Constructions and transformation of HdSTM in H. difformis

To generate the HdSTM-RNAi construct, the 271-bp sense and antisense fragments from the 5′-end of HdSTM cDNA (5–275 bp) were amplified using gene-specific primers containing XhoI (5′-end)/KpnI (3′-end) and XbaI (5′-end)/HindIII (3′-end) sites. The two fragments were separately inserted into the pHANNIBAL vector (Wesley et al., 2003). The entire RNAi cassette was subcloned into the pCAMBIA1300 binary vector through the SacI and PstI cleavage sites to construct the HdSTM-RNAi plasmid. The recombinant HdSTM-RNAi construct was transferred into Agrobacterium strain LBA4404 and transformed into H. difformis (Li et al., 2020). The full-length coding sequence of HdSTM was amplified and cloned into the pMYC vector (Heenatigala et al., 2020) through the PstI and SalI cleavage sites to generate the overexpression construct (35S::HdSTM). The 35S::HdSTM constructs were transformed into H. difformis (Li et al., 2020). Primer information is given in Supplemental Table S2.

For morphological analysis, leaves were photographed with a Canon EOS80D camera, and all light microscopy observations were performed under a Sunny EX20 light microscope and photographed with a ToupCam TP605100A digital camera. The images were integrated using MvImage media software (ToupCam). Leaf complexity was estimated based on the DI, calculated as previously described (Li et al., 2017). All calculations were performed using ImageJ 1.47v (http://rsb.info.nih.gov/ij/). Statistical differences were determined using Student’s t test.