sympodial shoot. This sequential process is repeated infinitely in indeterminate cultivars or finitely in determinate cultivars [2,3].
A complex gene regulatory network underlies the development of the reproductive meristems. To induce the floral transition, SINGLE FLOWER TRUSS (SFT, homolog of FLOWERING LOCUS T (FT)) and FALSIFLORA (FA, homolog of LEAFY (LFY)) are essential. SFT encodes the florigen, a systemic signal that moves from the leaves to the SAM to induce flowering in primary and sympodial shoots [4,5]. In a parallel pathway, FA also induces flowering by regulating FM identity. Consequently, flowering is abolished when disrupting both SFT and FA [4,6]. Other promoters of the floral transition, albeit having a mild role compared to SFT and FA, are the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) homologs TOMATO MADS 3 (TM3) and SISTER OF TM3 (STM3) [7], and the FRUITFULL (FUL) homologs FUL2 and MADS-BOX PROTEIN 20 (MBP20) [8].
After the initiation of flowering, a switch to IM and FM identity is required for inflorescence development. To confer reproductive fate, IM identity is promoted by MACROCALYX (MC, homolog of APETALA 1 (AP1)) [9]. Concomitantly, several genes promote the development of IMs into FMs, thereby repressing inflorescence branching. These genes include COMPOUND INFLORESCENCE (S, homolog of WUSCHEL-RELATED HOMEOBOX 9 (WOX9)) and ANANTHA (AN, homolog of UNUSUAL FLORAL ORGANS (UFO)), and the SEPALLATA 4 (SEP4) homologs JOINTLESS 2 (J2) and ENHANCER OF J2 (EJ2), of which the mutants have highly branched inflorescences [3,10,11]. Similar to j2 ej2, mutations in the floral regulators FUL1, FUL2 and MBP20 result in enhanced inflorescence branching [8].
Given that SOC1 is a master regulator of flowering time in Arabidopsis [12] and has conserved functions in a wide range of other species [13,14,15,16,17,18,19], there is still little known about its role in tomato. Two tomato SOC1 homologs, TM3 and STM3, are located in a highly repetitive and complex genomic region [7]. In addition, no research has yet been performed to elucidate the function of putative other tomato SOC1-like genes, and a complete phylogenetic analysis to identify and characterize all tomato SOC1-like genes is still lacking.
MADS-domain transcription factors (TFs) are involved in numerous developmental processes and often function as heterodimeric or -tetrameric complexes [20,21]. Recent publications have unveiled functions of individual MADS proteins in tomato flowering, including SOC1-, FUL- and SEP-like genes [7,8,9,10,11,22,23]. However, it is still largely unclear in which complexes these proteins interact to regulate the different steps during the floral transition and inflorescence development. In Arabidopsis, SOC1 and FUL form a strong heterodimer. The proteins interact in yeast [24] and they bind to the same promoter elements [25], preferentially as a heterodimer or -tetramer and not as homodimers [26]. Moreover, genetic experiments show that SOC1 and FUL function partially redundantly in flowering time [27]. Together, this indicates that SOC1 and FUL physically interact in Arabidopsis to control several developmental processes, including the floral transition.
It is likely that tomato SOC1 homologs genetically and physically interact with the aforementioned FUL and SEP4 homologs to regulate target gene expression in the reproductive meristems. Previous publications showed that the highly branching inflorescence phenotype of the j2 ej2 mutant can be largely suppressed by a knock-out of TM3 and STM3 [7,22,28], and that STM3 and J2 compete for binding at the FUL1 promoter [28]. These findings indicate that tomato SOC1 homologs can counteract the function of J2 and EJ2. Furthermore, FUL2 can interact with TM3, STM3, J2 and EJ2 [8]. STM3 can also interact with J2 [28], supporting that these TFs form heteromeric complexes to fulfill their functions. Strikingly, it was also reported that STM3 activates FUL1 in the apex [22,28] and that a knock-out of FUL1 can, like TM3 and STM3, repress the highly branching phenotype of j2 ej2 [22]. This apparently contradicts the repressive function in inflorescence branching of FUL homologs in a WT background [8], showing that the interactions of MADS-domain proteins during reproductive meristem development are still far from understood. Thus, it is clear that SOC1, FUL, and SEP4 homologs play a role in the regulation of the floral transition and inflorescence development in tomato, but their independent and combinatorial functions are still unclear. Here, we shed more light on the genetic and physical interactions of SOC1 and FUL homologs, and the functions of their encoded proteins during the floral transition and inflorescence development.
We characterized the developmental roles of the meristem-expressed SOC1 homologs TM3, STM3, MBP23 and MBP18 during reproductive meristem development in the indeterminate cultivar Moneyberg. Using CRISPR/Cas9 mutagenesis and whole-transcriptome profiling, we show that of these four homologs, mainly TM3 and STM3 are important regulators of the floral transition and inflorescence development, while MBP18 appears to play a minor role. We found that TM3/STM3 most likely control the floral transition by interacting with FUL2/MBP20 in a protein complex and together repress cytokinin inhibitors. Subsequently, TM3/STM3 and FUL1/FUL2/MBP20 have antagonistic functions in specifying FM and IM identity, which is required for normal inflorescence development.

2. Results

2.1. Identification and Characterization of Tomato SOC1 Homologs

2.1.1. Phylogenetic Analysis of the Solanaceae SOC1-like Proteins

To characterize the complete SOC1-like clade in tomato, a phylogenetic tree was built based on related proteins in several Solanaceae species and Arabidopsis (Figure 1a). The tree shows two clades of homologous proteins that are evolutionary most closely related to AtSOC1. Most analyzed Solanaceae have a single homolog in the first clade, including the FBP28-like proteins from Petunia. The tomato protein in this clade is encoded by Solyc10g017640 (coding for the MADS domain) and Solyc10g017630 (coding for the rest of the protein), of which the latter locus has been named MBP23 [29]. Homologs from Capsicum annuum, Nicotiana benthamiana and N. attenuata are absent in this clade. The other clade closely related to AtSOC1 contains the tomato paralogs TM3 and STM3. Tomato, eggplant, pepper, N. attenuata, N. tabacum and N. sylvestris each have two paralogs in this clade, which are separated in TM3- and STM3-like clusters. Potato and N. benthamiana do not have TM3-like proteins, while the Petunia FBP20 proteins group outside of the TM3/STM3 clade. More distantly related to AtSOC1 is a clade with the tomato homologs MBP13 and MBP14, which form separate clusters. Tomato, pepper, most Nicotiana species and Petunia have at least one homolog in both clusters, while eggplant and potato only have MBP13-like homologs.
To identify all proteins that potentially function as SOC1-like TFs in the regulation of tomato flowering, homologs of the related Arabidopsis flowering genes AGAMOUS-LIKE 42 (AGL42), AGL71 and AGL72, which emerged from a duplication of the ancestral SOC1 gene, were included in the phylogenetic analysis [30]. For most Solanaceae analyzed here, a single homolog is present in the clade containing AGL42, AGL71 and AGL72. Only for S. lycopersicum, two proteins, MBP18 and MBP19, group to this clade. Finally, floral regulators from the SEP family were used to root the tree.
It has recently become clear that structural variation, often involving gene copy number variation, is a common phenomenon within the germplasm of crops [7,31,32,33]. In tomato, TM3 and STM3 are located in a repetitive genomic region that is variable in STM3 copy number across tomato cultivars [7]. In our study, we used the indeterminate variety Moneyberg (a TMV-resistant version of Moneymaker [34]). A recent genome assembly of the cultivar Moneymaker contains a single copy of TM3 and two copies of STM3 [35], indicating that Moneyberg also carries two STM3 copies (Supplementary Figure S1a). The coding sequences of TM3 and STM3 are highly similar and their MADS domains are even identical at the nucleotide level (Supplementary Figure S1b). Consequently, we can distinguish TM3 from STM3, but cannot discriminate between the two STM3 copies on a DNA or RNA level.

2.1.2. Expression of Tomato SOC1 Homologs Peaks during the Primary Floral Transition

To select the homologs that potentially fulfil the flowering function of AtSOC1 in tomato, we characterized the expression patterns of the identified tomato SOC1-like genes during reproductive meristem development using qPCR on cDNA from three different meristem stages: VM, TM and combined FM/IM of the primary inflorescence (Figure 1b,c). We found a clear peak in expression in the TM for MBP23, TM3, STM3 and MBP18. Of these genes, STM3 stood out because of its extremely high expression levels. In contrast, expression was weak or decreasing during reproductive meristem development for MBP14, MBP13 and MBP19. Therefore, based on phylogenetics combined with considerable and dynamic expression in the TM, we selected MBP23, TM3, STM3 and MBP18 as targets to further study the role of SOC1-like genes during reproductive meristem development in tomato.

2.2. Functional Characterization of Tomato SOC1 Homologs in Flowering

2.2.1. TM3 and STM3 Display a Broad Protein–protein Interaction Profile, in Contrast to MBP23 and MBP18

To gain more insight into the overlapping and specific functions of the four selected tomato SOC1 homologs, we investigated their interactions with several other tomato MADS-domain proteins linked to flowering [8,9,10,11,36,37]. Yeast two-hybrid (Y2H) assays showed a broad and largely overlapping protein–protein interaction profile for TM3 and STM3, which was clearly distinguishable from the interaction profiles of MBP23 and MBP18 (Figure 2a and Supplementary Figure S2). TM3 and STM3 interacted with all screened flowering regulators from the AP1/FUL, SEP, AGAMOUS (AG) and SHORT-VEGETATIVE PHASE (SVP)/AGL24 TF families. Only the varying ability to heterodimerize with other SOC1 homologs illustrates minor differences between TM3 and STM3. Although both TM3 and STM3 could homodimerize in yeast, interaction was much stronger for STM3. These extensive interaction profiles indicate a broad role for TM3 and STM3 in reproductive development. Furthermore, our assays showed that MBP23 and MBP18 have a more limited number of protein interaction partners. Their interactions are very similar, except that MBP18 interacted with the important flowering regulators FUL2 and MC, while MBP23 did not. Both MBP23 and MBP18 were unable to form homodimers; thus, these proteins most likely depend on interaction with other MADS-domain proteins to fulfil their functions.

