3.4 Transcriptomic and metabolomic differentiation between CH and CL flowers

In addition to open and closed pollination of these two types of flowers, CH and CL flowers also have contrasting shapes, colors, and nectaries (L. Yang et al., 2011). The MADS-box gene family plays an essential role in floral organ development in all angiosperms (Ng & Yanofsky, 2001). We first examined their expressions in both dimorphic flowers. A total of 59 MADS-box genes were identified in S. tetraptera . They could further be classified into 12 clades, covering all of the identified clades inO. sativa and A. thaliana (Figure 3a, Figure S20) (Arora et al., 2007; Par̆enicová et al., 2003). Compared to O. sativa and A. thaliana , a conserved copy number of A-, B-, C- and E-class genes were detected inS. tetraptera , including six copies of AP1 s (class A), two copies of AP3 s and one copy of PI (class B), three copies of AG s (class C), and one copy of SEP1 and SEP3(class E) (Figure 3a). Their conserved copy numbers indicated that they may play a key role in floral organ development (ES & EM, 1991; van Tunen, Eikelboom, & Angenent, 1993). However, the genes of theAGL15/18 clade were highly expanded in S. tetraptera(Figure 3a) and a total of 21 more copies were identified from the tandem duplication.
Gibberellin (GA), Jasmonate acid (JA), and auxin (IAA) have been reported to play essential roles in regulating flower development (Ishiguro, Kawai-Oda, Ueda, Nishida, & Okada, 2001; Jibran, Tahir, Cooney, Hunter, & Dijkwel, 2017; Nagpal et al., 2005; Teotia & Tang, 2015). Our metabolomics analysis revealed the content of each differed significantly between CH and CL flowers (Figure 3b, Table S23). The contents of GA and JA in CHs were both significantly higher than those in CLs. We further assessed the expression level of genes related to GA, JA, and IAA regulated pathways, many of which belong to the MADS-box gene family (Table S24). AGAMOUS (AG ) (E class of MADS-box gene family) can bind to the promoter of DEFECTIVE IN ANTHER DEHISCENCE 1 (DAD1 ) and further positively regulate the content of JA (Ito et al. , 2007; Hu et al. , 2017). Both AGand DAD1 were highly expressed in CH flowers, probably contributing to their significantly higher JA content (Figure 3c). JA is probably involved in flowering by regulating AGL15/18 genes (MADS-box gene family) (Ishiguro et al., 2001; Jibran et al., 2017).
AGL15/18 genes have been reported to promote the expression of Gibberellin 2-oxidase 6 (GA2ox6 ) (Zheng, Zheng, Ji, Burnie, & Perry, 2016) and directly reduce the level of bioactive GAs by catalyzing their immediate precursors or inactive forms (Y.-X. Hu et al., 2017). The overexpression of AGL15 can delay blooming inA. thaliana (Adamczyk, Lehti-Shiu, & Fernandez, 2007). We foundAGL15 and GA2ox6 had lower expressions in CH flowers (Figure 3c), in which a substantially higher concentration of GA accumulated than in CL flowers (Figure 3b). GA may induce flowering by up-regulating SQUAMOSA promoter binding protein-like3(SPL3 ), SPL4 , and SPL5 genes and further promote flowering by targeting with FRUITFUL (FUL ), LEAFY (LFY) , and APETALA1 (AP1 ). FUL (A class of MADS-box gene family) can also promote the expression of SPL4 to control flower formation (Torti et al., 2012). These genes (SPL3 ,SPL4 , SPL5 , AP1 , LFY , and FUL ) were highly expressed in CH flowers (Figure 3c). In addition, the dosage ofAUXIN RESPONSE FACTOR6 (ARF6 ) and ARF8 could quantitatively affect the timing of flower maturation by regulating JA accounts (Nagpal et al., 2005). They are inhibited by AGL15/18and increased IAA (Yang et al., 2006; Zheng et al., 2016), while IAA could also delay flowering (Ke et al., 2018; Lu et al., 2018). The expression of ARF6/8 is also consistent with the IAA content in CL flowers (Figure 3b and 3c), which suggests that IAA may play a role in flower blooming through ARF6/8 .
Moreover, CH flowers are significantly larger than CLs (P <0.01) (Figure 4a, Table S25). Cytokinin (CTK) may regulate floral organ size by catalyzing itself with cytokinin oxidase/dehydrogenase 3 (CKX3 ) and CKX5 (Bartrina, Otto, Strnad, Werner, & Schmülling, 2011). We found both CTK content and the expression level of CKX3 and CKX5 were distinctly different between CH and CL flowers. A significantly higher CTK content was detected in CH flowers than in CLs (Figure 4a). The expression levels of CKX3 and CKX5 in CH flowers were lower than those in CLs (Figure 4b). These differences may contribute to the contrasting sizes of CH and CL flowers.
In addition, the petals of CH flowers are more colorful and brighter than CLs to attract pollinators to S. tetraptera (He et al., 2013). The gene expression levels involved in biosynthesized carotenoids, anthocyanin, and flavonoids generally engaged in petal coloring were obviously different between the two dimorphic flowers, with high expression in CHs (Figure 4c and Table S26). Furthermore, petals of CH flowers have nectaries (Figure 4d), which secrete nectar to attract insects for pollination (He et al., 2013). We identified thesugars-will-eventually-be-exported-transporters (SWEET ) gene family in the S. tetrapteragenome (Figure S21), and it has been suggested that the SWEET9 homolog is a significant sugar efflux transporter in plants (Lin et al., 2014). Most SWEET genes (including SWEET9 ) in S. tetraptera showed a higher expression level in CH than CL flowers (Figure 4d). However, threeSWEET genes were highly expressed in CLs (Figure 4d). In fact, two of them, SWEET17 and SWEET2, exhibited high expression levels in all tissues (Figure 4d; Figure S22), indicating that these two genes may be involved in the sugar efflux transporters in the whole plant (Guo et al., 2014; Klemens et al., 2013). The third gene, researched on SWEET8 , was highly expressed in pistil donor ligand (S.-Y. Kim, Yu, Hong, Woo, & Ahn, 2013), which may contribute to the potential pistil differences between CH and CL flowers in S. tetraptera (Figure 4b; Figure S22). Previous studies have reported thatblock of cell proliferation 1 (BOP1 ), BOP2, andCRABS CLAW (CRC ) are involved in nectary development (Kram & Carter, 2009). All of these genes were highly expressed in CHs, suggesting their essential roles in the nectary development of these flowers (Figure 4d, Table S24).