Abstract
Inflorescence branch number is a yield-related trait controlled by cell fate determination in meristems. Two MADS-box transcription factors (TFs)—SISTER OF TM3 (STM3) and JOINTLESS 2 (J2)—have opposing regulatory roles in inflorescence branching. However, the mechanisms underlying their regulatory functions in inflorescence determinacy remain unclear. Here, we characterized the functions of these TFs in tomato (Solanum lycopersicum) floral meristem and inflorescence meristem (IM) through chromatin immunoprecipitation and sequencing analysis of their genome-wide occupancy. STM3 and J2 activate or repress the transcription of a set of common putative target genes, respectively, through recognition and binding to CArG box motifs. FRUITFULL1 (FUL1) is a shared putative target of STM3 and J2 and these TFs antagonistically regulate FUL1 in inflorescence branching. Moreover, STM3 physically interacts with J2 to mediate its cytosolic redistribution and restricts J2 repressor activity by reducing its binding to target genes. Conversely, J2 limits STM3 regulation of target genes by transcriptional repression of the STM3 promoter and reducing STM3-binding activity. Our study thus reveals an antagonistic regulatory relationship in which STM3 and J2 control tomato IM determinacy and branch number.
The MADS-box transcription factors SISTER OF TM3 and JOINTLESS 2 affect each other’s binding to FRUITFULL1 in an antagonistic regulatory module controlling tomato inflorescence branch number.
IN A NUTSHELL.
Background: The number of branches in the inflorescence affects yield in food crops and the aesthetics of ornamental plants. Two MADS-box transcription factors, SISTER OF TM3 (STM3) and JOINTLESS2 (J2), are tomato homologs of Arabidopsis SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 and SEPALLATA 4, respectively, and have opposing regulatory functions in controlling inflorescence branching.
Question: How do STM3 and J2 antagonistically regulate inflorescence branching in tomato?
Findings: We used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and electrophoretic mobility shift assays to identify and confirm a set of common putative target genes that are directly bound by STM3 and J2 via the CArG box motif in their promoter regions. One of these direct targets, FRUITFULL1 (FUL1), is antagonistically regulated by STM3 and J2 during inflorescence development. STM3 forms a complex with J2 and mediates its translocation from the cytosol to the nucleus, thus restricting J2 repressor activity. J2 also limits STM3 function by repressing STM3 expression and inhibits its binding activity. Our results suggest an antagonistic regulatory mode in which STM3 and J2 exert flexible control over inflorescence meristem determinacy and branch number.
Next steps: We and others aim to identify additional factors affecting tomato inflorescence development and the environmental adaptability of inflorescence branching.
Introduction
Inflorescence branch number is a yield-determining trait for food crops and an aesthetic feature of ornamental plants. These plants exhibit a variety of inflorescence architectures progressing in complexity from a solitary flower to structures that contain multiple branches due to the initiation and development of different inflorescence meristems (IMs). In monopodial species, such as Arabidopsis thaliana, the apical meristem remains indeterminate, and upon floral induction, the secondary inflorescence branches are initiated in a basipetal direction.
Tomato (Solanum lycopersicum) remains the most well-studied model of sympodial plants, which include many tree species and a variety of other perennial plants. In sympodial species, the apical meristem terminates in the floral meristem (FM), while the lateral meristem becomes an IM. This meristem then repeats the process by terminating in an FM, with its lateral meristems becoming IMs (Hake 2008; Wang et al. 2018). Thus, the continual termination and reactivation of meristems and temporal changes in the acquisition of floral fate determine the diversity of inflorescence branches and flower numbers (Lippman et al. 2008). Consistent with these differences in inflorescence development, some genes required for flowering and FM/IM determination have functionally diverged between monopodial and sympodial species (Wang et al. 2018). Therefore, the developmental patterns specific to sympodial inflorescence require flexible regulatory mechanisms to control repeated occurrence and determination of IM.
In tomato, several molecular switches have been implicated in determining inflorescence architecture. For example, mutations in CLAVATA pathway genes, such as SlCLV3, FASCIATED AND BRANCHED (FAB), and FASCIATED INFLORESCENCE (FIN), cause meristems to enlarge, leading to increased inflorescence branching (Xu et al. 2015). ANANTHA (AN, homolog of Arabidopsis UNUSUAL FLORAL ORGANS), FALSIFLORA (FA, homolog of Arabidopsis LEAFY), and COMPOUND INFLORESCENCE (S, homolog of Arabidopsis WOX9) have also been identified as key factors promoting the progression of an IM to floral specification, and mutation of these genes leads to delayed FM maturation, ultimately resulting in highly branched inflorescences (Allen and Sussex 1996; Lippman et al. 2008; Hendelman et al. 2021). By contrast, the terminating flower (tmf) mutant converts the multiflowered inflorescence into a solitary flower in tomato due to precocious activation of the AN–FA complex (MacAlister et al. 2012). The other single-flower tomato mutant, single-flower truss (sft), disrupts normal sympodial growth, reverting reproductive inflorescence development towards vegetative growth after the initiation of one or a few flowers (Lifschitz et al. 2014; Park et al. 2014). Thus, inputs from multiple internal signaling pathways are integrated to decide the switch from vegetative to IM identity and from IM to floral specification that controls tomato inflorescence branch number.
Some MADS-box transcription factors (TFs) have been described to function as key switches in meristem identity during phase transition. In particular, MACROCALYX (MC) is essential for the transition to flowering in tomato and control of FM identity, similar to its Arabidopsis homolog, APETALA1 (AP1) (Nakano et al. 2012; Liu et al. 2014; Yuste-Lisbona et al. 2016). FRUITFULL (FUL), in the AP1/FUL subfamily, performs pleiotropic roles in the regulation of flowering and inflorescence identity, together with AP1 or SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) in Arabidopsis (Balanza et al. 2018). The 3 homologs of FUL in tomato, FUL1, FUL2, and MADS-box PROTEIN 20 (MBP20), promote flowering and repress inflorescence branching by delaying FM maturation (Jiang et al. 2022), while the 2 SEPALLATA 4 (SEP4)-like genes, JOINTLESS2 (J2) and ENHANCER-OF-JOINTLESS 2 (EJ2), activate the conversion from IM to FM, the mutation of which increases inflorescence branching in tomato (Soyk et al. 2017). Moreover, the homolog of Arabidopsis SOC1, SISTER OF TM3 (STM3), is a main regulator to promote the transition to flowering and inflorescence branching redundantly with Tomato MADS-box gene 3 (TM3) in tomato (Alonge et al. 2020; Wang et al. 2021). STM3 and J2 have been shown to play opposing regulatory roles in inflorescence branching, and tomato accessions with different allelic combinations of STM3 and J2 produce inflorescences with different degrees of branching (Alonge et al. 2020). However, the regulatory mechanisms underlying STM3 and J2 activity in inflorescence development are still unknown.
Here, to better understand the functional interactions between STM3 and J2, we performed chromatin immunoprecipitation and sequencing (ChIP-seq) to identify their putative targets. The results revealed a set of common putative target genes, harboring CArG box motifs, that are bound by both of these MADS-box TFs. Further, we show that STM3 and J2 antagonistically regulate the transcription of putative inflorescence development-related genes within this subset in tissues of IM and FM. Immunofluorescent staining assays showed that STM3 mediates cytosolic redistribution of J2, while ChIP-seq and qPCR showed that J2 is a repressor of STM3 transcription. STM3 and J2 thus mutually negatively affect each other's binding activity and regulatory activity, revealing a mechanism of antagonistic co-regulation of their shared putative target genes, mediated by STM3–J2 complex formation. This work thus expands our understanding of the mechanisms controlling inflorescence branching in tomato.
Results
Genome-wide identification of STM3 and J2-binding sites
SISTER OF TM3 (STM3) and JOINTLESS 2 (J2), 2 key MADS-box TFs, have demonstrated function as antagonistic inflorescence-branching regulators (Soyk et al. 2017; Alonge et al. 2020). To explore the regulatory mechanisms by which STM3 and J2 participate in controlling tomato inflorescence branching, we performed ChIP-seq to identify the binding sites of STM3 and J2 across the tomato genome. For ChIP-seq, we used HA antibody and IM and FM tissues of the STM3OE1 (Pro35S:STM3-YFP-HA) plants in the ST147 background (Wang et al. 2021) and the ProJ2:J2-HA complemented transgenic lines in the ST024 background. ST147 and ST024 are 2 stable recombinant inbred lines (RILs) generated by crossing S. lycopersicum var. cerasiforme LA1310 (CC) with S. lycopersicum Moneymaker (MM) and have a mutated J2 (i.e. j2del, 5 bp deletion in the 4th exon of J2) and 1 copy of STM3 or STM3 duplication (STM3-dup), respectively (Zhang et al. 2018) (Supplemental Table S1). Compared with ST024 plants, which exhibit jointless pedicels and highly branched inflorescence (>200 inflorescence branches), the flower abscission zone was partially restored and the inflorescence branch number was dramatically decreased in the ProJ2:J2-HA complemented transgenic plants (Fig. 1A). These observed phenotypic effects thus suggested that STM3 and J2 function in the transgenic plants was similar to its function in a wild-type background.
Figure 1.
Identification of loci bound by STM3 and J2 proteins. A) The inflorescence phenotypes and quantification of the inflorescence branch number of the ST024 plants and the ProJ2:J2-HA complementation transgenic lines. ST024, an RIL with a 5 bp deletion in the 4th exon of J2. The lefthand panel shows representative inflorescence images of the ST024 and ProJ2:J2-HA plants. The righthand panel shows quantification data for the inflorescence branch number of ST024 and ProJ2:J2-HA plants. The % of total inflorescences represents the proportion of the number of inflorescences within a certain range of inflorescence branches to the total number of inflorescences. The white asterisk indicates the joint. Scale bars = 1 cm. B) and C) Pie charts displaying the genomic distribution of STM3 (B) and J2 (C) binding sites in the IM and FM tissues of STM3OE1 and ProJ2:J2-HA by ChIP-seq. Promoter, −5 kb to +0 bp upstream of the ATG, down region, 0 bp to +1 kb downstream of the stop codon. D) and E) The most significant STM3 (D) and J2 (E) binding motifs produced by MEME. The bottom graph shows the distribution of the motif corresponding to the −150 to +150 bp region flanking the binding-site peaks. The x-axis indicates the position of the binding motif in the sequence, and the y-axis indicates the probability of the identified motif. F) Venn diagram showing the overlapping peaks between the STM3-binding loci and the J2-binding loci. G) Heatmap showing the density of overlapping regions bound by J2 and STM3. The x-axis indicates the distance from the central position of STM3 peak regions, and the y-axis indicates counts per million. H) and I) EMSA of the MBP-STM3 (H) and MBP-J2 (I) proteins interacting with the biotin-labeled DNA fragment containing the CArG box motif of the AP2c promoter. MBP protein is used as a negative control. Biotin-unlabeled probes are applied as competitors. pAP2cmu probe was the mutated probe. Arrows indicate the STM3-DNA (H), J2-DNA (I) complex, and the free probes. J) GO enrichment analysis of putative common target genes bound by STM3 and J2. The top 8 enriched biological process categories with P-values from the statistical overrepresentation test are shown.