2.2.2. TM3 and STM3 Promote the Primary and Sympodial Transition to Flowering

To further characterize the functions of the four selected tomato SOC1-like TFs in planta, we generated single- and higher-order mutants in the cultivar Moneyberg by CRISPR/Cas9 mutagenesis. We targeted the MADS-box sequence using two sgRNAs per gene and sgRNAs for multiple genes were combined in transformations to generate higher-order mutants with independent mutations. Of the quadruple mutant mbp23 tm3 stm3 mbp18, an additional independent line was generated by crossing mbp23 tm3 stm3 with tm3 stm3 mbp18 and selecting homozygous F2 mbp23 tm3 stm3 mbp18 plants. TM3 and STM3 were targeted simultaneously in higher-order mutants, as their MADS boxes are identical. The single mutants tm3 and stm3 were obtained by targeting their unique second exon. Primary transformants with deleterious mutations in all targeted genes were selected, and T1/T2 offspring with homozygous mutations in MBP23, TM3 and MBP18, and homozygous or multiallelic mutations in the four alleles of STM3 were further analyzed. The obtained genotypes are shown in Supplementary Figure S3.
Phenotyping of the selected mutants revealed that TM3 and STM3 promote the primary transition to flowering (Figure 2b and Supplementary Figure S4). Initially we disrupted these two genes simultaneously, which delayed flowering time by three leaves, confirming the observation of Alonge et al. [7] in the determinate cultivar M82. The newly characterized SOC1-like genes MBP23 and MBP18 hardly affected flowering time. We did not observe statistically significant effects on flowering time for the double mutant, nor additively when stacking mutations in the tm3 stm3 mutant. Nevertheless, there was a trend towards mildly delayed flowering in mbp23 mbp18. To compare flowering times more precisely, we performed a second screening in a growth chamber with more stable and controlled conditions than in the greenhouse of the initial screening. We characterized the single mutants tm3, stm3 and mbp18, and the double mutant mbp23 mbp18 (Figure 2c). This revealed that TM3, and more strongly also STM3, promote flowering independently, and that MBP18 mildly contributes to the primary floral transition, while we cannot exclude there is a mild role for MBP23 as well.
Similar to the primary transition, the sympodial transition to flowering was delayed in tm3 stm3 mutants (Figure 2d,e). While the number of leaves per sympodial unit (SU) is consistently three in the Moneyberg WT, the tm3 stm3 mutants displayed a mild but significant increase in leaf number in the SUs. MBP23 and MBP18 did not affect sympodial leaf number, indicating that these genes have a minor role in the primary, but not in the sympodial transition to flowering.

2.3. SlSOC1 and SlFUL Genes Regulate a Set of Common Target Genes during the Floral Transition

To explain the delayed flowering in tm3 stm3, we performed an RNA-Seq experiment on VM, TM, and FM/IM tissues of WT and tm3 stm3. For each biological replicate, meristems from multiple plants at specific stages were harvested (as shown in Figure 1b) and pooled for RNA extraction. Expression of 288 marker genes for VM, TM, FM and IM tissue [38] showed a clear separation between the different meristematic tissues, indicating that the dissection of the different meristems was accurate (Supplementary Figure S5). The list of differentially expressed genes (DEGs) did not contain any obvious flowering regulator such as SFT or FA. Because the delayed flowering in tm3 stm3 is very similar to the reported phenotypes of ful2 mbp20 and ful1 ful2 mbp10 mbp20 mutants (hereafter referred to as q-ful mutants, for ‘quadruple ful’) [8], and TM3 and STM3 can heterodimerize with the SlFULs (Figure 2a), we hypothesized that the TFs act together to regulate the floral transition. Overlapping the DEGs of tm3 stm3 and q-ful meristems from all stages showed a significant enrichment for shared target genes (P = 2.2 × 10−23), in which 55 out of 328 DEGs of tm3 stm3 overlap with q-ful targets (Figure 3a and Supplementary Table S1). As the majority of the shared targets (73%) is upregulated in both mutants, the putative SlSOC1/SlFUL complex probably acts predominantly as a repressive complex.
The large majority of overlapping DEGs showed a similar expression pattern in both the tm3 stm3 and q-ful mutants. Among these DEGs were several TFs involved in plant development (Figure 3b and Supplementary Figure S7). For example, the tomato ortholog of AHL15, a gene that delays axillary meristem maturation in Arabidopsis [26], was significantly upregulated in both tm3 stm3 and q-ful, suggesting that a complex of TM3/STM3/FUL2/MBP20 represses AHL15 in the tomato apical meristems. The list of overlapping DEGs also contained two B3-domain TFs, including a putative close ortholog of Arabidopsis REM16 according to Plaza5.0 [39]. Several B3-domain TFs, including REM16, have been linked to the vegetative-to-reproductive phase transition in Arabidopsis [40,41] and, possibly, this tomato B3-domain TF (Solyc01g108940) is also involved in flowering time regulation. Interestingly, the shared DEGs also included several WRKYs, which are usually associated with a response to environmental conditions, but have recently also been linked to regulating flowering time [42]. In particular, WRKY28 was strongly upregulated in both mutants. Notably, we also found that the SOC1-like gene MBP13 was upregulated in tm3 stm3 and q-ful, and we observed the same trend for MBP14, together possibly alleviating the flowering phenotype of the mutants (Figure 3c). AHL15 and MBP13 have perfect CArG boxes in their promoters, and MBP13 is bound by STM3 in the apex [28], supporting direct regulation by the SOC1- and FUL-like TFs.
Most interesting from the transcriptomic analysis was the overlap in cytokinin regulators as targets of SlSOC1 and SlFUL genes. In q-ful, disturbed cytokinin signaling has been associated with its delayed flowering [8], and our RNA-Seq revealed that CYTOKININ OXIDASE/DEHYDROGENASE 4 (CKX4) and CKX6 were not only upregulated in q-ful, but also in tm3 stm3 (Figure 3d). The increased expression of these cytokinin inhibitors suggests that in tm3 stm3 the delay in flowering is also, at least partially, linked to disturbed cytokinin levels. Cytokinin enhances the activity of meristems, and thereby the rate of cell divisions and differentiation [43,44]. In line with expectations, we observed a slower development of the TM in both tm3 stm3 and ful2 mbp20 compared to WT (Supplementary Figure S6). The microscopic analyses of the meristems during the floral transition revealed that the delayed doming seemed to be caused both by a later initiation of differentiation towards the TM, and by a slower development of the TM itself.

2.4. TM3/STM3 Does Not Completely Depend on the SlFULs to Regulate the Floral Transition

To further investigate the genetic interaction between TM3/STM3 and FUL2/MBP20, we studied the flowering time in tm3 stm3 ful2 mbp20 mutants (Figure 3e). This quadruple mutant flowered even later than the tm3 stm3, mbp23 tm3 stm3 mbp18, or ful2 mbp20 mutants, indicating that the SlSOC1 and SlFUL genes do not act completely redundantly in the floral transition.
The additive effect of TM3/STM3 and FUL2/MBP20 on flowering time is also visible in the transcriptome data. In addition to the genes coregulated by TM3/STM3 and the SlFULs, we identified genes uniquely regulated by TM3/STM3 (Figure 3f and Supplementary Figure S7). For example, expression of AP2c was increased in the VM of tm3 stm3, but not in q-ful, and the same trend was visible for its close homolog AP2b. It is probable that AP2b and AP2c contributed to the delayed flowering of tm3 stm3, as they are close homologs of Arabidopsis AP2-like floral repressors [45,46,47,48]. Most likely, STM3 directly inhibits AP2b and AP2c, as they were identified as STM3 targets in the apex by ChIP-Seq [28], and their promoters contain multiple imperfect but probably functional CArG boxes (e.g., CC[AT]6CG). Furthermore, some DEGs were weakly expressed in WT reproductive meristems and upregulated in tm3 stm3, suggesting that TM3/STM3 represses genes in the meristem that are functional in other tissues. Among others, an AP2-like ERF (Solyc06g068570) was strongly upregulated, which is involved in drought tolerance in leaves [49]. Other DEGs are expressed during reproductive meristem development and were increased in tm3 stm3, such as CONSTANS (CO) interacting factor 2a. Next to the association of this TF to drought tolerance [49], it may also play a role in flowering control by interacting with CO, a central player in the photoperiod flowering pathway [50]. We also identified several C2H2- and CCCH-type zinc finger TFs that were upregulated independently of the SlFULs (a.o. Solyc06g065440), which are both classes of TFs with a broad role in plant development [51,52]. These expression data indicate that TM3 and STM3 are involved in an extensive network of TFs to regulate flowering time and probably other plant developmental processes.

