To identify the LBD proteins in the genome of D. catenatum, a local BLAST search of the hidden Markov models (HMMs) of the SMART and Pfam databases was conducted. In total, 24 LBD genes from the complete D. catenatum genome were isolated. All of the deduced LBD proteins possessed a conserved LOB domain. We reconstructed a phylogenetic tree from an alignment of LBD protein sequences from Arabidopsis thaliana and D. catenatum. The DcaLBD genes were annotated based on the similarity of the protein sequences to those of A. thaliana (Figure 1).

On the basis of the phylogenetic analysis, the LBD proteins of D. catenatum were resolved into two monophyletic groups (classes I and II), of which 21 LBD proteins belonged to class I and three LBD proteins belonged to class II. Sequence analysis revealed that AtLBD1, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 23, 25, 36, 37, 38, 41, and LOB have orthologs in D. catenatum of which DcaLBD10, 12, and 22 comprised multiple orthologs.

The length of the DcaLBD proteins ranged from 148 to 307 aa, the molecular weight ranged from 17.06 to 34.33 KDa, the theoretical isoelectric point value ranged from 4.61 to 9.33, and the calculated grand average of hydrophobicity value ranged from −0.529 to 0.114. Most of the proteins were hydrophobic with the exception of DcaLBD4 and DcaLBD16 (Table 1).

To further explore the diversity of LBD genes in D. catenatum, the conserved motifs of the DcaLBD proteins were analyzed. A neighbor-joining (NJ) tree was constructed based on a multiple alignment of the DcaLBD protein sequences. The MEME online tool was used to predict the conserved motif composition of the DcaLBD proteins. The number of motifs ranged from three to seven. A LOB domain, motif 2, was detected in all DcaLBDs. In addition, motif 3 was detected in all class I members, indicating that the class I proteins have a specific structure (Figure 2).

On this basis, we further analyzed the DcaLBD protein sequences using the DNAMAN tool. The multiple sequence alignment indicated that a sequence comprising more than 100 amino acids was conserved in all DcaLBDs (Figure 3). For the class I proteins, a string consisting of a C-block, GAS-block, and L-rich block was detected. The C-block in DcaLBDs can be summarized as CX₂CX₆CX₃C. The GAS-block began with a FX₂V/AH motif and ended with a DPV/IYG motif. All class II DcaLBDs contained the conserved C-block similar to class I proteins and lacked the GAS-block and Leu-zipper-like domain.

We predicted that the LBD gene family were transcription factors localized in the nucleus to play roles in regulating plant growth and development. To support this hypothesis, we conducted a subcellular localization analysis for DcaLBD6 and DcaLBD18 from class I and DcaLBD37 and DcaLBD41 from class II. The respective DcaLBD gene was fused with the green fluorescent protein (GFP) gene and co-injected into Nicotiana benthamiana leaves. The transfected plants were cultured in a greenhouse for 3 days. The fluorescence in the injected leaves was observed under a microscope (Olympus FV1000, Tokyo, Japan) after 4′,6-diamidino-2-phenylindole (DAPI) injection. Fluorescent signal from the DcaLBD–GFP protein was detected in the nucleus, consistent with the proteins functioning as transcription factors (Figure 4).

Previous studies have shown that members of the LBD family have a positive feedback-regulatory effect on the master regulator VASCULAR-RELATED NAC-DOMAIN 7 (VND7) [27]. Therefore, we speculated that the DcaLBD family members may also regulate the expression of VND7. To test this hypothesis, we co-injected the ProAtVND7:LUC promoter with class I (35S:DcaLBD6 and 35S:DcaLBD18) or class II (35S:DcaLBD37 and 35S:DcaLBD41) gene constructs (Figure 5). When the ProAtVND7:LUC reporter plasmid was co-injected with the 35S:DcaLBD37 or 35S:DcaLBD41 effector plasmid, intense luciferase (LUC) fluorescence signal was detected. These results indicated that DcaLBD37 and DcaLBD41 directly activated expression of AtVND7. In contrast, when the ProAtVND7:LUC reporter plasmid was co-injected with the 35S:DcaLBD6 effector plasmid, faint LUC fluorescence signal was detected, but when injected without the effector plasmid the LUC fluorescence signal was weaker. Thus, DcaLBD6 restrained expression of AtVND7. Co-injection of the ProAtVND7:LUC reporter plasmid with the 35S:DcaLBD18 effector plasmid resulted in LUC fluorescence signal similar to that of the ProAtVND7:LUC. These results are consistent with previous studies of LBD family members in suggesting that DcaLBD family members exhibit different functions or even antagonistic roles [28].

To investigate the function of LBD genes in D. catenatum, we used transcriptomic data to determine changes in the expression of the DcaLBD family genes under different phytohormone treatments.

DcaLBD13 showed the highest relative expression level without treatment (Figure 6). The expression level of DcaLBD13 decreased to varying degrees under the various phytohormone treatments. After treatment with indole-3-acetic acid (IAA) for 3 h, DcaLBD6 showed the highest expression level. After treatment with IAA for 6 h, the expression level of DcaLBD18 was the highest. Among class II genes, DcaLBD38 showed the highest expression level without treatment. After treatment with jasmonic acid (JA) for 3 h, the expression level of DcaLBD38 was increased. The expression levels of DcaLBD37 were increased after phytohormone treatment. DcaLBD41 showed the highest expression level after treatment with salicylic acid (SA) for 3 h.

Heat maps were generated to indicate the relative expression of the DcaLBD family members in different organs (Figure 7). Among class I genes, DcaLBD22b showed the highest expression level in the pollinium. DcaLBD6 was more highly expressed in the pollinium than in other organs. The expression of DcaLBD18 was higher in the white portion of the root than in other organs. Among class II genes, DcaLBD38 showed the highest expression level in the gynostemium. The expression level of DcaLBD37 and DcaLBD41 in the pollinium and green root tip were higher than that in other organs.