A total of 676 overlapping peaks (repeat 1:1,129 peaks, repeat 2:1,070 peaks) were identified by ChIP-seq from 2 biological replicates in the STM3OE1 plants and enriched more than 2.5-fold over that in the IgG negative control samples (Supplemental Fig. S1A; Supplemental Table S2). Among them, 293 peaks (∼43%) were located in promoter regions (<5 kb upstream of the start codon ATG) (Fig. 1B). The 676 detected peaks were assigned to 587 adjacent genes (i.e. the peaks were located in the promoter region, exon, intron, or 3′UTR region, 1 kb downstream of the stop codon) (Supplemental Data Set 1). ChIP-seq from 2 replicates of the ProJ2:J2-HA plants identified a total of 960 overlapping peaks (repeat 1:4,138 peaks, repeat 2:2,004 peaks) with 2.5-fold enrichment over that in control samples. More than 43% of overlapping peaks were located in promoter regions, and these binding peaks corresponded to 726 putative target genes (Fig. 1C; Supplemental Fig. S1B; Supplemental Data Set 2; Supplemental Table S2).
Although most plant MADS-domain proteins recognize the canonical CArG box motif, CC(A/T)6GG, variations in the distribution of A and T nucleotides in the A/T stretch, or the length of this nucleotide region, can still differ among various MADS-domain proteins (Tang and Perry 2003; Muino et al. 2014). In order to determine the motifs recognized by J2 and STM3, we performed de novo motif discovery using MEME software (Machanick and Bailey 2011). The results showed that the most significantly enriched motif recognized by STM3 was the CCAAAAAWRGAAA sequence (E = 5.5e−33), containing the CC(A/T)6GG recognition motif with a 3′ AAA extension (Fig. 1D; Supplemental Fig. S1C) that was similar to the sequence(s) recognized by Arabidopsis SOC1 (Immink et al. 2012; Tao et al. 2012). In contrast with this STM3 recognition sequence, J2 binding was associated with a CCWTATAWGG sequence (E = 7.1e−12), which is a canonical CArG box (Fig. 1E; Supplemental Fig. S1D). These results confirmed that these 2 MADS-box TFs, STM3 and J2, could directly bind putative target genes via CArG box recognition in the tomato genome.
Identification of genes targeted by both STM3 and J2
Since STM3 and J2 are both known to regulate inflorescence branching in tomato, we compared their ChIP-seq datasets to test whether these 2 MADS-box TFs also target the same sites for transcriptional regulation. A total of 253 peaks were identified as binding targets of both STM3 and J2 (Fig. 1F). Moreover, this set of common binding peaks had high overlap in their co-occupancy regions, with more than 80% containing CArG boxes, and approximately 45% of the peaks located within promoter regions (Fig. 1G, Supplemental Fig. S1E).
To next determine whether STM3 and J2 could directly interact with these DNA sequences, we performed electrophoretic mobility shift assays (EMSAs) using a DNA fragment of the AP2c promoter that contained a CArG box located in the common binding region as the probe, with recombinant STM3 and J2 proteins carrying an N-terminal MBP tag (i.e. MBP-STM3 and MBP-J2). The results showed that MBP-STM3 and MBP-J2 proteins, but not MBP alone, could interact with the DNA probes. Furthermore, the addition of increasing amounts of competitors into the reaction mixture led to attenuated binding of these proteins with the DNA probes (Fig. 1, H and I). These results thus showed that STM3 and J2 proteins could both directly interact with the same DNA sequence(s).
The 253 common binding peaks were assigned to 203 adjacent genes, representing 35% (203/587) and 28% (203/726) of the STM3 and J2 putative target genes, respectively (Supplemental Data Set 3). We performed gene ontology (GO) analysis of these common putative target genes to explore the developmental/biological processes potentially co-regulated by these 2 MADS-box TFs. This analysis revealed high functional enrichment for terms such as “cell differentiation,” “response to red light,” and “flower development” (Fig. 1J). Notably, several orthologs of Arabidopsis flowering or FM and IM developmental regulators were included among the common putative target genes, such as APETALA2 (AP2) family TF genes, AP2a, AP2b, AP2c, and AP2e, and some MADS-box genes, AGAMOUS (AG), AGAMOUS-LIKE 104 (AGL104), and FUL1 (Supplemental Data Set 3) (Bowman et al. 1991; Chen 2004; Adamczyk and Fernandez 2009; Wollmann et al. 2010; Dinh et al. 2012; Huang et al. 2017; Balanza et al. 2018). These results suggested that STM3 and J2 likely directly bind a shared subset of putative target genes through CArG box recognition to mediate their respective functions in tomato inflorescence development.
STM3 and J2 exhibit antagonistic co-regulation of target gene transcription
According to our ChIP-seq data, J2 could also directly bind FUL1, a known downstream target of STM3 (Fig. 2A) (Wang et al. 2021). Subsequently, ChIP-qPCR confirmed that J2 bound at the P4 region of the FUL1 promoter, which was the same region bound by STM3 (Fig. 2B). We found that FUL1 transcription was decreased in the IM and FM tissues of ProJ2:J2-HA plants and enhanced in that of j2cr knockout mutant, which was generated in a S. pimpinellifolium accession PI365967 (PP) using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated genome editing (Supplemental Fig. S2A), indicating that J2 repressed FUL1 transcription, in contrast to the activation of FUL1 transcription by STM3 (Fig. 2, C and D) (Wang et al. 2021). Co-expression of J2-FLAG and LUC driven by the FUL1 promoter in Nicotiana benthamiana leaves resulted in significantly decreased luminescence intensity compared with that in the empty vector control, further demonstrating the direct repression of FUL1 via J2 binding (Fig. 2E).
Figure 2.
STM3 and J2 antagonistically regulate the transcription of common target genes. A) Chromatin binding profiles of STM3 and J2 at the FUL1 promoter. The short black lines P1 and P4 represent the regions for ChIP-qPCR. B) ChIP-qPCR analysis of binding regions at the FUL1 promoter. P1 and P4 represent the regions for ChIP-qPCR. IgG is used as a negative control. The DNA fragment at the centromere region of Chromosome 02 is used as an internal control. Two-tailed Student's t-tests were used for statistical analysis (**P < 0.01). Error bar, SD. C and D) Expression levels of FUL1 in tissues of IM and FM in the ProJ2:J2-HA plants (C) and the j2cr mutants (D). PP, PI365967.UBIQUITIN3 is used as the internal control. Student's t-tests are used for statistical analysis (**P < 0.01). Error bar, SD. E) Representative dual-luciferase reporter assay in N. benthamiana co-expressing Pro35S:J2-FLAG or Pro35S:FLAG and ProFUL1:luc-Pro35S:Rluc. Co-expressing ProFUL1:luc-Pro35S:Rluc and Pro35S:FLAG is used as the control (CK). The righthand panel shows representative images of leaves 48 h after infiltration. Renilla luciferase (REN) is an internal control. The ratio of LUC/REN represents the relative activity of promoters. Two-tailed Student's t-tests were used for statistical analysis (*P < 0.05). Error bar, SD. F) The inflorescence phenotypes and quantification data of the inflorescence branch number of the j2del, j2del ful1cr3, and j2del ful1cr4 mutants. The lefthand panel presents inflorescence images of the j2del, j2del ful1cr3, and j2del ful1cr4 plants. The righthand panel presents the quantification of the inflorescence branch number of these plants. The white triangles indicate the inflorescence branch. The % of total inflorescences represents the proportion of the number of inflorescences within a certain range of inflorescence branches to the total number of inflorescences. Scale bars = 1 cm. G–R) Chromatin binding profiles of STM3 and J2 proteins at the promoters of AP2a(G), AP2c(J), AG(M), and AGL104(P), and the expression levels of AP2a(H and I), AP2c(K and L), AG (N and O), and AGL104 (Q and R) in IM and FM tissues of the ProJ2:J2-HA plants and stm3cr mutants in the background of ST024, and the j2cr mutants and STM3OE5 lines in the background of PP. ST024, an RIL, taking a 5 bp deletion at the 4th exon of J2. PP, PI365967, UBIQUITIN3 is used as the internal control. Two-tailed Student's t-tests were used for statistical analysis (**P < 0.01, *P < 0.05). Error bar, SD.
We then mutated the FUL1 gene using CRISPR/Cas9-mediated genome editing in a j2del background (carrying a 5 bp deletion in the 4th exon of J2 and 1 copy of STM3). Two null alleles were generated, ful1cr-3 and ful1cr-4, which, respectively, harbored a 1 bp insertion or 1 bp deletion that resulted in early termination of FUL1 transcription (Supplemental Fig. S2B). Phenotypic analysis showed that inflorescence branching was obviously reduced in these j2del ful1cr-3 and j2del ful1cr-4 mutants compared with that in the j2del control plants (Fig. 2F). Therefore, disruption of FUL1 could rescue the inflorescence phenotypes of j2del mutants, indicating that J2 in tomato inflorescences branching was at least partially mediated through its target gene FUL1. Therefore, STM3 and J2 antagonistically regulate FUL1 transcription in tomato inflorescence development.
To investigate whether STM3 and J2 also antagonistically regulate the transcriptions of other common putative target genes, we then detected the relative expression levels of the putative common genes, AP2a, AP2c, AG, and AGL104, in IM and FM tissues of ProJ2:J2-HA and a stm3cr knockout mutant, which was generated in ST024 using CRISPR/Cas9-mediated genome editing (Wang et al. 2021) and j2cr knockout mutant and STM3 overexpression line STM3OE5, which were generated in PP (Supplemental Fig. S3; Supplemental Table S1). Our results showed that the expression levels of all these genes were significantly decreased in IM and FM tissues of stm3cr mutants and ProJ2:J2-HA, but enhanced in those of the j2cr mutants and STM3OE5 compared with their corresponding control plants (Fig. 2, G to R). These results indicate that the transcriptions of some common putative target genes can be repressed by J2 and activated by STM3. Based on this finding, we, therefore, hypothesized that STM3 and J2 have antagonistic roles in the transcriptional regulation of their partial common putative target genes.