2.5. TM3/STM3 and FUL2/MBP20 Destabilize FM/IM Development, but in Opposite Ways

It has been reported that mutating TM3 and STM3 in the j2 ej2 mutant background alleviates the branching phenotype of the j2 ej2 mutant [7,22,28]. Interestingly, J2/EJ2 and FUL2/MBP20 most likely form a complex to regulate FM maturation and both double mutants show increased inflorescence branching [8,11]. Given that TM3/STM3 can heterodimerize with both J2/EJ2 and FUL2/MBP20 (Figure 2a), but appears to counteract the function of J2/EJ2 in the FM, we set out to elucidate the interactions between the MADS-domain TFs in reproductive meristem specification further.
To start, we studied the expression of the tomato SOC1, FUL and SEP homologs in Moneyberg WT sympodial FM (sFM) and sympodial IM (sIM) tissue separately (Figure 4a). As for the primary reproductive meristems, we pooled 20–50 meristems of visually the same stage per biological replicate prior to RNA extraction. The expression of AN and UF, marker genes for FM and IM identity, respectively [3,53], showed a clear separation of these meristems. The data revealed that the SlSOC1 and SlFUL genes have similar expression patterns and are expressed in the sFM and sIM at equal levels. This suggests that the SOC1 and FUL homologs play a role in both IM and FM development. On the contrary, SEP-like expression (J2, EJ2, TM5) was specific for the sFM and much lower in the sIM. J2 and EJ2 were especially highly expressed in the sFM, indicating that they are required for FM development and have a minor contribution to the establishment of IM cell fate.
To further unravel what the role is of SlSOC1 and SlFUL genes in FM and IM specification, we phenotyped the inflorescences of single up to quadruple slsoc1 mutants. We observed enormous variation in inflorescence traits, including branching, revertance to vegetative growth and flower number (Supplementary Figure S8). The large variation in phenotypes hindered the pinpointing of the exact result of the mutations, but indicated that the usually strict regulation of meristem development was disrupted in the mutants. To acquire a better understanding of the effects of the tm3 stm3 mutations on the inflorescence phenotype, and investigate their effect on the increased branching phenotype of ful2 mbp20, we grew several mutant combinations in parallel. Categorizing the inflorescences on multiple traits revealed that inflorescence development was more disturbed in the slsoc1 mutants than in WT (Figure 4b). In WT, 62% of inflorescences was normal, i.e., being unbranched, having no revertance to vegetative growth and 7 to 10 flowers. Inflorescences were quantified in two separate screenings, and while the fraction of abnormal inflorescences was consistent in WT, the type of destabilization differed over the screenings, indicating that the environment affects the type of destabilization. In the slsoc1 mutants, inflorescence development was disturbed, as 17 to 40% of inflorescences was normal and the majority of inflorescences deviated from this standard by showing single or multiple deviating traits.
To better understand the role of TM3/STM3 in inflorescence development and their interaction with FUL2/MBP20, we compared the inflorescence phenotypes of the double and combinatorial quadruple mutants (Figure 4b). Inflorescence development was disturbed in both tm3 stm3 and ful2 mbp20. Notably, these mutants showed distinct abnormal phenotypes. In tm3 stm3, the majority of abnormal inflorescences had less than 7 or more than 10 flowers without branching or revertance, and 18% of all inflorescences was branched. In ful2 mbp20, 58% of all inflorescences showed a typical phenotype that is branched and has more than 10 flowers. Combining these mutations in tm3 stm3 ful2 mbp20 resulted in an alleviation of the ful2 mbp20 branching phenotype, as the main category “branched and aberrant flower number” was strongly reduced from 58% to 14%. Furthermore, the proportion of normal inflorescences was increased to 33%, similar to tm3 stm3. These results indicate that TM3/STM3 and FUL2/MBP20 have opposite roles in inflorescence development.

3. Discussion

3.1. The Role of SlSOC1 Genes in the Floral Transition

Regulation of flowering time is crucial for plant reproductive success. The underlying gene regulatory network, including integration of flowering signals by SOC1, is largely conserved between the Brassicaceae and Asteraceae families [54]. Here, we showed that, also in tomato, several SOC1 homologs have an evolutionarily conserved function in regulating flowering. The broad conservation of SOC1’s role in flowering regulation across angiosperms highlights the general importance of this gene.
We identified four SOC1 homologs in tomato that are expressed in the SAM upon the transition to flowering. CRISPR mutagenesis of these four genes revealed that tm3 and stm3 mutants display a significant delay in flowering in the indeterminate Moneyberg variety, which is in line with the previously reported role of TM3 and STM3 in the determinate cultivar M82 [7]. In addition to regulating the floral transition in the primary shoot, we show here that TM3 and STM3 also regulate the sympodial flowering time in Moneyberg. Our yeast protein–protein interaction data showed largely overlapping protein–protein interaction profiles for TM3 and STM3, suggesting that the paralogs act in a largely redundant manner. However, STM3 contributes more strongly to the floral transition than TM3, most likely because its expression in the reproductive meristems is about tenfold higher, partially due to its gene duplication. Thus, TM3 and STM3 probably promote flowering initiation in a redundant, but dose-dependent manner.
Next to TM3 and STM3, MBP23 and MBP18 are also related to AtSOC1 and dynamically expressed during the floral transition. However, their mutants did not show strong flowering phenotypes. Flowering was mildly delayed in the mbp18 single mutants, which only became clear in a growth chamber with a controlled environment. There was no visible flowering-time phenotype for mbp23 mutants, nor did its mutation enhance the phenotype of the other mutants. It is possible, however, that MBP23 is only induced under certain conditions that were not applied in our experiments. Another explanation for the mild or absent flowering phenotypes is a compensation effect of other SOC1-like proteins. For example, in tm3 stm3, the SOC1-like genes MBP13 and MBP14 were upregulated, possibly alleviating the flowering phenotype. A similar compensation effect by SOC1-like genes can explain the minor phenotypes in mbp23 and mbp18. In Arabidopsis, the SOC1-like genes AGL14, AGL19, AGL42, AGL71 and AGL72 regulate the floral transition together with SOC1 [30,55,56]. It is thus plausible that also in tomato, SOC1-like genes function partially redundantly and compensate for each other. Alternatively, MBP23 and/or MBP18 may have a more prominent role in other tissues. For example, the tomato eFP browser [57] shows that MBP23 is most highly expressed in the roots, and may thus play a role in root development. Interestingly, AtSOC1 is also expressed in the root, influences the number of lateral roots and is upregulated in response to nitrate deprivation [58]. A role for tomato SOC1 homologs in root growth is therefore not unlikely.
In Arabidopsis, SOC1 is a true floral integrator gene, integrating signals from the different flowering pathways [12]. We found that in tomato, TM3 and STM3 clearly promote the transition to flowering, but they are not essential. The tm3 stm3 mutants still flower, although later, and their phenotype is relatively mild compared to the severely delayed flowering phenotype of atsoc1 [27]. Nevertheless, in all slsoc1 mutants, we observed an increased variation in flowering time, which appeared to depend on the environmental conditions. This indicates that in the absence of TM3/STM3, the strict regulation of flowering time is lost and environmental pathways (e.g., light quality, temperature) become more pronounced. Possibly, the integrator function of SOC1 is shared among multiple SlSOC1 homologs, all responding to different signals, and together determining when the plant makes the transition to flowering, suggesting subfunctionalization within the SlSOC1 clade. The upregulation of MBP13 and MBP14 in the tm3 stm3 mutant may hint at this direction.

3.2. SlSOC1 and SlFUL Proteins Interact during the Floral Transition

To explain the delayed flowering of tm3 stm3, we performed an RNA-Seq experiment of reproductive meristems. The major highlight of the transcriptomic analysis was the significant overlap in target genes of TM3/STM3 and the SlFULs, which included cytokinin regulators. Considering that TM3/STM3 can physically interact with FUL2/MBP20 (Figure 2a), that MADS-domain proteins often function in heteromeric complexes [20], and that all four TFs are important for the floral transition (Figure 3), it is likely that the upregulation of CKXs in tm3 stm3 and q-ful is due to the alleviated binding of the tetramer TM3/STM3/FUL2/MBP20 to the CKX4/6 promoters. TM3/FUL2 and TM3/MBP20 have already been shown in in vitro assays to bind as tetramers to the promoter of CKX6 [8]. Since TM3 and STM3 have highly similar protein sequences, they are probably interchangeable in the tomato SOC1/FUL tetramer. Like CKX6, CKX4 has a CArG box in its promoter, providing a binding site for the heterotetrameric MADS complex. These results suggest that the delayed flowering of tm3 stm3 and q-ful is due to increased CKX expression, resulting in lower cytokinin levels, a less active SAM [43,44], and consequently a slower doming of the TM.
While tm3 stm3 and q-ful are both delayed in flowering and share many target genes, the quadruple mutant tm3 stm3 ful2 mbp20 is even more delayed in flowering (Figure 3e). This additive effect of mutations in SlSOC1 and SlFUL indicates that these genes act partially, but not completely, redundantly, like in Arabidopsis [27]. Such independent functions of SlSOC1 and SlFUL genes in the floral transition were further supported by the differential regulation of target genes between the mutants (Figure 3f and Supplementary Figure S7). For example, in q-ful, but not tm3 stm3, CKX5 and CKX8 were also upregulated, indicating that cytokinin regulation was more disturbed in q-ful than in tm3 stm3. On the other hand, other putative negative flowering regulators, including AP2b and AP2c, were more strongly upregulated in tm3 stm3 than in q-ful. Several members of the AP2-like family in Arabidopsis function as floral repressors by promoting the vegetative phase [45,46,47,59]. In tomato, TM3/STM3 are expressed earlier in the SAM than FUL2/MBP20, and may thus be more important for terminating vegetative fate via the repression of AP2b/AP2c. In line with this hypothesis, FUL2/MBP20 repress AP2b/AP2c later during secondary FM/IM development [8].
The subtle difference in flowering regulation between tm3 stm3 and q-ful indicates that different MADS-domain protein complexes have varying DNA-binding affinities. Indeed, changes in binding specificity are a common mechanism underlying differential gene expression during development [60,61,62]. Next to the regulation of target genes by TM3/STM3-FUL2/MBP20, other MADS-domain protein complexes can be formed, especially when the native complex in the mutants is not functional anymore. TM3 and STM3 can also bind to the DNA as homodimers (Figure 2 and [8]). FUL2 and MBP20 cannot homodimerize and depend on interaction with other MADS-domain TFs, potentially J, which is highly expressed in reproductive meristems (Supplementary Figure S9) and can interact with FUL2 and MBP20 [8]. Therefore, it is likely that the tetrameric SlSOC1/SlFUL complex competes for binding with other MADS-domain protein complexes including tomato SOC1 or FUL homologs (e.g., MBP13, MBP14, FUL1), resulting in different transcriptomic changes in tm3 stm3 and q-ful. Collectively, these changes in either mutant result in a similar cell identity shift and phenotype, namely, a delay of the floral transition.

