The WRKY gene family plays important roles in plant growth and development, as well as in the responses to biotic and abiotic stresses. Loropetalum chinense var. rubrum has high ornamental and medicinal value. However, few WRKY genes have been reported in this plant, and their functions remain unknown. To explore the roles that the WRKY genes play in L. chinense var. rubrum, we identified and characterized 79 LcWRKYs through BLAST homology analysis and renamed them (as LcWRKY1–79) based on their distribution on the chromosomes of L. chinense var. rubrum. In this way, according to their structural characteristics and phylogenetic analysis, they were divided into three groups containing 16 (Group I), 52 (Group II), and 11 (Group III) WRKYs, respectively. LcWRKYs in the same group have similar motifs and gene structures; for instance, Motifs 1, 2, 3, 4, and 10 constitute the WRKY domain and zinc-finger structure. The LcWRKY promoter region contains light response elements (ACE, G-box), stress response elements (TC-rich repeats), hormone response elements (TATC-box, TCA-element), and MYB binding sites (MBS, MBSI). Synteny analysis of LcWRKYs allowed us to establish orthologous relationships among the WRKY gene families of Arabidopsis thaliana, Oryza sativa, Solanum lycopersicum L., Vitis vinifera L., Oryza sativa L., and Zea mays L.; furthermore, analysis of the transcriptomes of mature leaves and flowers from different cultivars demonstrated the cultivar-specific LcWRKY gene expression. The expression levels of certain LcWRKY genes also presented responsive changes from young to mature leaves, based on an analysis of the transcriptome in leaves at different developmental stages. White light treatment led to a significant decrease in the expression of LcWRKY6, 18, 24, 34, 36, 44, 48, 61, 62, and 77 and a significant increase in the expression of LcWRKY41, blue light treatment led to a significant decrease in the expression of LcWRKY18, 34, 50, and 77 and a significant increase in the expression of LcWRKY36 and 48. These results enable a better understanding of LcWRKYs, facilitating the further exploration of their genetic functions and the molecular breeding of L. chinense var. rubrum.

The basic helix–loop–helix (bHLH) transcription factor family is the second-largest transcription factor family in plants. Members of this family are involved in the processes of growth and development, secondary metabolic biosynthesis, signal transduction, and plant resistance. Loropetalum chinense var. rubrum is a critical woody plant with higher ornamental and economic values, which has been used as ornamental architecture and traditional Chinese herbal medicine plants. However, the bHLH transcription factors in Loropetalum chinense var. rubrum (L. chinense var. rubrum) have not yet been systematically demonstrated, and their role in the biosynthesis of anthocyanin is still unclear. Here, we identified 165 potential LcbHLHs genes by using two methods, and they were unequally distributed on chromosomes 1 to 12 of the genome of L. chinense var. rubrum. Based on an evolutionary comparison with proteins from Arabidopsis and Oryza sativa, these bHLH proteins were categorized into 21 subfamilies. Most LcbHLHs in a particular subfamily had similar gene structures and conserved motifs. The Gene Ontology annotation and Cis-elements predicted that LcbHLHs had many molecular functions and were involved in processes of plant growth, including the biosynthesis of flavonoids and anthocyanins. Transcriptomic analysis revealed different expression patterns among different tissues and cultivars of L. chinense var. rubrum. Many LcbHLHs were expressed in the leaves, and only a few genes were highly expressed in the flowers. Six LcbHLHs candidate genes were identified by bioinformatics analysis and expression analysis. Further Real-time quantitative PCR analysis and protein interaction network analysis showed that LcbHLH156, which is one of the candidate proteins belonging to the IIIf subfamily, could interact with proteins related to anthocyanin synthesis. Therefore, LcbHLH156 was transiently expressed in L. chinense var. rubrum to verify its function in regulating anthocyanin synthesis. Compared with the control group, red pigment accumulation appeared at the wound after injection, and the total anthocyanin content increased at the wound of leaves. These results lay a foundation for the research of the regulation mechanism of leaf colors in L. chinense var. rubrum and also provide a basis for the function of the LcbHLH family.

