Identification of the slf mutant
In order to study the mechanism regulating floral organ identity in tomato, we screened tomato EMS-mutant library and identified a mutant (TOMJPG2637-1) with identity defects in stamens and pistils, while the identity and number of sepals and petals were unchanged compared with wild type (WT) (Fig. 1a-c, e, g). In the mutant, stamens showed severely carpelloid in the third whorl (Fig. 1a). From the longitudinal sections, we verified that the pistil-like structures were formed in the third whorl (Fig. 1a, b). Only a few flowers (~22.95%) have stamen-like structure remaining in the third whorl of the mutant based on the transverse sections of flowers (Fig. 1b, d). The carpelloid stamens of the mutant developed into irregular fruits with more locules and the vestigial stamen structures were later formed radial cracks on the fruit surface (Fig. 1c). As this phenotype is similar to previously reported stamenless mutant [11, 12], we thus named the mutant stamenless like flower (slf).
Besides, our histological analyses showed that new shoot/floral meristems instead of carpel primordium formed in the fourth whorl of the mutant (Fig1. g). The ectopic shoot/floral meristem in the slf mutant produced ectopic aberrant foliage and flowers in the fourth whorl, indicating that the normal floral determinacy was lost (Fig1. g). As a result, the carpelloid stamen in slf mutant developed into the parthenocarpic fruit without seeds (Fig. 1c, d, Fig. S1c, d). Interestingly, in the carpal-like structure, the ovule development seemed normal in slf mutant. Therefore, we attempted to use WT pollen grains for the cross-pollination in slf mutant, and only small amount of seeds were obtained. This result may be due to the abnormal pistil-like structures that hindered the pollen-ovule process (Fig. S 1e, g). All these results indicated that the slf mutant was almost sterile.
SlGT11 gene encodes a regulator involved in floral organ identity
To identify the causal gene in slf mutant, we first conducted a genetic analysis by crossing the mutant to the WT. In the F2 segregated population, we found 92 progenies resembling the WT and 28 progenies with slf phenotypes, which were close to the 3:1 Mendelian segregation rule, indicating that the phenotypes in slf were caused by a recessive mutation at a single locus. Through bulked segregant analysis sequencing (BSA-Seq), we identified a signal peak on chromosome 3 (Fig. 2a). Further SNP analysis assigned the causal mutation to the gene Solyc03g006900 which encodes a nucleus-localized Trihelix transcription factor named SlGT11 previously , containing a putative GT1 DNA-binding domain and a PKc kinase domain (Fig. S3). The A to T substitution at the 2195bp position identified forms the termination codon TAG and mRNA level of the SlGT11 gene in the mutant was significantly decreased (Fig. 2b, f). Further sequencing analyses verified that the base substitution occurred in all 28 F2 progenies with slf phenotypes (Fig. 2c). Subcellular localization in tobacco leaves showed that SlGT11-GFP was located in the nucleus, consistent with the presence of DNA-binding domain (Fig. 2d).
To further verify the SlGT11 function, we transformed WT tomato with an RNA interference (RNAi) plasmid targeting the C-terminus of the SlGT11. The phenotypes of 5 independent transgenic RNAi lines were consistent with the slf mutant. qRT-PCR verified the significant reduced expression of SlGT11 in RNAi lines (Fig. 2f). The observed phenotypes including carpelloid stamens in the third whorl and new meristem formation from the fourth whorl in RNAi lines (#1 and #6) indicated that SlGT11 was the gene causing the developmental defects of stamens and carpels in slf (Fig. 2e). In addition, abnormal fruits were also found in transgenic lines #1 and #6, indicating that SlGT11 plays an important role in regulating the floral identity and floral meristem termination (Fig. 2g).
Phylogenetic analysis showed that all the SlGT11 homologous genes in solanaceae were grouped into the same cluster, while Arabidopsis homologous gene At5g51800 belonged to a less related cluster (Fig. S2). Consistent with this phylogenetic distance, At5g51800 mutation does not cause the similar floral phenotype, indicating the function of this gene is not completely conserved among different species. The comparative analysis of the amino acid sequence of SlGT11 in solanaceae showed that the N-terminal GT1 domain and the C-terminal PKc kinase domain are highly conserved (Fig. S3).
Spatial and temporal expression pattern of SlGT11 in tomato
To examine the expression pattern of SlGT11, we performed qRT-PCR in different tomato tissues including roots, hypocotyls, cotyledons, stems, leaves, flowers and fruits. The expression of SlGT11 was highly enriched in the flowers (Fig. 3a). Then RNA was extracted from different parts of flowers at anthesis for qRT-PCR and we found that SlGT11 was predominantly expressed in stamens, indicating that SlGT11 could be important for stamen development (Fig. 3b). Furthermore, we analyzed the temporal expression trend of SlGT11 during the floral development. qRT-PCR showed that SlGT11 expression was time-specific, with high expression levels from 6 days to 2 days before flowering (at stage12-18) (Fig. 3c).