3.3. SlSOC1 and SlFUL Genes Have Opposite Roles in Inflorescence Development

Besides the delayed floral transition, tm3 stm3 and ful2 mbp20 mutants also show an increase in abnormal inflorescences. When taking a closer look, the destabilization of inflorescence development in these mutants is clearly different. Interestingly, mutating TM3 and STM3 suppressed the inflorescence-branching phenotype of ful2 mbp20, suggesting that TM3/STM3 and FUL2/MBP20 have opposite roles in inflorescence development. TM3/STM3 have already been shown to act antagonistically to J2/EJ2 in inflorescence branching [7,22,28], and TM3/STM3 may have a similar role in counteracting FUL2/MBP20. FUL2/MBP20/J2/EJ2, together, seem to regulate inflorescence branching by promoting FM maturation, while TM3/STM3 have an opposite role in this process.
The question that remains is how TM3/STM3 can function antagonistically to FUL2/MBP20, although these four TFs seem to be spatiotemporally similarly expressed and can regulate target genes together. One possible explanation is that the resolution of our experiments was insufficient to capture spatial differences between TM3/STM3 and FUL2/MBP20. We successfully separated FM and IM tissue and TM3/STM3 and FUL2/MBP20 were similarly expressed in either meristem type. Still, there can be differences in expression in cell layers of these meristems, as is also the case for other MADS-domain proteins that give rise to the different floral organs by being active in specific cell layers [63]. So far, the transcriptomics of tomato meristems has not exceeded a single-meristem resolution [38]. A more detailed expression profile, either by transcriptional reporters or the novel stereo-seq [64], can answer whether TM3/STM3 and FUL2/MBP20 are truly co-expressed.
Another explanation why TM3/STM3 and FUL2/MBP20 have opposite effects on the developing inflorescence is that they compete in regulating target genes. FUL2/MBP20 and J2/EJ2 both promote FM maturation and can interact [8,10], and are thus likely to regulate targets together as a tetrameric complex. Alternatively, TM3/STM3 forms tetrameric complexes with either FUL2/MBP20 or J2/EJ2, thereby competing with the FM-promoting complex FUL2/MBP20/J2/EJ2. TM3/STM3, which can also bind to the DNA as homodimer [8], and therefore regulate targets independently. If TM3/STM3 interferes with the regulation of FUL2/MBP20/J2/EJ2 targets, this explains why TM3/STM3 and FUL2/MBP20 function antagonistically in inflorescence development. This hypothesis is supported by the observation that STM3 and J2 compete for binding at target genes [28]. However, overlapping SlFUL targets in the sFM/sIM [8] with J2/EJ2 targets in the FM [10] did not provide promising candidates (e.g., B- and C-class flowering genes), possibly because slightly different tissues of different cultivars were compared.

3.4. The Role of SlSOC1 and SlFUL Genes in the Tomato Flowering Network

In this study, we investigated the function of SlSOC1 genes in the floral transition and inflorescence development. We demonstrate that the tomato SOC1 homologs TM3 and STM3 interact with the FUL homologs FUL2 and MBP20 to promote the floral transition, while TM3/STM3 and FUL2/MBP20 probably have opposite roles in subsequent inflorescence development (Figure 5). Moreover, TM3/STM3 regulates target genes in either process independently of the SlFULs, thereby adding an additional layer of floral activation to the TM, and allowing this complex to act antagonistically of the SlFULs in FM/IM development. While several open questions remain that will need to be addressed in other studies, this work resolves a piece of the puzzle of reproductive meristem development in tomato.

4. Materials and Methods

4.1. Plant Materials and Growing Conditions

Solanum lycopersicum L. cultivar Moneyberg was used as the WT. Plants were grown on rockwool blocks under a 16 h light/8 h dark cycle. Seedlings were grown in a growth chamber at 21 °C and watered with 1 g/L hyponex solution. After 5–8 weeks, the plants were transferred to a greenhouse, watered by fertigation and grown under natural light supplemented with sodium lights. The mutants ful2 mbp20 and q-ful were obtained from Jiang et al. [8], and tm3 stm3 ful2 mbp20 was obtained by crossing tm3 stm3 mbp18 with ful2 mbp20 and selecting F2 homozygous tm3 stm3 ful2 mbp20 mutants.

4.2. Phylogenetic Analysis of SOC1 Homologs

For phylogenetic analysis, 69 amino acid sequences from SOC1-like proteins were retrieved from the Sol Genomics Network [65] and the National Center for Biotechnology Information [66]. All amino acid sequences are listed in Supplementary Table S2. Sequences were analyzed in MEGA X 10.1.7 [67] and aligned with the MUSCLE algorithm, using default settings [68]. Evolutionary history was inferred using the maximum likelihood method and JTT matrix-based model [69]. The bootstrap consensus tree [70] inferred from 5000 replicates was taken to represent the evolutionary history of the analyzed taxa. Branches were collapsed if they corresponded to partitions in less than 50% of bootstrap replicates. Initial trees for the heuristic search were obtained automatically by applying Neighbour-Join and BioNJ algorithms to a matrix of pairwise distances, which were estimated using a JTT model. The phylogenetic tree giving the superior log likelihood value was selected. All positions with less than 95% site coverage were eliminated, i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option).

4.3. Meristem Imaging

Meristem images were taken with a stereomicroscope (Stemi 508, Zeiss, Oberkochen, Germany) coupled to a camera (Axiocam 105 color, Zeiss, Oberkochen, Germany). Prior to imaging, the meristems were exposed by removing older leaves using forceps.

4.4. Meristem Transcriptome Profiling by RT-qPCR and RNA-Seq

For gene expression analysis, RNA was isolated from the VM, TM and FM/IM (primary reproductive meristems) or separate sFM and sIM (reproductive meristem of first sympodial unit). Meristems were hand-dissected under a stereomicroscope, directly frozen in liquid nitrogen, and RNA was extracted using the Arcturus PicoPure RNA Extraction kit (ThermoFisher Scientific, Waltham, MA, USA). Primary reproductive meristems were sampled in triplicate and sFM and sIM in quadruplicate, meaning that three and four independent batches of WT and mutant plants were grown, respectively. For RNA extraction, 11 to 52 meristems of the same stage were pooled per biological replicate, yielding 0.27 to 1.43 µg RNA. The VM was sampled just before the visible doming of the meristem. The TM was harvested when the meristem had domed, but had not bifurcated yet. The first formed FM and IM of the inflorescence were sampled before the visible formation of flower organs, either together for primary FM/IM samples, or separately for sFM and sIM samples.
For qPCR analysis, DNA was removed from the samples using the TURBO DNA-free Kit (ThermoFisher Scientific, MA, USA). cDNA was synthesized using the iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). Real-time qPCR was performed with iQ SYBR Green Supermix (BioRad, CA, USA) in a CFX Connect Real-Time machine (BioRad, CA, USA). CAC was used as reference gene. All used primers are listed in Supplementary Table S3.
For the RNA-seq, library preparation and sequencing were performed at BGI Genomics. RNA quality was validated using the Agilent 2100 Bioanalyzer and transcripts were sequenced using 2 × 150 bp paired-end DNB Sequencing. Adapter sequences, contamination and low-quality reads were removed from the raw data. Filtered reads were mapped to the tomato SL4.0 genome using HISAT2 [71] with default parameters. Counts per gene were generated using StringTie in expression-estimation mode [72] (Supplementary Table S4). DEGs were calculated using DESeq2 [73], while only considering genes with at least 10 read counts (Supplementary Table S1). A hypergeometric test was used to calculate the significance of the overlapping DEGs between datasets.
The raw data from the two RNA-Seq experiments are publicly available in the Sequence Read Archive (SRA, ncbi.nlm.nih.gov/sra) with the accession number PRJNA980935.

4.5. Yeast Two-Hybrid (Y2H)

To study protein–protein interactions, a Y2H screening using the GAL4 system was performed according to De Folter and Immink [74]. The full-length coding sequences were amplified from IM cDNA and cloned into pDEST32 and pDEST22 vectors, respectively, for bait and prey proteins. The expression vectors were transformed into the yeast strains PJ69-A (pDEST22) and PJ69-4α (pDEST32). Protein–protein interaction was screened on -LWH dropout medium containing 3 mM 3-amino-1,2,4-triazole (3-AT), or on -LWA dropout medium. Plates were incubated at 20 °C for five days. The screening was performed twice in both reciprocal directions (AD × BD and BD × AD). All primers used for cloning are listed in Supplementary Table S3.

4.6. CRISPR Construct Generation and Stable Tomato Transformation

CRISPR sgRNAs were designed on CRISPOR [75]. CRISPR constructs were assembled using the MoClo toolkit and Golden Gate cloning [76,77]. Each sgRNA was expressed under an Arabidopsis U6-26 promoter, and hCas9 was driven by 2 × proCaMV35S as described by Slaman et al. [78]. 35S::GFP and NOS::NPTII were used as selection markers. All primers used for cloning are listed in Supplementary Table S3.
Agrobacterium tumefaciens strain C58C1 was used for stable tomato transformations according to Van Roekel et al. [79], except that 2 mg/L zeatin was used instead of zeatin riboside, and that medium B did not contain IAA, but was supplemented with 0.05 mg/L 2,4-D. Explants were cultured in a growth chamber at 25 °C with 16 h light, at an intensity of 60 µE at plate level, and 8 h dark. Transgenic seedlings were tested for ploidy with Iribov Analytical Services B.V. Diploid plants with deleterious mutations according to PROVEAN [80] due to a frameshift leading to a premature stop codon or due to a deletion of the essential amino acids that were selected.