Interaction of J2 and STM3 mediates the mutual inhibition of their binding to the common targets
MADS-box TFs often physically interact and form multimeric complexes to cooperatively regulate plant development (Smaczniak et al. 2012a, 2012b). Since both STM3 and J2 recognize the CArG box motif and bind many common genomic regions, we next examined whether they can directly interact and form a complex as part of a shared regulatory function. To this end, we performed yeast two-hybrid assays that showed J2 could interact with STM3 in yeast cells (Fig. 3A). We confirmed their interactions using Co-immunoprecipitation (Co-IP) assays through transient co-expression of STM3-YFP-HA and J2-FLAG in N. benthamiana leaves using co-expression of J2-FLAG and YFP-HA as a control. More specifically, J2-FLAG was immunoprecipitated by HA antibody when it was co-expressed with STM3-YFP-HA, but not with YFP-HA (Fig. 3B). These results indicated that STM3 physically interacts with J2 to form a complex.
Figure 3.
Interaction of J2 and STM3 mediates the mutual inhibition of their binding to the common targets. A) Yeast two-hybrid analysis of the protein interaction between STM3 and J2. Yeast cells were grown on SD/-Leu/-Trp (SD/-LT) plates and SD/-Leu/-Trp/-Ade/-His (SD/-LTHA) plates (or containing X-α-gal) for 3 d. Positive interactions were determined by the appearance of colonies on quadruple dropout agar medium SD/-LTHA. Negative controls contained empty bait and/or prey vectors. B) In vivo interaction between STM3 and J2 determined by Co-IP in N. benthamiana leaves. Protein samples obtained from the transient expressions of STM3-YFP-HA and J2-FLAG in N. benthamiana leaves are immunoprecipitated with HA antibody. Immunoblots of the total protein extracts (input) and the anti-HA-bound target proteins are performed using an anti-FLAG antibody. C) The subcellular localization of STM3 and J2 in N. benthamiana leaves by expressing STM3-RFP and YFP, J2-YFP and RFP, or STM3-RFP and J2-YFP. Scale bars = 20 µm. D) The abundance of STM3 and J2 proteins in the nucleus and cytoplasm by expressing STM3-YFP-HA and J2-FLAG, STM3-YFP-HA and FLAG, or YFP-HA and J2-FLAG in N. benthamiana leaves. N, nucleus; C, cytoplasm. HSP70 and histone H3 as cytoplasmic and nuclear fraction markers, respectively. E) BiFC visualization of the interaction between J2 and STM3 in the nuclei and cytoplasm of N. benthamiana epidermal cells. The N-terminal fragment of YFP (nYFP) and the C-terminal fragment of YFP (cYFP) are fused to the C-terminus of STM3 and J2, respectively. Combinations of nYFP or cYFP with the corresponding STM3 and J2 constructs are used as negative controls. Bars = 20 μm. F) Diagram of truncated J2 and STM3 proteins. STM3M or J2M, containing MADS-domain and I region of STM3 or J2. STM3K or J2K, containing I, K, and C region of STM3 or J2. MADS-domain (MADS), intervening (I), keratin-like (K), and C-terminal domains (C). G) BiFC visualization of the interaction between J2 and STM3M or STM3K, STM3 and J2M or J2K in N. benthamiana epidermal cells. The N-terminal fragment of YFP (nYFP) and the C-terminal fragment of YFP (cYFP) are fused to the C-terminus of STM3, J2, STM3M, STM3K, J2M, and J2K, respectively. Bars = 20 μm. H) Binding efficiency assay of HiS-STM3 and MBP-J2 proteins with the biotin-labeled DNA fragment containing the CArG box motif of the FUL1 promoter by EMSA. Arrows indicate the STM3-DNA, J2-DNA complex, and the free probes. I) Binding efficiency assay of MBP-J2 or MBP-STM3 proteins with the biotin-labeled DNA fragment containing the CArG box motif of the FUL1 promoter as an increasing amount of truncated protein STM3M and STM3K or J2M and J2K by EMSA. Arrows indicate the STM3-DNA, J2-DNA complex, and the free probes.
Interactions between different MADS-domain proteins often result in intracellular redistribution of the interacting partners and thus affect their functions (Lee et al. 2008; Nayar et al. 2014; Paul et al. 2020). To investigate the subcellular localization of STM3 and J2, we transiently expressed the coding sequence (CDS) of STM3 and J2 fused with red fluorescent protein (RFP) or yellow fluorescent protein (YFP), respectively (i.e. STM3-RFP and J2-YFP), in N. benthamiana leaf epidermal cells. When STM3-RFP and J2-YFP were expressed individually, STM3-RFP could be detected in the nucleus and cytoplasm, whereas J2-YFP was observed exclusively in the nucleus. However, under co-expression of these 2 proteins in N. benthamiana leaf epidermal cells, the fluorescence signals of STM3-RFP and J2-YFP were both detectable in the cytoplasm and nucleus (Fig. 3C), suggesting that STM3 could be responsible for the cytoplasmic localization of J2. We then quantified the levels of these 2 proteins in the nucleus and cytoplasm by immunoblot analysis. Consistent with the fluorescence signals, STM3 proteins were detected in the nucleus and cytoplasm. However, we also observed that J2 proteins can be detected in the cytoplasm, although it was much less abundant than in the nucleus. When we co-expressed STM3 and J2, the interaction between STM3 and J2 nearly has no effect on the cell distribution of STM3 proteins, but the amount of J2 was significantly decreased in the nucleus and increased in the cytoplasm, which was the same as the fluorescence signal we observed (Fig. 3D).
Further bimolecular fluorescence complementation (BiFC) assays using transient expression of STM3-nYFP or STM3-cYFP together with J2-cYFP or J2-nYFP show that the patterns of STM3–J2 interaction in the nucleus and cytoplasm appeared similar to those observed for STM3–STM3 interactions, but different from the specific fluorescence signal resulting from J2–J2 interactions in the nucleus (Fig. 3E), further confirmed that cytoplasmic localization of J2 was accumulated by interaction with STM3. To determine which domain mediates the interaction of J2 and STM3, we transiently expressed the J2M and STM3M (M, without K-domain and C-terminal domains) or J2K and STM3K (K, without MADS-domain) with STM3 or J2 in N. benthamiana leaves, respectively. Fluorescence signals revealed that all these truncated proteins could interact with the J2 or STM3 (Fig. 3, F and G; Supplemental Fig. S4). To investigate whether the DNA-binding activity of J2 and STM3 is affected by their interaction, we analyzed the binding efficiency of HIS-STM3 and MBP-J2 proteins with the biotin-labeled DNA fragment containing the CArG box motif of the FUL1 promoter by EMSA. The result shows that with the increase of the STM3 proteins, the shift bands of the combination of J2 and FUL1 probe were weakened and vice versa, indicating mutual competition between J2 and STM3 to bind common putative targets in a complex (Fig. 3H).
To further validate their competition of interaction with the FUL1 probe, we performed the EMSA and found that STM3K and J2K could not bind DNA, but with the increase of STM3K and J2K proteins, the binding efficiency of J2 or STM3 to the probe, respectively, was dramatically reduced. Different from STM3K and J2K, STM3M and J2M proteins could interact with the FUL1 probe, and more shift bands make it difficult to evaluate the effects of STM3M and J2M proteins on the binding efficiency of J2 and STM3 (Fig. 3I). These results demonstrated that the interaction between J2 and STM3 affects the subcellular translocation of J2 from the cytoplasm to the nucleus and the mutual competition between STM3 and J2 proteins binding on the putative common targets is due to their protein interaction.
STM3 represses J2 binding to its target genes
The subcellular redistribution and binding activity of J2 mediated by interaction with STM3 prompted us to examine the possibility that STM3 could affect the transcriptional regulatory activity of J2 in regulating inflorescence branching. To investigate this hypothesis, we generated ProJ2:J2-HA stm3cr plants by crossing ProJ2:J2-HA plants with stm3cr mutants in ST024 (Wang et al. 2021). Phenotypic analysis showed that the number of inflorescence branches in the resulting ProJ2:J2-HA stm3cr plants was lower than that in the ProJ2:J2-HA parental line (Fig. 4A). Consistent with the well-established activation of FUL1 by STM3, FUL1 expression levels were decreased in the IM and FM tissues of ProJ2:J2-HA stm3cr plants compared with those in the corresponding tissues of ProJ2:J2-HA plants (Fig. 4B). We then checked for differences in J2 target site binding activity between ProJ2:J2-HA and ProJ2:J2-HA stm3cr plants using ChIP-qPCR. In IM and FM tissues, the J2-binding signal was approximately 2-fold higher at the FUL1 promoter in ProJ2:J2-HA stm3cr plants compared with that in ProJ2:J2-HA plants, as well as for other putative target genes, such as AP2a, AP2c, AG, and AGL104 (Fig. 4C). These results indicate that the redistribution of J2 proteins and the weakened binding activity on its putative target genes via interaction with STM3 led to J2 playing a stronger inhibitory function for regulating inflorescence branching when STM3 is mutated.
Figure 4.
STM3 represses J2 binding to its target genes. A) The inflorescence phenotypes and quantification of the inflorescence branch number of the ProJ2:J2-HA and ProJ2:J2-HA stm3cr plants. The lefthand panel presents representative inflorescence images of ProJ2:J2-HA and ProJ2:J2-HA stm3cr plants. The righthand panel presents the quantification of the inflorescence branch number of these plants. Scale bars = 1 cm. B) Expression levels of FUL1 in tissues of IM and FM in the ProJ2:J2-HA and ProJ2:J2-HA stm3cr plants. UBIQUITIN3 is used as the internal control. Student's t-tests are used for statistical analysis (**P < 0.01). Error bar, SD. C) ChIP-qPCR analysis of the DNA enrichment at the binding regions of FUL1, AP2a, AP2c, AG, and AGL104 by J2 proteins in the ProJ2:J2-HA and ProJ2:J2-HA stm3cr plants. The DNA fragment at the centromere region of Chromosome 02 is used as an internal control. Two-tailed Student's t-tests are used for statistical analysis (**P < 0.01). Error bar, SD.