Light quality is a vital environmental signal used to trigger growth and to develop structural differentiation in plants, and it influences morphological, physiological, and biochemical metabolites. In previous studies, different light qualities were found to regulate the synthesis of anthocyanin. However, the mechanism of the synthesis and accumulation of anthocyanins in leaves in response to light quality remains unclear. In this study, the Loropetalum chinense var. rubrum “Xiangnong Fendai” plant was treated with white light (WL), blue light (BL), ultraviolet-A light (UL), and blue light plus ultraviolet-A light (BL + UL), respectively. Under BL, the leaves were described as increasing in redness from “olive green” to “reddish-brown”. The chlorophyll, carotenoid, anthocyanin, and total flavonoid content were significantly higher at 7 d than at 0 d. In addition, BL treatment also significantly increased the accumulation of soluble sugar and soluble protein. In contrast to BL, ultraviolet-A light increased the malondialdehyde (MDA) content and the activities of three antioxidant enzymes in the leaves, including catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), in varying degrees over time. Moreover, we also found that the CRY-like gene, HY5-like gene, BBX-like gene, MYB-like gene, CHS-like gene, DFR-like gene, ANS-like gene, and UFGT-like gene were significantly upregulated. Furthermore, the SOD-like, POD-like, and CAT-like gene expressions related to antioxidase synthesis were found under ultraviolet-A light conditions. In summary, BL is more conducive to reddening the leaves of “Xiangnong Fendai” and will not lead to excessive photooxidation. This provides an effective ecological strategy for light-induced leaf-color changes, thereby promoting the ornamental and economic value of L. chinense var. rubrum.

The basic helix-loop-helix (bHLH) transcription factor family is the second-largest transcription factor in plants. Members of this family are involved in the process of growth and development, secondary metabolic biosynthesis, signal transduction, and plant resistance. Loropetalum chinense var. rubrum is a critical woody plant with higher ornamental and economic values, which used as ornamental architecture and traditional Chinese herbal medicine plant. However, the bHLH transcription factor in the L. chinense var. rubrum (Loropetalum chinense var. Rubrum) have yet to be systematically demonstrated, and their role in anthocyanin biosynthesis remains secret. Here, we identified 165 potential LcbHLHs genes by two methods, and they were unequally distributed on chromosomes 1 to 12 of the L. chinense genome. Based on a phylogenetic comparison with proteins from Arabidopsis, these bHLH proteins were categorized into 21 subfamilies. Most LcbHLHs in a given subfamily had similar gene structures and conserved motifs. The gene ontology annotation and cis-elements predicted that the LcbHLHs had many molecular functions and were involved in plant growth processes, including flavonoid and anthocyanin biosynthetic processes, etc. Transcriptomic analysis revealed different expression patterns among different tissues and cultivars in L. chinense. Many LcbHLHs were expressed in leaves, and only a few genes were highly expressed in flowers. Six LcbHLH genes differentially expressed between species and periods in color variation, which may play a key role in anthocyanin synthesis. These further functional predictions of LcbHLHs were established by quantitative real-time PCR analysis and protein-protein interaction network. This study identified the six potential bHLH genes related to anthocyanin in L. chinense and created a solid framework for future research on the function and evolution of bHLHs.

Loropetalum chinense is a significant small tree and ornamental shrub known for its colorful foliage and is widely used in landscaping in tropical and subtropical regions. This study aimed to establish an efficient, tissue culture-independent genetic transformation system for L. chinense. Cuttings from two varieties, ‘Xiangnong Xiangyun’ and ‘Hei Zhenzhu’, were infected with different strains of Agrobacterium rhizogenes. The results showed that the K599 strain significantly induced hairy roots in both varieties, with ‘Xiangnong Xiangyun’ demonstrating a higher survival rate (60%), rooting rate (51.66%), and hairy root induction efficiency (45%) compared to ‘Hei Zhenzhu’. Based on these findings, ‘Xiangnong Xiangyun’ and the K599 strain were selected for further optimization through an orthogonal L9 (3³) experiment, which focused on optimizing the infection solution composition, bacterial concentration, and infection duration, Finally, the genetic transformation system established at the beginning of the experiment was validated on ‘Xiangnong Xiangyun’ plants using the pre-screening LcDREB-43 gene of our group. Among these factors, infection duration was identified as the most influential for improving transformation efficiency. The optimal conditions were determined as an infection solution containing MES solution, a bacterial concentration of OD₆₀₀ = 0.8, and a 15 min infection duration. Under these optimized conditions, the survival rate, rooting rate, induction efficiency, and transformation efficiency reached 86.67%, 70%, 61.67%, and 43.33%, respectively. Furthermore, the transgenic plants with LcDREB-43 overexpression and pCAMBIA1305-GFP were obtained through the established transformation system, the authenticity of the system was proved, and the production application was carried out through phenotypic observation, molecular identification, and auxiliary verification of physiological indicators.