4.7. Phenotyping

Flowering time was phenotyped in the greenhouse in four independent screenings in spring or winter, or in a growth chamber. The primary floral transition was determined by counting the number of leaves until the primary inflorescence, the sympodial flowering time by the number of leaves in at least five SUs. The average size of the sympodial units was used to test the statistical significance. Inflorescence architecture was scored in completely developed inflorescences. Statistical significance was determined by one-way ANOVA and Tukey post-test (α = 0.05). Per genotype, two to five transgenic lines from independent transformations with independent mutations were phenotyped. Only for mbp23, one independent transgenic line was obtained, with two different homozygous alleles in the T2 generation.

4.8. Accession Numbers

AHL15, Solyc12g087950; AP2b, Solyc02g064960; AP2c, Solyc02g093150; AP2-like ERF, Solyc06g068570; B3 domain TF, Solyc01g108940; B3 domain TF (2), Solyc01g108930; CKX4, Solyc04g080820; CKX5, Solyc04g016430; CKX6, Solyc12g008900; CKX8, Solyc10g017990; CO-interacting protein 2a, Solyc06g072040; EJ2 (MADS1), Solyc03g114840; ERF1a, Solyc04g014530; ERF2, Solyc11g011740; FUL1, Solyc06g069430; FUL2, Solyc03g114830; J, Solyc11g010570; J2 (MBP21), Solyc12g038510; MADS-RIN, Solyc05g012020; MBP10, Solyc02g065730; MBP12, Solyc12g088090; MBP13, Solyc08g080100; MBP14, Solyc12g056460; MBP18 (FYFL), Solyc03g006830; MBP19, Solyc06g035570; MBP20, Solyc02g089210; MBP22, Solyc11g005120; MBP23, Solyc10g01640/30; MBP24, Solyc01g105800; MBP9, Solyc04g076680; MC, Solyc05g056620; STM3, Solyc01g092950; TAG1, Solyc02g071730; TM3, Solyc01g160330/Solyc01g093965; TM5, Solyc05g015750; WRKY28, Solyc12g011200; WRKY44, Solyc10g084380; Zinc finger TF, Solyc06g065440; Zinc finger TF 34, Solyc04g064770.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/plants12152754/s1. Figure S1: Sequence characteristics of SOC1-like genes and proteins; Figure S2: Y2H screening showing interactions of SOC1-like proteins with several MADS-domain TFs; Figure S3: Mutations of SlSOC1 genes generated by CRISPR/Cas9 mutagenesis; Figure S4: Quantification of the primary transition to flowering in slsoc1 mutants under greenhouse conditions; Figure S5: Scaled expression of 288 marker genes for VM, TM and FM stages identified by Meir et al. [38] in WT and tm3 stm3 RNA-Seq samples; Figure S6: Stereomicroscope images of reproductive meristem development in WT, tm3 stm3 and ful2 mbp20; Figure S7: Expression of several genes of interest in VM, TM and FM/IM of tm3 stm3 and q-ful; Figure S8: Quantification of inflorescence traits in several slsoc1 and ful2 mbp20 mutants; Figure S9: Expression of J during reproductive meristem development; Table S1: Overview of DEGs of RNA-Seq data; Table S2: Protein sequences used to build the phylogenetic tree in Figure 1a; Table S3: List of primers; Table S4: Overview of read counts of RNA-Seq data.

Author Contributions

Conceptualization, I.E.Z., G.C.A. and M.B.; formal analysis; I.E.Z. and M.B.; investigation, I.E.Z. and C.R.; supervision, G.C.A. and M.B.; writing—original draft, I.E.Z.; writing—review and editing, G.C.A. and M.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by a graduate-program grant from the Experimental Plant Sciences (EPS) graduate school. C.R. was funded by a grant from NWO-TTW (#17873).