J2 hinders STM3 function and prevents accessing target genes
Although STM3 could affect J2 function in regulating downstream genes, high expression of STM3 resulting from STM3 duplication in the tomato genome could induce greater inflorescence branching that is only observed in j2TEej2w double mutants (M82 background), but not in tomato plants carrying the wild-type J2EJ2 (Alonge et al. 2020). Similarly, we found that the STM3-dup could significantly increase inflorescence branching of j2del RILs but not the RILs with wild-type J2 lines in the RIL population (Supplemental Fig. S5). These results led us to propose a hypothesis that the J2 exerts inhibitory effects on STM3 regulatory functions in inflorescence branching.
To this end, we generated a series of transgenic lines in a uniform background PP (J2 and 1 copy of STM3). Firstly, we overexpressed STM3 by transforming the Pro35S:STM3-FLAG vector construct into the wild-type PP tomato accession and obtained 9 independent STM3-overexpressing (STM3OE) transgenic lines. Relative expression levels of STM3 in the STM3OE4 and STM3OE5 lines were increased by several dozen-fold over those in wild-type PP. No obvious effects on inflorescence-branching phenotype were observable in either of the 2 STM3OE lines (Supplemental Fig. S3). We then crossed STM3OE5 plants with j2cr mutants to obtain STM3OE5/j2cr plants in wild-type PP, which exhibited more inflorescence branching than that of j2cr mutants (Fig. 5A). In addition, FUL1 was highly expressed in the IM and FM tissues of STM3OE5/j2cr plants, but was only slightly upregulated in STM3OE5 plants (Fig. 5B) that was in agreement with the inflorescence phenotypes of these plants. These results thus supported the likelihood that the presence of J2 protein hindered STM3 regulatory function in inflorescence-branching development.
Figure 5.
J2 inhibits STM3 regulation of target genes by limiting its binding. A) The inflorescence phenotypes and quantification data of the inflorescence branch number of the wild-type PP, j2cr, STM3OE5, and STM3OE5/j2cr plants. The lefthand panel presents representative inflorescence images of wild-type PP, j2cr, STM3OE5, and STM3OE5/j2cr plants. The white triangles indicate the inflorescence branch. The righthand panel is the quantification of the inflorescence branch number of these plants. The % of total inflorescences represents the proportion of the number of inflorescences within a certain range of inflorescence branches to the total number of inflorescences. PP, PI365967. Scale bars = 1 cm. B) Expression levels of FUL1 in tissues of IM and FM of the wild-type PP, STM3OE5, and STM3OE5/j2cr. UBIQUITIN3 is used as the internal control. Student's t-tests are used for statistical analysis (**P < 0.01). Error bar, SD. C) ChIP-qPCR analysis of the DNA enrichment at the binding regions of FUL1, AP2a, AP2c, AG, and AGL104 by STM3 proteins in the STM3OE5 and STM3OE5/j2cr plants. A DNA fragment at the centromere region of Chromosome 02 is used as an internal control. Two-tailed Student's t-tests are used for statistical analysis (**P < 0.01). Error bar, SD. D) Representative of dual-luciferase reporter assay in N. benthamiana co-expressing ProFUL1:luc-Pro35S:Rluc and Pro35S:STM3-FLAG individually or in a combination of Pro35S:STM3-FLAG and Pro35S:J2-FLAG. Co-expressing ProFUL1:luc-Pro35S:Rluc and Pro35S:FLAG is used as the control (CK). Representative images of N. benthamiana leaves 48 h after infiltration are shown in the righthand panel. Renilla luciferase (REN) is used as an internal control. The ratio of LUC/REN represents the relative activity of promoters. Tukey's multiple comparisons test is used for statistical analysis (P < 0.05), Error bar, SD.
To confirm that interaction with J2 imposed any inhibitory effects on STM3 binding to recognition sequences, we carried out ChIP-qPCR and compared the binding efficiency of STM3 on the FUL1 promoter and other putative target genes in the tissues of IM and FM of STM3OE5 and STM3OE5/j2cr plants. The results showed a marked increase in DNA enrichment for the STM3-bound FUL1 promoter and the promoters of other putative target genes in STM3OE5/j2cr plants, compared with that in STM3OE5 plants (Fig. 5C), suggesting that J2 inhibits STM3 binding to common putative target genes.
To further verify that J2 directly hinders STM3 accessing target genes, we transiently expressed STM3 protein alone, or co-expressed STM3 and J2 proteins with LUC driven by the FUL1 promoter in N. benthamiana leaves. The luminescence intensity driven by FUL1 promoter activity was obviously enhanced upon expressed STM3. However, the luminescence intensity was substantially lower under co-expression of STM3 and J2 than that under the expression of STM3 alone, similar to that in empty vector control, indicating STM3-binding activity of downstream genes was inhibited in the presence of J2 that is consistent with the EMSA assay (Figs. 5D and 3, H and I). Taken together, these results demonstrated that J2 hinders STM3 function in the transcriptional regulation of putative inflorescence-branching-related target genes.
J2 directly binds the STM3 promoter to repress its function
In addition, our ChIP-seq dataset revealed that J2 could bind the STM3 promoter, and the significant peak observed at the STM3 promoter region in IM and FM tissues (Fig. 6A) suggested that J2 might directly regulate STM3 expression during inflorescence development. In particular, our ChIP-qPCR assays showed that J2 recognized and bound the P1 DNA fragment around the STM3 promoter region (Fig. 6B). Moreover, co-expression of J2 protein with LUC driven by the STM3 promoter in N. benthamiana leaves resulted in significantly decreased luminescence compared with that in empty vector controls, further confirming that J2 interaction with the STM3 promoter led to its transcriptional repression (Fig. 6C). To further verify this transcriptional repression in tomato, we detected STM3 expression levels in the IM and FM tissues of j2cr mutants and wild-type PP. The expression levels of STM3 were significantly higher in j2cr mutant plants compared with those in controls, supporting that J2 interaction with the STM3 promoter represses its transcription in IM and FM tissues (Fig. 6D).
Figure 6.
J2 directly binds at STM3 promotor and represses its transcription. A) Chromatin binding profiles of J2 at the promoter of STM3. ChIP-seq reads were visualized using the IGV. The short black lines P1 and P2 represent the regions for ChIP-qPCR. B) ChIP-qPCR analysis of the DNA enrichment at the P1 or P2 regions bound by J2 proteins. The anti-HA antibody is used for DNA immunoprecipitation from the IM and FM tissues of the ProJ2:J2-HA plants. The DNA fragment at the centromere region of Chromosome 02 is used as an internal control. Two-tailed Student's t-tests are used for statistical analysis (**P < 0.01). Error bar, SD. C) Representative of dual-luciferase reporter assay in N. benthamiana co-expressing Pro35S:J2-FLAG and ProSTM3:luc-Pro35S:Rluc. Co-expressing ProSTM3:luc- Pro35S:Rluc and Pro35S:FLAG is used as the control (CK). The righthand panel shows representative images of leaves 48 h after infiltration. Renilla luciferase (REN) is used as an internal control. The ratio of LUC/REN represents the relative activity of promoters. Two-tailed Student's t-tests were used for statistical analysis (*P < 0.05). Error bar, SD. D) Expression levels of STM3 in tissues of IM and FM of the wild-type PP and j2cr mutants. PP, PI365967. UBIQUITIN3 is used as the internal control. Student's t-tests are used for statistical analysis (**P < 0.01). Error bar, SD. E) Inflorescence phenotypes and quantification data of the inflorescence branch number of the wild-type PP, j2cr, stm3 tm3, and stm3 tm3 j2 mutants. The lefthand panel presents representative inflorescence images of wild-type PP, j2cr, stm3 tm3, and stm3 tm3 j2 mutants. The righthand panel is the quantification of the inflorescence branch number of these plants. PP, PI365967. Scale bars = 1 cm. F) A working model of the STM3–J2 module defines tomato inflorescence branching. In wild-type plants (middle panel), STM3 and J2 can both recognize the CArG box motif of some putative common target genes in antagonistic regulation of tomato inflorescence branching. J2 could bind the STM3 promoter to repress its expression. Moreover, interaction with STM3 mediates J2 translocation from the cytosol to the nucleus. STM3 hinders J2 function by preventing accessing targets and vice versa. When STM3 is mutated (lefthand panel), more J2 proteins in the nucleus bind at some putative common target promoters to repress their transcriptions resulting in less inflorescence branch number than that of wild-type plants. When J2 is mutated (righthand panel), STM3 is highly expressed and more STM3 proteins could bind some putative targets to activate their transcriptions leading to more inflorescence branches than that of wild-type plants.
We then obtain stm3 tm3 j2 triple mutants in the PP background by crossing j2cr plants with stm3 tm3 double mutants, which were generated in PP by CRISPR/Cas9-mediated genome editing (Supplemental Fig. S6). STM3 and TM3 genes as 2 SOC1 homologs redundantly regulate tomato inflorescence branching (Wang et al. 2021). Phenotypic analysis showed that the j2cr mutants exhibited more inflorescence branching compared with wild type (PP), while the inflorescence branch number of the stm3 tm3 double mutants were slightly less than that of wild-type (PP) plants, supporting their opposite roles in inflorescence branching. Notably, the stm3 tm3 j2 triple mutants displayed less inflorescence branches than that of the j2cr mutants, indicating that mutation of STM3 could partially rescue the highly branched inflorescence phenotype of the j2cr mutants (Fig. 6E). Taken together, these results supported that the STM3 is a downstream target gene of J2 in inflorescence development.
Discussion
In tomato, the MADS-box TFs J2 and STM3 are known to function as negative and positive regulators, respectively, in the control of inflorescence branching (Soyk et al. 2017; Alonge et al. 2020; Wang et al. 2021). Here, using ChIP-seq, we identified the binding sites of J2 and STM3 throughout the genome and found that these 2 TFs both recognize CArG box motifs and that they physically interact to form a complex in the co-regulation of shared putative target genes. However, in complex, J2 and STM3 mutually hinder each other's transcriptional regulation of FUL1 either through translocation of J2 from the cytoplasm to the nucleus, or by preventing STM3 or J2 accessing FUL1 promoter. In addition, J2 also represses STM3 transcription through direct binding with its promoter region (Fig. 6F). Our findings thus highlight the complexity of the regulatory network responsible for tomato inflorescence development downstream of J2-STM3 and reveal the mechanisms by which MADS-box TFs antagonistically co-regulate the transcription of their direct targets.
STM3 is a homolog of Arabidopsis SOC1, and functions as a flowering integrator, promoting flowering in tomato similar to that of SOC1 in Arabidopsis (Lee and Lee 2010; Alonge et al. 2020; Wang et al. 2021). However, STM3 acts as a positive regulator to promote tomato inflorescence branching (Alonge et al. 2020), unlike SOC1, which negatively regulates inflorescence branching by interaction with AP1 (Teo et al. 2014). These various findings indicate functional divergence between STM3 and SOC1 in regulating inflorescence branching of tomato and Arabidopsis. Our results reveal that STM3 physically interacts with J2, the homolog of SEP4, to form a complex to regulate FUL1 transcription during tomato inflorescence development. By contrast, SOC1 interacts with AP1 to repress TERMINAL FLOWER 1 (TFL1) transcription, redundantly with SEP4, AGL24, and SHORT VEGETATIVE PHASE (SVP), to control Arabidopsis inflorescence branching (Teo et al. 2014). Thus, STM3 and SOC1 control inflorescence branching through largely distinct regulatory pathways in Arabidopsis and tomato. Understandably, regulatory mechanisms of SEP4 and J2 in inflorescence development may be also functionally divergent in Arabidopsis and tomato, although they are both negative regulators of inflorescence branching.
Our genome-wide identification of J2 and STM3 putative target genes in IM and FM tissues indicated that these 2 proteins can recognize and bind a large suite of common putative target genes that carry promoter region CArG box motifs. Although the enriched DNA sequences recognized by STM3 and J2 in ChIP-seq are not exactly the same, they are the same as the DNA sequences recognized by their respective homologs, SOC1 and SEP4, in Arabidopsis (Immink et al. 2012; Tao et al. 2012; Liu et al. 2013). In addition, the homologs of some STM3 and J2 putative target genes, such as AP2s, are also direct targets of SOC1 in Arabidopsis (Immink et al. 2012; Tao et al. 2012). Therefore, STM3 and SOC1 show conservation in their respective regulatory networks. However, no homolog of TFL1, which is directly repressed by SOC1 in Arabidopsis meristems to control inflorescence branching, has been identified among the putative target genes of STM3 and J2 in tomato (Liu et al. 2013). Instead, STM3 and J2 control tomato inflorescence branching through FUL1, rather than TFL1. Mutation of FUL1 results in partial restoration of the increased inflorescence-branching phenotype induced by high STM3 expression or J2 knockout (Wang et al. 2021). More than FUL1, one recent work indicates that FUL2 and MBP20 also regulate inflorescence branching (Jiang et al. 2022). However, FUL2 and MBP20 are not direct targets of STM3 and J2 according to our ChIP-seq data (Supplemental Fig. S7), suggesting that they are involved in different regulatory pathways during inflorescence development.
Several reports have shown that MADS-domain proteins can form dimers or tetramers to regulate the transcription of putative target genes (Smaczniak et al. 2012a, 2012b; Liu et al. 2013; Liu et al. 2014; Muino et al. 2014; Jiang et al. 2022). In Arabidopsis, SOC1 interacts with AGL24 to promote flowering via transcriptional activation of LFY (Lee et al. 2008; Liu et al. 2008), but interacts with AP1 to suppress TFL1 transcription in FM (Gregis et al. 2009). These contrasting functions of SOC1 as an activator or suppressor of transcription may reflect the influence of its various interaction partners in different regulatory pathways. In our study, STM3 directly heterodimerizes with J2 to regulate the transcription of downstream genes. In this complex, STM3 positively regulates FUL1 expression, as well as other targets, while J2 represses their expression, thus performing counteracting regulatory functions in tomato inflorescence branching. Furthermore, mutation of either J2 or STM3 results in significantly increased binding of the unmutated partner with putative target genes and consequently enhanced positive or negative regulatory effects, suggesting that they antagonistically regulate their common putative target genes.
Our finding that STM3 mediates J2 redistribution from the cytoplasm to the nucleus, accompanied by a decrease in its regulatory activity, supports J2 function as a TF in the nucleus. Other TFs have also been shown to undergo subcellular redistribution as part of a transcriptomic reprogramming mechanism (Lee et al. 2008; Cui et al. 2016). Different from J2 inhibition by more protein retaining in the cytoplasm, the increased binding of STM3 in the j2cr mutant does not appear to be an effect of subcellular redistribution, supported by the fact that STM3 is itself translocated into the nucleus. However, inflorescence-branching number is not significantly increased by either higher STM3 copy number or STM3 overexpression when J2 is present (Soyk et al. 2019), suggesting that J2 reciprocally inhibits STM3 function in inflorescence development. Our results showing increased DNA enrichment for STM3 at the FUL1 promoter and other common putative target genes in j2 mutants indicate that interaction between J2 and STM3 hinders STM3 binding to target gene promoters same as other complexes of TFs (Tao et al. 2013; Mateos et al. 2015). Moreover, J2 can also directly bind and repress STM3 transcription in the IM and FM tissues, and STM3 expression levels are significantly enhanced in the absence of J2 (i.e. in the j2cr mutant). Our findings demonstrate that J2 and STM3, in a complex, exert their multilevel antagonism of regulatory function that enables flexibility and specificity in modulating the expression of their shared target genes responsible for inflorescence branching in tomato.
Materials and methods
Plant materials and growth conditions
ST024 is a stable line with the genotype of j2del (i.e. j2del, 5 bp deletion in the 4th exon of J2) and STM3-dup from the RILs generated from a cross of S. lycopersicum var. cerasiforme LA1310 (CC) and S. lycopersicum Moneymaker (MM) (Zhang et al. 2018). S. pimpinellifolium accession PI365967 (PP) is a wild tomato species and has the wild-type J2 and STM3. J2del plants have 1 copy of STM3 and wild-type FUL1. The stm3 tm3 j2 triple mutant was generated in the PP background by crossing the j2cr mutant with the stm3 tm3 double mutant and identifying the homozygous plants in the F2 generation using PCR-based markers for each mutant. The stm3cr mutants generated in ST024 background were described in the previously published paper (Wang et al. 2021). ProJ2:J2-HA stm3cr was generated by crossing the ProJ2:J2-HA transgenic line with the stm3cr mutant in ST024 and validating the genotypes of ProJ2:J2-HA stm3cr plants in the F2 generation by PCR. STM3OE5/j2cr was generated by crossing the STM3OE5 transgenic line with the j2cr mutant in the PP background, and the genotypes of STM3OE5/j2cr were confirmed in the F2 generation by PCR and sequencing. The genotypes of plant materials used are listed in Supplemental Table S1 and all primers used are listed in Supplemental Data Set 4.
Tomato plants were grown in a greenhouse at the Shunyi experimental station in Beijing, China. Tomato seeds were sown in 32-cell plastic flats with soil (vermiculite:peat:pearlite was 2:1:1). Thirty-day-old seedlings were transplanted into the natural soil and grown in a naturally illuminated greenhouse. More than 5 plants of each line or each transgenic line were used for inflorescence-branching examination; 5 to 8 inflorescences of each plant were evaluated. N. benthamiana were grown in the pot with soil (vermiculite:peat:pearlite was 2:1:1) in a naturally illuminated culture frame and maintained at 60% relative humidity under 12 h of light (200 µE m−2 s−1) at 26 °C ± 3 °C and 12 h of dark at 20 °C ± 2 °C.
Plasmid construction and plant transformation
The CRISPR/Cas9 vectors targeted sites at the exons of the J2, STM3, TM3, and FUL1 genes were designed using the CRISPR-P v2.0 tool (http://cbi.hzau.edu.cn/CRISPR2/). The vectors were constructed according to the previously published method (Song et al. 2020). Primers containing sgRNAs and BsaI recognition sites were used to amplify the sgRNAX_U6-26t_SlU6p_sgRNAX fragments using a pCBC_DT1T2_SlU6p vector as the template, after which the fragments were purified and cloned into pTX041 at the BsaI sites (Deng et al. 2018), primer P1 was used for J2, primer P2 was used for STM3 and TM3, and primer P9 was used for FUL1. All plasmids were validated by sequencing and then transformed into the Agrobacterium tumefaciens strain AGL1. CRISPR/Cas9 vectors targeting J2 were transformed into PP, CRISPR/Cas9 vectors targeting STM3 TM3 were transformed into ST024 and PP, and CRISPR/Cas9 vectors targeting FUL1 were transformed into a j2del plants, which have 1 copy of STM3 and wild-type FUL1. The positive transgenic lines were validated by PCR using primer P3. Genomic DNA samples extracted from T0 positive plants were used as templates for PCR, and P4 (j2cr), P5 and P6 (stm3 tm3), and P10 (ful1cr) primers were used to amplify the fragment containing the target site for Sanger sequencing to obtain homozygous genome-edited plants.
The ProJ2:J2-HA complementation vector was constructed using the native J2 promoter (containing 3.28 kb DNA sequence upstream of the ATG) and the J2 genomic DNA without the first intron. Two DNA fragments including the promoter with the first exon and the other region of J2 genomic DNA amplified using primer P7 and the genomic DNA of wild-type PP as the template were then cloned into the vector pCAMBIA2300-HA (XmaI) using the In-Fusion cloning system kit (Clontech, 639648). The ProJ2:J2-HA complementation vector was validated by sequencing and then transformed into the A. tumefaciens strain AGL1. The complementation plasmid was transformed into the ST024. The transgenic lines were validated by PCR using P8.
The Pro35S:STM3-FLAG vector was constructed using the full-length CDS of STM3, which was amplified with the primer P11 and MM cDNA as the template, and then cloned into the pCAMBIA2300-Pro35S:FLAG (BamHI and EcoRI) vector. The Pro35S:STM3-FLAG vector was validated by sequencing and transformed into the A. tumefaciens strain AGL1. The Pro35S:STM3-FLAG plasmid was transformed into PP, producing 9 independent T0 transgenic plants. The STM3OE4 and STM3OE5 are 2 overexpression lines with higher STM3 expression.
ChIP-seq and ChIP-qPCR
ChIP was performed using 0.5 g hand-dissected IM and FM tissues of STM3OE1, ProJ2:J2-HA, ProJ2:J2-HA stm3cr, STM3OE5/PP, and STM3OE5/j2cr seedlings (Wang et al. 2021). The collected plant tissues were completely ground in liquid nitrogen and cross-linked in 1% (v/v) formaldehyde (Sigma, 104003) for 10 min at 4 °C, followed by the addition of glycine to a concentration of 0.1 M, which was infiltrated for 5 min at 4 °C. Chromatin was sheared using a Diagenode Bioruptor Plus instrument to obtain ∼300 bp DNA fragments. STM3OE1 and ProJ2:J2-HA plants were immunoprecipitated with anti-HA antibody (Sigma, H6908), and STM3OE5 and STM3OE5/j2cr were immunoprecipitated with anti-FLAG antibody (Sigma, F1804). The DNA isolated by immunoprecipitated was used for Illumina sequencing or qPCR analysis. ChIP-seq libraries were prepared using the NEXTflex Rapid DNA-Seq Kit for Illumina (NOVA, 5144-08) according to the manufacturer's protocol. The 150-bp single-end reads were mapped to tomato reference genome (SL4.0) using Bowtie2 (version 2.3.5; parameters: -p 10 -5 15 -3 85 -phred 33) (Langmead and Salzberg 2012). Duplicated and low-quality mapped reads were identified and removed by SAMtools (SAMtools view -F 1028 -Sb -q 10) (Acosta et al. 2009). Enriched binding peaks were then identified using MACS2 v.2.1 (Zhang et al. 2008). The overlapping peaks in 2 biological replicates were identified by a custom Perl script. MEME-ChIP and Homer were used for Motif discovery and peak summits (±150 bp) (Heinz et al. 2010; Machanick and Bailey 2011). For normalization and visualization, the filtered and sorted bam files were converted to bigwig format using the “bamCoverage” function in deepTools v3.3.0 (Ramírez et al. 2016). GO analyses of putative target genes were performed using their closest homologs in Arabidopsis with DAVID (The Database for Annotation, Visualization and Integrated Discovery, https://david.Ncifcrf.gov/). ChIP-qPCR was performed using EvaGreen 2×qPCR MasterMix-No Dye (Abm, MasterMix-S) on a Bio–Rad CFX-96 Real-Time PCR instrument with the following program: 3 min at 95 °C followed by 50 cycles of 20 s at 95 °C, 30 s at 60 °C, and 20 s at 72 °C. The DNA fragment at the centromere region of Chromosome 02 was used as an internal control. All primers used are listed in Supplemental Data Set 4.
Tissue collection, RNA extraction, and gene expression analysis
Meristems of about 4-wk-old seedlings were dissected within 2 h and immediately fixed in 100% acetone, followed by vacuum infiltration (0.06 MPa, 40 min) (Park et al. 2012). About 200 tissues of IM and FM were microdissected by tweezers under a stereomicroscope and dried for 3 min at room temperature to remove the remaining acetone, and then immediately stored in liquid nitrogen. The materials were thoroughly ground in a mixer mill MM300 (Retsch) with a tungsten bead (3 mm diameter). Total RNA was extracted from each collection using the PicoPure RNA Extraction kit (Thermo-Fisher, 12204-01). First-strand cDNA synthesis was performed using 100 ng of total RNA with the TransScript II One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen). Reverse transcription-quantitative PCR (RT-qPCR) was performed with EvaGreen 2×qPCR MasterMix-No Dye (Abm, MasterMix-S) on a Bio–Rad CFX-96 Real-Time PCR instrument using the following program: 3 min at 95 °C followed by 40 cycles of 20 s at 95 °C, 30 s at 60 °C, and 20 s at 72 °C. UBI3 (Solyc01g056840) was used as the internal control for RT-qPCR. The primers used for RT-qPCR are listed in Supplemental Data Set 4.
Electrophoretic mobility shift assay
The full-length CDSs of STM3 and J2 genes were amplified and then ligated into the MBP-pMCSG7 and HIS-pMCSG7 plasmid containing a polyhistidine (6×His) sequence (Eschenfeldt et al. 2009). All recombinant proteins were expressed and purified from Escherichia coli strain BL21. The MBP-STM3, MBP-J2, and MBP proteins were induced by 0.2 mM isopropylthio-b-D-galactoside (IPTG), and HIS and HIS-STM3 proteins were induced by 0.125 mM IPTG at 16 °C for 16 h and purified by Ni-NTA agarose (QIAGEN, 1018244) according to the manufacturer's instructions. The 5′ biotin-labeled DNA probes and mutated probe (the binding motif was deleted) were synthesized and annealed. DNA gel shift assays were performed using the LightShift Chemiluminescent EMSA kit (Thermo-Fisher, 20148). Each EMSA binding reaction (20 μL) contained 1 μg purified recombinant protein, 4 μL biotin-labeled DNA probe (100 pmol), 2 μL binding buffer, and 1 μL poly (dI-dC). For the competition experiment, EMSA binding reaction (10 μL) contained 0.5 μg purified HIS-STM3 protein with MBP-J2 protein (0.5, 1, and 2 μg), 2 μg purified MBP-J2 protein with HIS-STM3 protein (0.5, 1, and 2 μg), 1 μL biotin-labeled DNA probe (100 pmol), and 1 μL binding buffer. Transferred DNA and purified proteins were cross-linked using a UV lamp at 312 nm. The bands were visualized on a Tanon-5200 Chemiluminescent Imaging System (Tanon Science and Technology).
Dual-luciferase transient transcriptional activity assay
The ProSTM3:luc-Pro35S:Rluc vector was constructed, in which a 3.2-kb DNA fragment of STM3 promoter upstream of ATG amplified using MM genomic DNA as a template was integrated into pluc-Pro35S:Rluc (EcoRI) using the In-fusion HD Cloning Kit (Clontech, 639649) (Hajdukiewicz et al. 1994). The ∼2.3 kb FUL1 promoter sequence was amplified using MM genomic DNA as a template and integrated into pluc-Pro35S:Rluc (EcoRI) using the In-fusion HD Cloning Kit to generate the ProFUL1:luc-Pro35S:Rluc vector (Clontech, 639649). The CDS of J2 were amplified using cDNA of MM as the template and was cloned into pCAMBIA2300-Pro35S:FLAG (SmaI) vector by using an In-fusion HD Cloning Kit to construct Pro35S:J2-FLAG vector. The ProSTM3:luc-Pro35S:Rluc and ProFUL1:luc-Pro35S:Rluc plasmids were transformed into A. tumefaciens EHA105 competent cells with Pro35S:STM3-FLAG, Pro35S:J2-FLAG, and Pro35S:FLAG (Wang et al. 2021). A single colony was cultured in Luria-Bertani medium until the OD600 value reached 1. The A. tumefaciens cells were collected by centrifugation and suspended using 10 mM MgCl2 and 150 μM acetosyringone. The cells containing expression plasmids, luciferase plasmids, and p19 plasmid were mixed in a volume ratio (2:1:3), and subsequently, infiltrated into N. benthamiana leaves using a syringe. Two days after infiltration, the leaves were harvested and ground in liquid nitrogen. The activities of firefly luciferase and Renilla luciferase were measured using a dual-luciferase reporter assay system (Promega E1910) on a Promega GLOMAX 20/20 LUMINOMETER. The ratio between LUC and REN activity was measured with 7 (STM3-FLAG or J2-FLAG and ProFUL1:luc) or 4 (J2-FLAG and ProSTM3:luc) biological replicates with 4 leaves.
Yeast two-hybrid
The CDSs of STM3 and J2 using MM cDNA as a template were cloned into bait or prey vectors, pGBKT7 and pGADT7, respectively. A pair of bait and prey plasmids were co-transformed into the AH109 yeast (Saccharomyces cerevisiae) strain according to the Clontech yeast protocol handbook instructions. The resultant strains were subsequently grown on plates lacking leucine and tryptophan for 3 d at 30 °C. The interaction was tested via growth assays on media lacking leucine, tryptophan, histidine, and adenine but containing X-alpha-Gal indicator within 3 to 5 d.
BiFC assays
The full-length and truncated CDSs of STM3 and J2 genes were amplified and then ligated into the pEarleyGate 201-YN or pEarleyGate 202-YC vectors (PacI and SpeI) (Lu et al. 2010) using the In-fusion HD Cloning Kit. Vectors were subsequently transformed into the A. tumefaciens EHA105 strain, and intact and truncated STM3 fused proteins were mixed with intact and truncated J2 fused proteins for injection into the leaves of 4-wk-old N. benthamiana plants in an equal volume of culture. Two days after injection, YFP fluorescence signals were observed using a confocal laser scanning microscope (Leica DM6 CS). We captured the images with a laser excitation wavelength of 514 nm and collection bandwidth range from 525 to 570 nm, intensity values of about 10, and gain value of around 500. All primers used are listed in Supplemental Data Set 4.
Co-IP assay
The Co-IP assay was performed as previously described (Wang et al. 2021). The Pro35S:STM3-YFP-HA and Pro35S:J2-FLAG plasmids were transformed into A. tumefaciens strain EHA105 and then co-infiltrated into the leaves of 4-wk-old N. benthamiana plants together with RNA silencing suppressor P19 (Lindbo 2007). Samples were collected 2 d after infiltration. Total protein extracted using IP-extraction buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.19% (v/v) CA630; 20% (v/v) glycerol; 5 mM DTT; and 1 tablet/50 ml of protease inhibitor cocktail) and then incubated overnight at 4 °C with the HA-binding beads (5 μg anti-HA (Sigma, H6908) antibody bound to 20 μl Dynabeads Protein G (Novex, 10001D)). The proteins were separated by electrophoresis in a 10% SDS–PAGE gel and transferred to a PVDF membrane (Immobilon-P, IPVH00010). Immunoblots were performed using an anti-FLAG antibody (MBL, M185-3L, 1:2000). The bands were visualized on a Tanon-5200 Chemiluminescent Imaging System (Tanon Science and Technology).
Subcellular localization
The Pro35S:STM3-RFP-FLAG and Pro35S:J2-YFP-HA vectors were constructed with the CDSs of STM3 and J2 using MM cDNA as a template fused to the N-terminus of RFP-FLAG or YFP-HA, respectively. These plasmids were transformed into A. tumefaciens strain EHA105 and then co-infiltrated into the leaves of 4-wk-old N. benthamiana plants together with RNA silencing suppressor P19 (Lindbo 2007). The localization of the fluorescent fusion proteins was analyzed by confocal scanning laser microscopy analyses (Leica DM6 CS). For YFP, we captured the images with laser excitation at 514 nm and collection bandwidth range from 525 to 570 nm, and gain value of around 600. For RFP, we used 514 nm for excitation and collection bandwidth of 585 to 625 nm, gain of around 700, and intensity values of about 5.
Cell fractionation assay
The CDSs of STM3 and J2 were fused to the N-terminus of FLAG or YFP-HA tags to construct Pro35S:STM3-YFP-HA and Pro35S:J2-FLAG vectors. These plasmids were transformed into A. tumefaciens strain EHA105 and then infiltrated into the leaves of 4-wk-old N. benthamiana plants together with P19. Equal numbers of samples were collected 2 d after infiltration, ground in liquid nitrogen, and then homogenized in 20 ml of ice-cold nuclei isolation buffer (1 M sucrose, 10 mM HEPES (PH 7.6), 5 mM MgCl2, 5 mM KCl, 5 mM EDTA (PH 8.0), and 1 tablet/50 ml of protease inhibitor cocktail (Roche, 04693132001)). The solution was mixed sufficiently and then centrifuged at 1,260 ×g for 10 min at 4 °C. Discard the supernatant and resuspend the pellet in 300 μl of nuclei isolation buffer with 0.6% Triton-X-100 and then centrifuged at 12,000 × g for 10 min at 4 °C. Collect the supernatant (cytoplasm) and resuspend the pellet in 300 μl of nuclei isolation buffer with 0.6% Triton-X-100. Carefully lay the supernatant nuclei pellet on 500 μl of nuclei separation solution (1 M sucrose,10 mM HEPES (PH 7.6), 5 mM MgCl2, 5 mM KCl, 5 mM EDTA (PH 8.0), 15% (v/v) Percoll, and 1 tablet/50 ml of protease inhibitor cocktail) and centrifuge at 3,000 × g for 10 min at 4 °C. Remove supernatant, and the precipitate is the nucleus. The proteins were separated by 15% SDS–PAGE and transferred to a PVDF membrane (Immobilon-P, IPVH00010). J2-FLAG protein was detected with anti-FLAG antibody diluted 1:2000 (MBL, M185) and STM3-YFP-HA protein was detected with anti-HA (Sigma, H6908) antibody diluted 1:2000. Anti-HSP70 (Agrisera, AS08 371) and anti-H3 antibodies (Abcam, ab1791) were used as cytoplasmic and nuclear markers, respectively.
Accession numbers
The ChIP sequencing datasets generated in this study have been deposited in the Sequence Read Archive (SRA) under the accession numbers PRJNA666805 and PRJNA777648. Sequence data from this article can be found in the Sol Genomics Network data libraries under the following accession numbers: STM3, Solyc01g092950; TM3, Solyc01g093965; J2, Solyc12g038510; FUL1, Solyc06g069430; UBI3, Solyc01g056940; AP2c, Solyc02g093150; AP2a, Solyc03g044300; AG, Solyc02g091550; AGL104, Solyc04g078300; UBI3, Solyc01g056840. Other data supporting our findings are available in the manuscript file or from the corresponding author upon request.
Supplementary Material
Contributor Information
Xiaotian Wang, State Key Laboratory of Vegetable Biobreeding, Sino-Dutch Joint Laboratory of Horticultural Genomics, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China; Guangdong Laboratory for Lingnan Modern Agriculture, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, South China Agricultural University, Guangzhou 510642, China.
Zhiqiang Liu, State Key Laboratory of Vegetable Biobreeding, Sino-Dutch Joint Laboratory of Horticultural Genomics, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Jingwei Bai, State Key Laboratory of Vegetable Biobreeding, Sino-Dutch Joint Laboratory of Horticultural Genomics, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Shuai Sun, State Key Laboratory of Vegetable Biobreeding, Sino-Dutch Joint Laboratory of Horticultural Genomics, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Jia Song, State Key Laboratory of Vegetable Biobreeding, Sino-Dutch Joint Laboratory of Horticultural Genomics, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Ren Li, State Key Laboratory of Vegetable Biobreeding, Sino-Dutch Joint Laboratory of Horticultural Genomics, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Xia Cui, State Key Laboratory of Vegetable Biobreeding, Sino-Dutch Joint Laboratory of Horticultural Genomics, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China; Key Laboratory of Quality and Safety Control for Subtropical Fruit and Vegetable, Ministry of Agriculture and Rural Affairs, College of Horticulture Science, Zhejiang A&F University, Hangzhou 311300, China.
Author contributions
X.W. carried out most of the experiments; Z.L. analyzed the ChIP-seq data; J.B., S. S., J. S., and R.L. helped to observe the phenotypes; X.C. conceived the project and designed research; and X.W. and X. C. wrote the manuscript.
Supplemental data
The following materials are available in the online version of this article.
Supplemental Figure S1 . Analysis of binding loci and the recognized motifs by STM3 and J2.
Supplemental Figure S2 . The j2cr and ful1cr knockout mutants generated by CRISPR/Cas9-mediated genome editing.
Supplemental Figure S3 . The inflorescence phenotypes of STM3-overexpressing transgenic lines in PP.
Supplemental Figure S4 . Bimolecular Fluorescence Complementation (BiFC) visualization of the interaction between STM3M, STM3K, J2M, J2K and corresponding contrast in N. benthamiana epidermal cells.
Supplemental Figure S5 . Quantification of inflorescence branch number of RILs with 4 different genotypes, STM3 J2, STM3-dup J2, STM3 j2del, and STM3-dup j2del.
Supplemental Figure S6 . The stm3 tm3 knockout mutants generated by CRISPR/Cas9-mediated genome editing in PP.
Supplemental Figure S7 . Chromatin binding profiles of J2 and STM3 in the promoter of FUL2 and MBP20.
Supplemental Table S1 . The genotypes of critical genes about inflorescence branch development in our all materials.
Supplemental Table S2 . ChIP sequence count.
Supplemental Data Set 1 . List of ChIP-Seq peaks bound by STM3.
Supplemental Data Set 2 . List of ChIP-Seq peaks bound by J2.
Supplemental Data Set 3 . The common putative target genes bound by STM3 and J2.
Supplemental Data Set 4 . List of primers used in the work.
Supplemental Data Set 5 . Summary of statistical analyses.
Funding
This work was supported by the National Natural Science Foundation of China (31930099), the National Key Research and Development Program of China (2022YFF1003002), and the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.
Data availability
The ChIP sequencing datasets generated in this study have been deposited in the Sequence Read Archive (SRA) under the accession number PRJNA666805 and PRJNA777648. Other data supporting our findings are available in the manuscript file or from the corresponding author upon request.
References
- Acosta IF, Laparra H, Romero SP, Schmelz E, Hamberg M, Mottinger JP, Moreno MA, Dellaporta SL. Tasselseed1 is a lipoxygenase affecting jasmonic acid signaling in sex determination of maize. Science. 2009:323(5911):262–265. 10.1126/science.1164645 [DOI] [PubMed] [Google Scholar]
- Adamczyk BJ, Fernandez DE. MIKC* MADS domain heterodimers are required for pollen maturation and tube growth in Arabidopsis. Plant Physiol. 2009:149(4):1713–1723. 10.1104/pp.109.135806 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Allen KD, Sussex IM. Falsiflora and anantha control early stages of floral meristem development in tomato (Lycopersicon esculentum Mill.). Planta. 1996:200(2):254–264. 10.1007/BF00208316 [DOI] [Google Scholar]
- 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(1):145–161.e23. 10.1016/j.cell.2020.05.021 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Balanza V, Martinez-Fernandez I, Sato S, Yanofsky MF, Kaufmann K, Angenent GC, Bemer M, Ferrandiz C. Genetic control of meristem arrest and life span in Arabidopsis by a FRUITFULL-APETALA2 pathway. Nat Commun. 2018:9(1):565. 10.1038/s41467-018-03067-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bowman JL, Drews GN, Meyerowitz EM. Expression of the Arabidopsis floral homeotic gene AGAMOUS is restricted to specific cell types late in flower development. Plant Cell. 1991:3:749–758. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen X. A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science. 2004:303(5666):2022–2025. 10.1126/science.1088060 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cui J, You C, Zhu E, Huang Q, Ma H, Chang F. Feedback regulation of DYT1 by interactions with downstream bHLH factors promotes DYT1 nuclear localization and anther development. Plant Cell. 2016:28(5):1078–1093. 10.1105/tpc.15.00986 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Deng L, Wang H, Sun C, Li Q, Jiang H, Du M, Li C-B, Li C. Efficient generation of pink-fruited tomatoes using CRISPR/Cas9 system. J Genet Genomics. 2018:45(1):51–54. 10.1016/j.jgg.2017.10.002 [DOI] [PubMed] [Google Scholar]
- Dinh TT, Girke T, Liu XG, Yant L, Schmid M, Chen XM. The floral homeotic protein APETALA2 recognizes and acts through an AT-rich sequence element. Development. 2012:139(11):1978–1986. 10.1242/dev.077073 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eschenfeldt WH, Lucy S, Millard CS, Joachimiak A, Mark ID. A family of LIC vectors for high-throughput cloning and purification of proteins. Methods Mol Biol. 2009:498:105–115. 10.1007/978-1-59745-196-3_7 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gregis V, Sessa A, Dorca-Fornell C, Kater MM. The Arabidopsis floral meristem identity genes AP1, AGL24 and SVP directly repress class B and C floral homeotic genes. Plant J. 2009:60(4):626–637. 10.1111/j.1365-313X.2009.03985.x [DOI] [PubMed] [Google Scholar]
- Hajdukiewicz P, Svab Z, Maliga P. The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol. 1994:25(6):989–994. 10.1007/BF00014672 [DOI] [PubMed] [Google Scholar]
- Hake S. Inflorescence architecture: the transition from branches to flowers. Curr Biol. 2008:18(23):R1106–R1108. 10.1016/j.cub.2008.10.024 [DOI] [PubMed] [Google Scholar]
- Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, Cheng JX, Murre C, Singh H, Glass CK. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B Cell identities. Mol Cell. 2010:38(4):576–589. 10.1016/j.molcel.2010.05.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hendelman A, Zebell S, Rodriguez-Leal D, Dukler N, Robitaille G, Wu X, Kostyun J, Tal L, Wang P, Bartlett ME, et al. Conserved pleiotropy of an ancient plant homeobox gene uncovered by cis-regulatory dissection. Cell. 2021:184(7):1724–1739.e16. 10.1016/j.cell.2021.02.001 [DOI] [PubMed] [Google Scholar]