Furthermore, we constructed a GUS reporter driven by SlGT11 promoter and transformed it into WT tomato (Fig. 3d). GUS staining showed that SlGT11 was expressed throughout the early stages of flowers and the expression became more specific to the stamen and carpel in later stages (Fig. 3e). The expression pattern of SlGT11 in inner two whorls of flower implies that it is probably involved in the regulation of tomato stamen and carpel development.
Stamen defects occur at the early stage
To investigate how SlGT11 affects stamens and carpels at different floral developmental stages , we used scanning electron microscopy (SEM) to visualize the floral development in WT, slf and SlGT11 RNAi line 6 (Fig. 4a-o). The early stages (before the stage 3 when sepal primordia and petal primordia were initiated) of floral development in slf and SlGT11 RNAi line 6 appeared to be similar to that of the WT (Fig. 4a-f). At stage 5, the differences between WT and slf or SlGT11 RNAi line 6 became more prominent. In the WT, six stamen primordia and one carpel primordium with four locules were initiated in the third and fourth whorl respectively (Fig. 4g). In contrast, the third and fourth floral organ primordia in the slf and SlGT11 RNAi line 6 were initiated in disorder (Fig. 4h, j). The defective floral organ identity became more severe in slf and SlGT11 RNAi line 6 at stages 6 and 9 (Fig. 4j, m). In the mutant, most stamens were transformed into carpel-like structures, and some ectopic meristems were produced in the central area of the flower (Fig. 4k, l, n and o). Combined with the spatiotemporal expression, we concluded that SlGT11 plays an essential role in the early development of floral organs.
Expression of floral development genes in slf mutant
Since the defects of stamens and carpels occurred at the early stages, we compared the expression of BCE genes that were previously reported to affect stamen and carpel identity in the floral buds at stage 1-6 between WT and slf . Consistent with the phenotypes, the BCE genes showed the distinct expression pattern between WT and slf mutant. Class B genes TAP3, TPI and TPIB, class C genes TAG1, TAGL1 and class E gene TM29, were all significantly down-regulated in slf. However, the expression level of the B-class gene TM6 and E class gene TM5 were not significantly affected in slf during the ﬂoral development (Fig. 5a).
We next analyzed the expression levels of some regulators involved in floral meristem identity and floral meristem termination. Since the ectopic floral meristem was repeatedly emerged in the later stages of floral development(Fig. 1g), we chose a set of essential genes for floral development including SlWUS, SlKNU, SlCLV3, SlCLV1, SlCLV2, FALSIFLORA (FA), SlULT1-like and SlRBL-like for transcriptional analysis at the later floral stage (stage 20). FA and SlWUS were up-regulated in slf, while SlKNU, SlCLV3, SlCLV1 and SlULT1-like appeared to be down-regulated in slf flowers (Fig. 5b, c). These results were consistent with the floral meristem termination defects in slf mutant.
High temperature inhibits the expression of SlGT11 and TM29
During the cultivation in the greenhouse where the temperature in summer was higher than the standard, we found that the phenotypes of slf became more severe, with stamens hardly visible and the defective flowers with ectopic floral meristem dramatically increased. As the temperature was reported previously to play a role in the floral development , we tested whether the SlGT11 function is also affected by temperature. To that end, we germinated the WT and slf mutant seeds at 25 °C for 4 weeks, and grew them in a heated incubator (37 °C daytime/ 28 °C at night) for 20 days. The flowers produced by the slf mutant grown at the higher temperature had more carpelloid structures and no stamen-like structures in the third whorl was visible (Fig. 6d). In addition, the petals seemed to partially acquire sepals identity by forming greenish petals with sepal structure (Fig. 6f). Furthermore, we found shoot/floral meristems were produced at the center of the mature flowers (Fig. 6d). Despite the carpelloid stamens and ectopic shoot/floral meristems were also produced in slf flowers at lower temperature (25 °C daytime/22 °C night), their occurrence frequency became significantly higher at higher temperature.
To further dissect the influence of the higher temperature on SlGT11, we performed qRT-PCR to analyze potential transcriptional change. The floral buds at early stages of WT and slf mutant grown at higher temperature were collected for RNA extraction and qRT-PCR. Our results showed that SlGT11 expression was inhibited by the higher temperature (Fig. 6g). We then examined the expression levels of BCE genes at 3 h, 7 h and 24 h after the high temperature treatment. Our results showed that E class gene TM29 was further significantly down-regulated by high temperature in slf mutant (Fig. 6h, i). Yeast-one-hybrid assay failed to detect the direct binding of SlGT11 to the TM29 promoter region. All results (Fig.5, Fig.6 g-i) indicate that SlGT11 indirectly activates TM29 transcription, and the high temperature further represses the transcription of SlGT11 and TM29 both in WT and slf.