Data Availability Statement

The data supporting the results presented in this article can be found in the main figures and Supplementary tables and figures. The raw data from the two RNA-Seq experiments are publicly available in the Sequence Read Archive (SRA, ncbi.nlm.nih.gov/sra) with the accession number PRJNA980935.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Fornara, F.; de Montaigu, A.; Coupland, G. SnapShot: Control of flowering in Arabidopsis. Cell 2010, 141, 550.e1–550.e2. [Google Scholar] [CrossRef] [Green Version]
  2. Park, S.J.; Jiang, K.; Schatz, M.C.; Lippman, Z.B. Rate of meristem maturation determines inflorescence architecture in tomato. Proc. Natl. Acad. Sci. USA 2012, 109, 639–644. [Google Scholar] [CrossRef] [PubMed]
  3. Lippman, Z.B.; Cohen, O.; Alvarez, J.P.; Abu-Abied, M.; Pekker, I.; Paran, I.; Eshed, Y.; Zamir, D. The making of a compound inflorescence in tomato and related nightshades. PLoS Biol. 2008, 6, e288. [Google Scholar] [CrossRef] [PubMed]
  4. Molinero-Rosales, N.; Latorre, A.; Jamilena, M.; Lozano, R. SINGLE FLOWER TRUSS regulates the transition and maintenance of flowering in tomato. Planta 2004, 218, 427–434. [Google Scholar] [CrossRef] [PubMed]
  5. Lifschitz, E.; Eviatar, T.; Rozman, A.; Shalit, A.; Goldshmidt, A.; Amsellem, Z.; Alvarez, J.P.; Eshed, Y. The tomato FT ortholog triggers systemic signals that regulate growth and flowering and substitute for diverse environmental stimuli. Proc. Natl. Acad. Sci. USA 2006, 103, 6398–6403. [Google Scholar] [CrossRef]
  6. Molinero-Rosales, N.; Jamilena, M.; Sergio, Z.; Gómez, P.; Capel, J.; Lozano, R. FALSIFLORA, the tomato orthologue of FLORICAULA and LEAFY, controls flowering time and floral meristem identity. Plant J. 1999, 20, 685–693. [Google Scholar] [CrossRef]
  7. Alonge, M.; Wang, X.; Benoit, M.; Soyk, S.; Pereira, L.; Zhang, L.; Suresh, H.; Ramakrishnan, S.; Maumus, F.; Ciren, D.; et al. Major impacts of widespread structural variation on gene expression and crop improvement in tomato. Cell 2020, 182, 145–161. [Google Scholar] [CrossRef]
  8. Jiang, X.; Lubini, G.; Hernandes-Lopes, J.; Rijnsburger, K.; Veltkamp, V.; de Maagd, R.A.; Angenent, G.C.; Bemer, M. FRUITFULL-like genes regulate flowering time and inflorescence architecture in tomato. Plant Cell 2022, 34, 1002–1019. [Google Scholar] [CrossRef]
  9. Yuste-Lisbona, F.J.; Quinet, M.; Fernandez-Lozano, A.; Pineda, B.; Moreno, V.; Angosto, T.; Lozano, R. Characterization of vegetative inflorescence (mc-vin) mutant provides new insight into the role of MACROCALYX in regulating inflorescence development of tomato. Sci. Rep. 2016, 6, 18796. [Google Scholar] [CrossRef] [Green Version]
  10. Soyk, S.; Lemmon, Z.H.; Sedlazeck, F.J.; Jimenez-Gomez, J.M.; Alonge, M.; Hutton, S.F.; Van Eck, J.; Schatz, M.C.; Lippman, Z.B. Duplication of a domestication locus neutralized a cryptic variant that caused a breeding barrier in tomato. Nat. Plants 2019, 5, 471–479. [Google Scholar] [CrossRef]
  11. Soyk, S.; Lemmon, Z.H.; Oved, M.; Fisher, J.; Liberatore, K.L.; Park, S.J.; Goren, A.; Jiang, K.; Ramos, A.; van der Knaap, E.; et al. Bypassing negative epistasis on yield in tomato imposed by a domestication gene. Cell 2017, 169, 1142–1155.e12. [Google Scholar] [CrossRef]
  12. Lee, J.; Lee, I. Regulation and function of SOC1, a flowering pathway integrator. J. Exp. Bot. 2010, 61, 2247–2254. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Kou, K.; Yang, H.; Li, H.; Fang, C.; Chen, L.; Yue, L.; Nan, H.; Kong, L.; Li, X.; Wang, F.; et al. A functionally divergent SOC1 homolog improves soybean yield and latitudinal adaptation. Curr. Biol. 2022, 32, 1728–1742.e6. [Google Scholar] [CrossRef] [PubMed]
  14. Liu, Z.; Wu, X.; Cheng, M.; Xie, Z.; Xiong, C.; Zhang, S.; Wu, J.; Wang, P. Identification and functional characterization of SOC1-like genes in Pyrus bretschneideri. Genomics 2020, 112, 1622–1632. [Google Scholar] [CrossRef] [PubMed]
  15. Tyagi, S.; Sri, T.; Singh, A.; Mayee, P.; Shivaraj, S.M.; Sharma, P.; Singh, A. SUPPRESSOR of OVEREXPRESSION of CONSTANS1 influences flowering time, lateral branching, oil quality, and seed yield in Brassica juncea cv. Varuna. Funct. Integr. Genom. 2019, 19, 43–60. [Google Scholar] [CrossRef]
  16. Lei, H.J.; Yuan, H.Z.; Liu, Y.; Guo, X.W.; Liao, X.; Liu, L.L.; Wang, Q.; Li, T.H. Identification and characterization of FaSOC1, a homolog of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 from strawberry. Gene 2013, 531, 158–167. [Google Scholar] [CrossRef]
  17. Hou, D.; Li, L.; Ma, T.; Pei, J.; Zhao, Z.; Lu, M.; Wu, A.; Lin, X. The SOC1-like gene BoMADS50 is associated with the flowering of Bambusa oldhamii. Hortic. Res. 2021, 8, 133. [Google Scholar] [CrossRef]
  18. Wang, Y.; Huang, X.; Huang, X.; Su, W.; Hao, Y.; Liu, H.; Chen, R.; Song, S. BcSOC1 promotes bolting and stem elongation in flowering Chinese cabbage. Int. J. Mol. Sci. 2022, 23, 3459. [Google Scholar] [CrossRef]
  19. Jaudal, M.; Zhang, L.; Che, C.; Li, G.; Tang, Y.; Wen, J.; Mysore, K.S.; Putterill, J. A SOC1-like gene MtSOC1a promotes flowering and primary stem elongation in Medicago. J. Exp. Bot. 2018, 69, 4867–4880. [Google Scholar] [CrossRef] [Green Version]
  20. De Folter, S.; Immink, R.G.H.; Kieffer, M.; Parenicova, L.; Henz, S.R.; Weigel, D.; Busscher, M.; Kooiker, M.; Colombo, L.; Kater, M.M.; et al. Comprehensive interaction map of the Arabidopsis MADS Box transcription factors. Plant Cell 2005, 17, 1424–1433. [Google Scholar] [CrossRef] [Green Version]
  21. Immink, R.G.H.; Tonaco, I.A.; de Folter, S.; Shchennikova, A.; van Dijk, A.D.; Busscher-Lange, J.; Borst, J.W.; Angenent, G.C. SEPALLATA3: The ‘glue’ for MADS box transcription factor complex formation. Genome Biol. 2009, 10, R24. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Wang, X.; Liu, Z.; Sun, S.; Wu, J.; Li, R.; Wang, H.; Cui, X. SISTER OF TM3 activates FRUITFULL1 to regulate inflorescence branching in tomato. Hortic. Res. 2021, 8, 251. [Google Scholar] [CrossRef]
  23. Liu, D.; Wang, D.; Qin, Z.; Zhang, D.; Yin, L.; Wu, L.; Colasanti, J.; Li, A.; Mao, L. The SEPALLATA MADS-box protein SlMBP21 forms protein complexes with JOINTLESS and MACROCALYX as a transcription activator for development of the tomato flower abscission zone. Plant J. 2014, 77, 284–296. [Google Scholar] [CrossRef]
  24. Immink, R.G.H.; Pose, D.; Ferrario, S.; Ott, F.; Kaufmann, K.; Valentim, F.L.; de Folter, S.; van der Wal, F.; van Dijk, A.D.J.; Schmid, M.; et al. Characterization of SOC1’s central role in flowering by the identification of its upstream and downstream regulators. Plant Physiol. 2012, 160, 433–449. [Google Scholar] [CrossRef] [Green Version]
  25. Balanza, V.; Martinez-Fernandez, I.; Ferrandiz, C. Sequential action of FRUITFULL as a modulator of the activity of the floral regulators SVP and SOC1. J. Exp. Bot. 2014, 65, 1193–1203. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Karami, O.; Rahimi, A.; Khan, M.; Bemer, M.; Hazarika, R.R.; Mak, P.; Compier, M.; van Noort, V.; Offringa, R. A suppressor of axillary meristem maturation promotes longevity in flowering plants. Nat. Plants 2020, 6, 368–376. [Google Scholar] [CrossRef] [PubMed]
  27. Melzer, S.; Lens, F.; Gennen, J.; Vanneste, S.; Rohde, A.; Beeckman, T. Flowering-time genes modulate meristem determinacy and growth form in Arabidopsis thaliana. Nat. Genet. 2008, 40, 1489–1492. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Wang, X.; Liu, Z.; Bai, J.; Sun, S.; Song, J.; Li, R.; Cui, X. Antagonistic regulation of target genes by the SISTER of TM3–JOINTLESS2 complex in tomato inflorescence branching. Plant Cell 2023, 35, 2062–2078. [Google Scholar] [CrossRef]
  29. Wang, Y.; Zhang, J.; Hu, Z.; Guo, X.; Tian, S.; Chen, G. Genome-wide analysis of the MADS-box transcription factor family in Solanum lycopersicum. Int. J. Mol. Sci. 2019, 20, 2961. [Google Scholar] [CrossRef] [Green Version]
  30. Dorca-Fornell, C.; Gregis, V.; Grandi, V.; Coupland, G.; Colombo, L.; Kater, M.M. The Arabidopsis SOC1-like genes AGL42, AGL71 and AGL72 promote flowering in the shoot apical and axillary meristems. Plant J. 2011, 67, 1006–1017. [Google Scholar] [CrossRef]
  31. Li, N.; He, Q.; Wang, J.; Wang, B.; Zhao, J.; Huang, S.; Yang, T.; Tang, Y.; Yang, S.; Aisimutuola, P.; et al. Super-pangenome analyses highlight genomic diversity and structural variation across wild and cultivated tomato species. Nat. Genet. 2023, 55, 852–860. [Google Scholar] [CrossRef]
  32. Qin, P.; Lu, H.; Du, H.; Wang, H.; Chen, W.; Chen, Z.; He, Q.; Ou, S.; Zhang, H.; Li, X.; et al. Pan-genome analysis of 33 genetically diverse rice accessions reveals hidden genomic variations. Cell 2021, 184, 3542–3558.e16. [Google Scholar] [CrossRef] [PubMed]
  33. Tang, D.; Jia, Y.; Zhang, J.; Li, H.; Cheng, L.; Wang, P.; Bao, Z.; Liu, Z.; Feng, S.; Zhu, X.; et al. Genome evolution and diversity of wild and cultivated potatoes. Nature 2022, 606, 535–541. [Google Scholar] [CrossRef] [PubMed]
  34. Van Rengs, W.M.J.; Schmidt, M.H.W.; Effgen, S.; Le, D.B.; Wang, Y.; Zaidan, M.W.A.M.; Huettel, B.; Schouten, H.J.; Usadel, B.; Underwood, C.J. A chromosome scale tomato genome built from complementary PacBio and Nanopore sequences alone reveals extensive linkage drag during breeding. Plant J. 2022, 110, 572–588. [Google Scholar] [CrossRef] [PubMed]
  35. Zhou, Y.; Zhang, Z.; Bao, Z.; Li, H.; Lyu, Y.; Zan, Y.; Wu, Y.; Cheng, L.; Fang, Y.; Wu, K.; et al. Graph pangenome captures missing heritability and empowers tomato breeding. Nature 2022, 606, 527–534. [Google Scholar] [CrossRef]
  36. Pnueli, L.; Hareven, A.D.; Rounsley, S.D.; Yanofsky, M.F.; Lifschitz, E. Isolation of the tomato AGAMOUS gene TAG1 and analysis of its homeotic role in transgenic plants. Plant Cell 1994, 6, 163–173. [Google Scholar] [CrossRef] [Green Version]
  37. Slugina, M.A.; Dyachenko, E.A.; Kochieva, E.Z.; Shchennikova, A.V. Structural and functional diversification of SEPALLATA genes TM5 and RIN in tomato species (Section Lycopersicon). Dokl. Biochem. Biophys. 2020, 492, 152–158. [Google Scholar] [CrossRef]