- Huang ZG, Shi T, Zheng BL, Yumul RE, Liu XG, You CJ, Gao ZH, Xiao LT, Chen XM. APETALA2 Antagonizes the transcriptional activity of AGAMOUS in regulating floral stem cells in Arabidopsis thaliana. New Phytol. 2017:215(3):1197–1209. 10.1111/nph.14151 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Immink RG, Pose D, Ferrario S, Ott F, Kaufmann K, Valentim FL, de Folter S, van der Wal F, van Dijk AD, 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(1):433–449. 10.1104/pp.112.202614 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jiang X, Lubini G, Hernandes-Lopes J, Rijnsburger K, Veltkamp V, de Maagd RA, Angenent GC, Bemer M. FRUITFULL-like genes regulate flowering time and inflorescence architecture in tomato. Plant Cell. 2022:34(3):1002–1019. 10.1093/plcell/koab298 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012:9(4):357–359. 10.1038/nmeth.1923 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee J, Lee I. Regulation and function of SOC1, a flowering pathway integrator. J Exp Bot. 2010:61(9):2247–2254. 10.1093/jxb/erq098 [DOI] [PubMed] [Google Scholar]
- Lee J, Oh M, Park H, Lee I. SOC1 translocated to the nucleus by interaction with AGL24 directly regulates leafy. Plant J. 2008:55(5):832–843. 10.1111/j.1365-313X.2008.03552.x [DOI] [PubMed] [Google Scholar]
- Lifschitz E, Ayre BG, Eshed Y. Florigen and anti-florigen—a systemic mechanism for coordinating growth and termination in flowering plants. Front Plant Sci. 2014:5:465. 10.3389/fpls.2014.00465 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lindbo JA. High-efficiency protein expression in plants from agroinfection-compatible Tobacco mosaic virus expression vectors. BMC Biotechnol. 2007:7(1):52. 10.1186/1472-6750-7-52 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lippman ZB, Cohen O, Alvarez JP, 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(11):e288. 10.1371/journal.pbio.0060288 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liu C, Chen H, Er HL, Soo HM, Kumar PP, Han JH, Liou YC, Yu H. Direct interaction of AGL24 and SOC1 integrates flowering signals in Arabidopsis. Development. 2008:135(8):1481–1491. 10.1242/dev.020255 [DOI] [PubMed] [Google Scholar]
- Liu C, Teo ZW, Bi Y, Song S, Xi W, Yang X, Yin Z, Yu H. A conserved genetic pathway determines inflorescence architecture in Arabidopsis and rice. Dev Cell. 2013:24(6):612–622. 10.1016/j.devcel.2013.02.013 [DOI] [PubMed] [Google Scholar]
- 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(2):284–296. 10.1111/tpj.12387 [DOI] [PubMed] [Google Scholar]
- Lu Q, Tang X, Tian G, Wang F, Liu K, Nguyen V, Kohalmi SE, Keller WA, Tsang EW, Harada JJ, et al. Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin. Plant J. 2010:61(2):259–270. 10.1111/j.1365-313X.2009.04048.x [DOI] [PubMed] [Google Scholar]
- MacAlister CA, Park SJ, Jiang K, Marcel F, Bendahmane A, Izkovich Y, Eshed Y, Lippman ZB. Synchronization of the flowering transition by the tomato TERMINATING FLOWER gene. Nat Genet. 2012:44(12):1393–1398. 10.1038/ng.2465 [DOI] [PubMed] [Google Scholar]
- Machanick P, Bailey TL. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics. 2011:27(12):1696–1697. 10.1093/bioinformatics/btr189 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mateos JL, Madrigal P, Tsuda K, Rawat V, Richter R, Romera-Branchat M, Fornara F, Schneeberger K, Krajewski P, Coupland G. Combinatorial activities of SHORT VEGETATIVE PHASE and FLOWERING LOCUS C define distinct modes of flowering regulation in Arabidopsis. Genome Biol. 2015:16(1):31. 10.1186/s13059-015-0597-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Muino JM, Smaczniak C, Angenent GC, Kaufmann K, van Dijk AD. Structural determinants of DNA recognition by plant MADS-domain transcription factors. Nucleic Acids Res. 2014:42(4):2138–2146. 10.1093/nar/gkt1172 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nakano T, Kimbara J, Fujisawa M, Kitagawa M, Ihashi N, Maeda H, Kasumi T, Ito Y. MACROCALYX And JOINTLESS interact in the transcriptional regulation of tomato fruit abscission zone development. Plant Physiol. 2012:158(1):439–450. 10.1104/pp.111.183731 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nayar S, Kapoor M, Kapoor S. Post-translational regulation of rice MADS29 function: homodimerization or binary interactions with other seed-expressed MADS proteins modulate its translocation into the nucleus. J Exp Bot. 2014:65(18):5339–5350. 10.1093/jxb/eru296 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Park SJ, Jiang K, Schatz MC, Lippman ZB. Rate of meristem maturation determines inflorescence architecture in tomato. Proc Natl Acad Sci USA. 2012:109(2):639–644. 10.1073/pnas.1114963109 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Park SJ, Jiang K, Tal L, Yichie Y, Gar O, Zamir D, Eshed Y, Lippman ZB. Optimization of crop productivity in tomato using induced mutations in the florigen pathway. Nat Genet. 2014:46(12):1337–1342. 10.1038/ng.3131 [DOI] [PubMed] [Google Scholar]
- Paul P, Dhatt BK, Miller M, Folsom JJ, Wang Z, Krassovskaya I, Liu K, Sandhu J, Yu H, Zhang C, et al. MADS78 And MADS79 are essential regulators of early seed development in rice. Plant Physiol. 2020:182(2):933–948. 10.1104/pp.19.00917 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ramírez F, Ryan DP, Grüning B, Bhardwaj V, Kilpert F, Richter AS, Heyne S, Dündar F, Manke T. Deeptools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 2016:44(W1):W160–W165. 10.1093/nar/gkw257 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smaczniak C, Immink RG, Angenent GC, Kaufmann K. Developmental and evolutionary diversity of plant MADS-domain factors: insights from recent studies. Development. 2012a:139(17):3081–3098. 10.1242/dev.074674 [DOI] [PubMed] [Google Scholar]
- Smaczniak C, Immink RG, Muiño JM, Blanvillain R, Busscher M, Busscher-Lange J, Dinh QD, Liu S, Westphal AH, Boeren S, et al. Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development. Proc Natl Acad Sci USA. 2012b:109(5):1560–1565. 10.1073/pnas.1112871109 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Song J, Zhang S, Wang X, Sun S, Liu Z, Wang K, Wan H, Zhou G, Li R, Yu H, et al. Variations in both FTL1 and SP5G, two tomato FT paralogs, control day-neutral flowering. Mol Plant. 2020:13(7):939–942. 10.1016/j.molp.2020.05.004 [DOI] [PubMed] [Google Scholar]
- Soyk S, Lemmon ZH, Oved M, Fisher J, Liberatore KL, Park SJ, 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(6):1142–1155.e12. 10.1016/j.cell.2017.04.032 [DOI] [PubMed] [Google Scholar]
- Soyk S, Lemmon ZH, Sedlazeck FJ, Jimenez-Gomez JM, Alonge M, Hutton SF, Van Eck J, Schatz MC, Lippman ZB. Duplication of a domestication locus neutralized a cryptic variant that caused a breeding barrier in tomato. Nat Plants. 2019:5(5):471–479. 10.1038/s41477-019-0422-z [DOI] [PubMed] [Google Scholar]
- Tang W, Perry SE. Binding site selection for the plant MADS domain protein AGL15: an in vitro and in vivo study. J Biol Chem. 2003:278(30):28154–28159. 10.1074/jbc.M212976200 [DOI] [PubMed] [Google Scholar]
- Tao Q, Guo D, Wei B, Zhang F, Pang C, Jiang H, Zhang J, Wei T, Gu H, Qu LJ, et al. The TIE1 transcriptional repressor links TCP transcription factors with TOPLESS/TOPLESS-RELATED corepressors and modulates leaf development in Arabidopsis. Plant Cell. 2013:25(2):421–437. 10.1105/tpc.113.109223 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tao Z, Shen L, Liu C, Liu L, Yan Y, Yu H. Genome-wide identification of SOC1 and SVP targets during the floral transition in Arabidopsis. Plant J. 2012:70(4):549–561. 10.1111/j.1365-313X.2012.04919.x [DOI] [PubMed] [Google Scholar]
- Teo ZW, Song S, Wang YQ, Liu J, Yu H. New insights into the regulation of inflorescence architecture. Trends Plant Sci. 2014:19(3):158–165. 10.1016/j.tplants.2013.11.001 [DOI] [PubMed] [Google Scholar]
- 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(1):251. 10.1038/s41438-021-00677-x [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang B, Smith SM, Li J. Genetic regulation of shoot architecture. Annu Rev Plant Biol. 2018:69(1):437–468. 10.1146/annurev-arplant-042817-040422 [DOI] [PubMed] [Google Scholar]
- Wollmann H, Mica E, Todesco M, Long JA, Weigel D. On reconciling the interactions between APETALA2, miR172 and AGAMOUS with the ABC model of flower development. Development. 2010:137(21):3633–3642. 10.1242/dev.036673 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Xu C, Liberatore KL, MacAlister CA, Huang Z, Chu YH, Jiang K, Brooks C, Ogawa-Ohnishi M, Xiong G, Pauly M, et al. A cascade of arabinosyltransferases controls shoot meristem size in tomato. Nat Genet. 2015:47(7):784–792. 10.1038/ng.3309 [DOI] [PubMed] [Google Scholar]
- Yuste-Lisbona FJ, 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(1):18796. 10.1038/srep18796 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang S, Jiao Z, Liu L, Wang K, Zhong D, Li S, Zhao T, Xu X, Cui X. Enhancer-promoter interaction of SELF PRUNING 5G shapes photoperiod adaptation. Plant Physiol. 2018:178(4):1631–1642. 10.1104/pp.18.01137 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, Nusbaum C, Myers RM, Brown M, Li W, et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 2008:9(9):R137–R137. 10.1186/gb-2008-9-9-r137 [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The ChIP sequencing datasets generated in this study have been deposited in the Sequence Read Archive (SRA) under the accession number PRJNA666805 and PRJNA777648. Other data supporting our findings are available in the manuscript file or from the corresponding author upon request.