  38. Meir, Z.; Aviezer, I.; Chongloi, G.L.; Ben-Kiki, O.; Bronstein, R.; Mukamel, Z.; Keren-Shaul, H.; Jaitin, D.; Tal, L.; Shalev-Schlosser, G.; et al. Dissection of floral transition by single-meristem transcriptomes at high temporal resolution. Nat. Plants 2021, 7, 800–813. [Google Scholar] [CrossRef]
  39. Van Bel, M.; Silvestri, F.; Weitz, E.M.; Kreft, L.; Botzki, A.; Coppens, F.; Vandepoele, K. PLAZA 5.0: Extending the scope and power of comparative and functional genomics in plants. Nucleic Acids Res. 2022, 50, D1468–D1474. [Google Scholar] [CrossRef] [PubMed]
  40. Yu, Y.C.; Qiao, L.F.; Chen, J.C.; Rong, Y.H.; Zhao, Y.H.; Cui, X.K.; Xu, J.P.; Hou, X.M.; Dong, C.H. Arabidopsis REM16 acts as a B3 domain transcription factor to promote flowering time via directly binding to the promoters of SOC1 and FT. Plant J. 2020, 103, 1386–1398. [Google Scholar] [CrossRef]
  41. Jing, Y.J.; Guo, Q.; Lin, R.C. The B3-Domain transcription factor VAL1 regulates the floral transition by repressing FLOWERING LOCUS T. Plant Physiol. 2019, 181, 236–248. [Google Scholar] [CrossRef] [Green Version]
  42. Yang, C.; Liu, C.; Li, S.; Zhang, Y.; Zhang, Y.; Wang, X.; Xiang, W. The transcription factors WRKY41 and WRKY53 mediate early flowering induced by the novel plant growth regulator guvermectin in Arabidopsis thaliana. Int. J. Mol. Sci. 2023, 24, 8424. [Google Scholar] [CrossRef] [PubMed]
  43. Bartrina, I.; Otto, E.; Strnad, M.; Werner, T.; Schmulling, T. Cytokinin regulates the activity of reproductive meristems, flower organ size, ovule formation, and thus seed yield in Arabidopsis thaliana. Plant Cell 2011, 23, 69–80. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Merelo, P.; Gonzalez-Cuadra, I.; Ferrandiz, C. A cellular analysis of meristem activity at the end of flowering points to cytokinin as a major regulator of proliferative arrest in Arabidopsis. Curr. Biol. 2022, 32, 749–762.e3. [Google Scholar] [CrossRef]
  45. Zhang, B.L.; Wang, L.; Zeng, L.P.; Zhang, C.; Ma, H. Arabidopsis TOE proteins convey a photoperiodic signal to antagonize CONSTANS and regulate flowering time. Genes Dev. 2015, 29, 975–987. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Aukerman, M.J.; Sakai, H. Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes. Plant Cell 2003, 15, 2730–2741. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Yant, L.; Mathieu, J.; Dinh, T.T.; Ott, F.; Lanz, C.; Wollmann, H.; Chen, X.; Schmid, M. Orchestration of the floral transition and floral development in Arabidopsis by the bifunctional transcription factor APETALA2. Plant Cell 2010, 22, 2156–2170. [Google Scholar] [CrossRef] [Green Version]
  48. Karlova, R.; Rosin, F.M.; Busscher-Lange, J.; Parapunova, V.; Do, P.T.; Fernie, A.R.; Fraser, P.D.; Baxter, C.; Angenent, G.C.; de Maagd, R.A. Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening. Plant Cell 2011, 23, 923–941. [Google Scholar] [CrossRef] [Green Version]
  49. Gong, P.; Zhang, J.; Li, H.; Yang, C.; Zhang, C.; Zhang, X.; Khurram, Z.; Zhang, Y.; Wang, T.; Fei, Z.; et al. Transcriptional profiles of drought-responsive genes in modulating transcription signal transduction, and biochemical pathways in tomato. J. Exp. Bot. 2010, 61, 3563–3575. [Google Scholar] [CrossRef]
  50. Yang, T.; He, Y.; Niu, S.; Yan, S.; Zhang, Y. Identification and characterization of the CONSTANS (CO)/CONSTANS-like (COL) genes related to photoperiodic signaling and flowering in tomato. Plant Sci. 2020, 301, 110653. [Google Scholar] [CrossRef]
  51. Wang, Q.; Song, S.F.; Lu, X.T.; Wang, Y.Q.; Chen, Y.; Wu, X.W.; Tan, L.; Chai, G.H. Hormone regulation of CCCH zinc finger proteins in plants. Int. J. Mol. Sci. 2022, 23, 14288. [Google Scholar] [CrossRef]
  52. Liu, Y.H.; Khan, A.R.; Azhar, W.; Wong, C.E.; Li, Y.L.; Huang, Y.; Cao, X.; Liu, Z.N.; Gan, Y.B. Cys2/His2-type zinc finger proteins regulate plant growth and development. CRC Crit. Rev. Plant Sci. 2022, 41, 351–363. [Google Scholar] [CrossRef]
  53. Dielen, V.; Quinet, M.; Chao, J.; Batoko, H.; Havelange, A.; Kinet, J.M. UNIFLORA, a pivotal gene that regulates floral transition and meristem identity in tomato (Lycopersicon esculentum). New Phytol. 2004, 161, 393–400. [Google Scholar] [CrossRef]
  54. Leijten, W.; Koes, R.; Roobeek, I.; Frugis, G. Translating Flowering Time from Arabidopsis thaliana to Brassicaceae and Asteraceae Crop Species. Plants 2018, 7, 111. [Google Scholar] [CrossRef] [Green Version]
  55. Perez-Ruiz, R.V.; Garcia-Ponce, B.; Marsch-Martinez, N.; Ugartechea-Chirino, Y.; Villajuana-Bonequi, M.; de Folter, S.; Azpeitia, E.; Davila-Velderrain, J.; Cruz-Sanchez, D.; Garay-Arroyo, A.; et al. XAANTAL2 (AGL14) is an important component of the complex gene regulatory network that underlies Arabidopsis shoot apical meristem transitions. Mol. Plant 2015, 8, 796–813. [Google Scholar] [CrossRef] [Green Version]
  56. Kang, M.J.; Jin, H.S.; Noh, Y.S.; Noh, B. Repression of flowering under a non-inductive photoperiod by the HDA9-AGL19-FT module in Arabidopsis. New Phytol. 2015, 206, 281–294. [Google Scholar] [CrossRef]
  57. Waese, J.; Fan, J.; Pasha, A.; Yu, H.; Fucile, G.; Shi, R.; Cumming, M.; Kelley, L.A.; Sternberg, M.J.; Krishnakumar, V.; et al. ePlant: Visualizing and exploring multiple levels of data for hypothesis generation in plant biology. Plant Cell 2017, 29, 1806–1821. [Google Scholar] [CrossRef] [Green Version]
  58. Alvarez-Buylla, E.R.; Garcia-Ponce, B.; Sanchez, M.P.; Espinosa-Soto, C.; Garcia-Gomez, M.L.; Pineyro-Nelson, A.; Garay-Arroyo, A. MADS-box genes underground becoming mainstream: Plant root developmental mechanisms. New Phytol. 2019, 223, 1143–1158. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  59. Zhou, B.; Luo, Q.; Shen, Y.; Wei, L.; Song, X.; Liao, H.; Ni, L.; Shen, T.; Du, X.; Han, J.; et al. Coordinated regulation of vegetative phase change by brassinosteroids and the age pathway in Arabidopsis. Nat. Commun. 2023, 14, 2608. [Google Scholar] [CrossRef] [PubMed]
  60. Van Mourik, H.; Chen, P.; Smaczniak, C.; Boeren, S.; Kaufmann, K.; Bemer, M.; Angenent, G.C.; Muino, J.M. Dual specificity and target gene selection by the MADS-domain protein FRUITFULL. Nat. Plants 2023, 9, 473–485. [Google Scholar] [CrossRef]
  61. Smaczniak, C.; Muino, J.M.; Chen, D.; Angenent, G.C.; Kaufmann, K. Differences in DNA binding specificity of floral homeotic protein complexes predict organ-specific target genes. Plant Cell 2017, 29, 1822–1835. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  62. Rieu, P.; Turchi, L.; Thevenon, E.; Zarkadas, E.; Nanao, M.; Chahtane, H.; Tichtinsky, G.; Lucas, J.; Blanc-Mathieu, R.; Zubieta, C.; et al. The F-box protein UFO controls flower development by redirecting the master transcription factor LEAFY to new cis-elements. Nat. Plants 2023, 9, 315–329. [Google Scholar] [CrossRef]
  63. Theissen, G. Development of floral organ identity: Stories from the MADS house. Curr. Opin. Plant Biol. 2001, 4, 75–85. [Google Scholar] [CrossRef]
  64. Xia, K.K.; Sun, H.X.; Li, J.; Li, J.M.; Zhao, Y.; Chen, L.C.; Qin, C.; Chen, R.Y.; Chen, Z.Y.; Liu, G.Y.; et al. The single-cell stereo-seq reveals region-specific cell subtypes and transcriptome profiling in Arabidopsis leaves. Dev. Cell 2022, 57, 1299–1310.e4. [Google Scholar] [CrossRef] [PubMed]
  65. Fernandez-Pozo, N.; Menda, N.; Edwards, J.D.; Saha, S.; Tecle, I.Y.; Strickler, S.R.; Bombarely, A.; Fisher-York, T.; Pujar, A.; Foerster, H.; et al. The Sol Genomics Network (SGN)-from genotype to phenotype to breeding. Nucleic Acids Res. 2015, 43, D1036–D1041. [Google Scholar] [CrossRef] [PubMed]
  66. Sayers, E.W.; Bolton, E.E.; Brister, J.R.; Canese, K.; Chan, J.; Comeau, D.C.; Connor, R.; Funk, K.; Kelly, C.; Kim, S.; et al. Database resources of the national center for biotechnology information. Nucleic Acids Res. 2022, 50, D20–D26. [Google Scholar] [CrossRef]
  67. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef]
  68. Edgar, R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32, 1792–1797. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  69. Jones, D.T.; Taylor, W.R.; Thornton, J.M. The rapid generation of mutation data matrices from protein sequences. Bioinformatics 1992, 8, 275–282. [Google Scholar] [CrossRef] [PubMed]
  70. Felsenstein, J. Confidence-limits on phylogenies—An approach using the bootstrap. Evolution 1985, 39, 783–791. [Google Scholar] [CrossRef]
  71. Kim, D.; Paggi, J.M.; Park, C.; Bennett, C.; Salzberg, S.L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 2019, 37, 907–915. [Google Scholar] [CrossRef] [PubMed]
  72. Pertea, M.; Pertea, G.M.; Antonescu, C.M.; Chang, T.C.; Mendell, J.T.; Salzberg, S.L. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 2015, 33, 290–295. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  73. Love, M.I.; Huber, W.; Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014, 15, 550. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  74. De Folter, S.; Immink, R.G.H. Yeast protein-protein interaction assays and screens. Methods Mol. Biol. 2011, 754, 145–165. [Google Scholar] [CrossRef]
  75. Concordet, J.-P.; Haeussler, M. CRISPOR: Intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 2018, 46, W242–W245. [Google Scholar] [CrossRef] [Green Version]
  76. Engler, C.; Youles, M.; Gruetzner, R.; Ehnert, T.M.; Werner, S.; Jones, J.D.; Patron, N.J.; Marillonnet, S. A golden gate modular cloning toolbox for plants. ACS Synth. Biol. 2014, 3, 839–843. [Google Scholar] [CrossRef]
  77. Weber, E.; Gruetzner, R.; Werner, S.; Engler, C.; Marillonnet, S. Assembly of designer TAL effectors by golden gate cloning. PLoS ONE 2011, 6, e19722. [Google Scholar] [CrossRef] [Green Version]
  78. Slaman, E.; Lammers, M.; Angenent, G.C.; de Maagd, R.A. High-throughput sgRNA testing reveals rules for Cas9 specificity and DNA repair in tomato cells. Front. Genome Ed. 2023, 5, 1196763. [Google Scholar] [CrossRef]
  79. Van Roekel, J.S.C.; Damm, B.; Melchers, L.S.; Hoekema, A. Factors influencing transformation frequency of tomato (Lycopersicon esculentum). Plant Cell Rep. 1993, 12, 644–647. [Google Scholar] [CrossRef]
  80. Choi, Y.; Chan, A.P. PROVEAN web server: A tool to predict the functional effect of amino acid substitutions and indels. Bioinformatics 2015, 31, 2745–2747. [Google Scholar] [CrossRef] [Green Version]
Plants 12 02754 g001 550
Figure 1. Characterization of tomato SOC1-like genes during the floral transition. (a) Phylogenetic tree of SOC1-like proteins from several Solanaceae species and Arabidopsis thaliana. Numbers at the branches indicate bootstrap values inferred from 5000 replicates. (b) Stereomicroscope images of reproductive meristem development. Dashed lines indicate hand-dissected tissue for transcriptome analysis. The scale bar is 200 µm. (c) Expression of the SOC1-like genes MBP23, TM3, STM3, MBP14, MBP13, MBP19 and MBP18 relative to CAC in WT VM, TM and FM/IM tissue (dCT values are shown per gene relative to the reference). Shown are the mean values ± SE of three biological replicates. L, leaf; VM, vegetative meristem; TM, transition meristem; FM/IM, floral and inflorescence meristem; SYM, sympodial shoot meristem.
Figure 1. Characterization of tomato SOC1-like genes during the floral transition. (a) Phylogenetic tree of SOC1-like proteins from several Solanaceae species and Arabidopsis thaliana. Numbers at the branches indicate bootstrap values inferred from 5000 replicates. (b) Stereomicroscope images of reproductive meristem development. Dashed lines indicate hand-dissected tissue for transcriptome analysis. The scale bar is 200 µm. (c) Expression of the SOC1-like genes MBP23, TM3, STM3, MBP14, MBP13, MBP19 and MBP18 relative to CAC in WT VM, TM and FM/IM tissue (dCT values are shown per gene relative to the reference). Shown are the mean values ± SE of three biological replicates. L, leaf; VM, vegetative meristem; TM, transition meristem; FM/IM, floral and inflorescence meristem; SYM, sympodial shoot meristem.
Plants 12 02754 g001
Plants 12 02754 g002 550
Figure 2. Functional characterization of SOC1 homologs in flowering control. (a) Y2H screening showing interactions of SOC1-like proteins with several MADS-domain TFs. Reciprocal interaction is shown by dark grey shading, one-way interaction by light grey shading, and blank cells indicate a failure to interact. MBP24 was only tested as prey (AD), not as bait (BD). (b) Quantification of the number of leaves to the first inflorescence, i.e., the primary transition to flowering, in higher-order slsoc1 mutants under greenhouse conditions. (c) Quantification of the primary transition to flowering in single and double slsoc1 mutants under growth-chamber conditions. (d) Quantification of the sequential sympodial shoot floral transitions in higher-order slsoc1 mutants under greenhouse conditions. Shown are mean values of the cumulative number of leaves per sympodial unit (SU) ± SE. The average size of the first five SUs per plant was used to calculate significance. (e) Representative sympodial shoots were from WT and tm3 stm3 plants. Leaves were bent to the right for visualization. L, leaf number. In (b–d), letters indicate statistical significance.
Figure 2. Functional characterization of SOC1 homologs in flowering control. (a) Y2H screening showing interactions of SOC1-like proteins with several MADS-domain TFs. Reciprocal interaction is shown by dark grey shading, one-way interaction by light grey shading, and blank cells indicate a failure to interact. MBP24 was only tested as prey (AD), not as bait (BD). (b) Quantification of the number of leaves to the first inflorescence, i.e., the primary transition to flowering, in higher-order slsoc1 mutants under greenhouse conditions. (c) Quantification of the primary transition to flowering in single and double slsoc1 mutants under growth-chamber conditions. (d) Quantification of the sequential sympodial shoot floral transitions in higher-order slsoc1 mutants under greenhouse conditions. Shown are mean values of the cumulative number of leaves per sympodial unit (SU) ± SE. The average size of the first five SUs per plant was used to calculate significance. (e) Representative sympodial shoots were from WT and tm3 stm3 plants. Leaves were bent to the right for visualization. L, leaf number. In (b–d), letters indicate statistical significance.
Plants 12 02754 g002
Plants 12 02754 g003 550
Figure 3. TM3/STM3 share targets with the SlFULs that also independently contribute to the floral transition. (a) Venn diagram showing the overlap of DEGs of tm3 stm3 and q-ful in VM, TM and FM/IM stages. Genes with padj < 0.01 in at least one stage were selected as DEG. (b) Expression of genes downstream of tm3 stm3 and q-ful during reproductive meristem development. (c) MBP13 and MBP14 expression in VM, TM and FM/IM of tm3 stm3 and q-ful. (d) CKX4 and CKX6 expression in VM, TM and FM/IM of tm3 stm3 and q-ful. (e) Quantification of the primary transition until flowering of tm3 stm3, mbp23 tm3 stm3 mbp18, ful2 mbp20 and tm3 stm3 ful2 mbp20 under greenhouse conditions. (f) Expression of TFs downstream of tm3 stm3 but not q-ful during reproductive meristem development. In (b–d,f), bars show mean FPKM values ± SEM. Asterisks indicate statistical significance according to the DESeq2 Padj values from the mutant compared to its respective WT (* Padj < 0.05, ** Padj < 0.01, *** Padj < 0.001). B3 domain TF, Solyc01g108940; AP2-like ERF, Solyc06g068570; Zinc finger TF, Solyc06g065440. In (e), different letters indicate statistically significant differences. FPKM, fragments per kilobase million; VM, vegetative meristem; TM, transition meristem; FM/IM, floral and inflorescence meristem.
Figure 3. TM3/STM3 share targets with the SlFULs that also independently contribute to the floral transition. (a) Venn diagram showing the overlap of DEGs of tm3 stm3 and q-ful in VM, TM and FM/IM stages. Genes with padj < 0.01 in at least one stage were selected as DEG. (b) Expression of genes downstream of tm3 stm3 and q-ful during reproductive meristem development. (c) MBP13 and MBP14 expression in VM, TM and FM/IM of tm3 stm3 and q-ful. (d) CKX4 and CKX6 expression in VM, TM and FM/IM of tm3 stm3 and q-ful. (e) Quantification of the primary transition until flowering of tm3 stm3, mbp23 tm3 stm3 mbp18, ful2 mbp20 and tm3 stm3 ful2 mbp20 under greenhouse conditions. (f) Expression of TFs downstream of tm3 stm3 but not q-ful during reproductive meristem development. In (b–d,f), bars show mean FPKM values ± SEM. Asterisks indicate statistical significance according to the DESeq2 Padj values from the mutant compared to its respective WT (* Padj < 0.05, ** Padj < 0.01, *** Padj < 0.001). B3 domain TF, Solyc01g108940; AP2-like ERF, Solyc06g068570; Zinc finger TF, Solyc06g065440. In (e), different letters indicate statistically significant differences. FPKM, fragments per kilobase million; VM, vegetative meristem; TM, transition meristem; FM/IM, floral and inflorescence meristem.
Plants 12 02754 g003
Plants 12 02754 g004 550
Figure 4. Functional characterization of SlSOC1 and SlFUL genes in inflorescence development. (a) Expression of tomato SOC1, FUL and SEP homologs in the first sFM or sIM of WT plants. Shown are the average FPKM values of four biological replicates. AN and UF are shown as marker genes as a control for sampling stages. The stereomicroscope image at the bottom shows the harvested tissue. The scale bar is 200 µm. (b) Quantification of inflorescence phenotypes. A normal inflorescence was defined as unbranched, having no revertance to vegetative growth and 7 to 10 flowers. Inflorescences deviating from this were categorized and representative inflorescences are shown at the bottom with color-coded borders. The mutants were phenotyped in two independent screenings with separate WT controls, which had a consistent fraction of normal inflorescences.
Figure 4. Functional characterization of SlSOC1 and SlFUL genes in inflorescence development. (a) Expression of tomato SOC1, FUL and SEP homologs in the first sFM or sIM of WT plants. Shown are the average FPKM values of four biological replicates. AN and UF are shown as marker genes as a control for sampling stages. The stereomicroscope image at the bottom shows the harvested tissue. The scale bar is 200 µm. (b) Quantification of inflorescence phenotypes. A normal inflorescence was defined as unbranched, having no revertance to vegetative growth and 7 to 10 flowers. Inflorescences deviating from this were categorized and representative inflorescences are shown at the bottom with color-coded borders. The mutants were phenotyped in two independent screenings with separate WT controls, which had a consistent fraction of normal inflorescences.
Plants 12 02754 g004
Plants 12 02754 g005 550
Figure 5. Model showing the proposed functions of SlSOC1 and SlFUL proteins in reproductive meristem development. White TFs interacting with FUL2/MBP20 are possibly J2/EJ2.
Figure 5. Model showing the proposed functions of SlSOC1 and SlFUL proteins in reproductive meristem development. White TFs interacting with FUL2/MBP20 are possibly J2/EJ2.
Plants 12 02754 g005
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Zahn, I.E.; Roelofsen, C.; Angenent, G.C.; Bemer, M. TM3 and STM3 Promote Flowering Together with FUL2 and MBP20, but Act Antagonistically in Inflorescence Branching in Tomato. Plants 2023, 12, 2754. https://doi.org/10.3390/plants12152754

AMA Style

Zahn IE, Roelofsen C, Angenent GC, Bemer M. TM3 and STM3 Promote Flowering Together with FUL2 and MBP20, but Act Antagonistically in Inflorescence Branching in Tomato. Plants. 2023; 12(15):2754. https://doi.org/10.3390/plants12152754

Chicago/Turabian Style

Zahn, Iris E., Chris Roelofsen, Gerco C. Angenent, and Marian Bemer. 2023. "TM3 and STM3 Promote Flowering Together with FUL2 and MBP20, but Act Antagonistically in Inflorescence Branching in Tomato" Plants 12, no. 15: 2754. https://doi.org/10.3390/plants12152754

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop