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Abstract Background Liriodendron chinense is a distinctive ornamental tree species due to its unique leaves and tulip-like flowers. The discovery of genes involved in leaf development and morphogenesis is critical for uncovering the underlying genetic basis of these traits. Genes in the AP2/ERF family are recognized as plant-specific transcription factors that contribute to plant growth, hormone-induced development, ethylene response factors, and stress responses. Results In this study, we identified 104 putative AP2/ERF genes in the recently released L. chinense genome and transcriptome database. In addition, all 104 genes were grouped into four subfamilies, the AP2, ERF, RAV, and Soloist subfamilies. This classification was further supported by the results of gene structure and conserved motif analyses. Intriguingly, after application of a series test of cluster analysis, three AP2 genes, LcERF 94, LcERF 96, and LcERF 98, were identified as tissue-specific in buds based on the expression profiles of various tissues. These results were further validated via RT-qPCR assays and were highly consistent with the STC analysis. We further investigated the dynamic changes of immature leaves by dissecting fresh shoots into seven discontinuous periods, which were empirically identified as shoot apical meristem (SAM), leaf primordia and tender leaf developmental stages according to the anatomic structure. Subsequently, these three candidates were highly expressed in SAM and leaf primordia but rarely in tender leaves, indicating that they were mainly involved in early leaf development and morphogenesis. Moreover, these three genes displayed nuclear subcellular localizations through the transient transformation of tobacco epidermal cells. Conclusions Overall, we identified 104 AP2/ERF family members at the genome-wide level and discerned three candidate genes that might participate in the development and morphogenesis of the leaf primordium in L. chinense.

BLADE-ON-PETIOLE 2 (BOP2) plays a pivotal role in leaf morphogenesis. Liriodendron tulipifera is a suitable model for exploring the molecular mechanisms underlying leaf serration formation, which are largely unknown. Here, we isolated the full-length LtuBOP2 gene and its promoter from L. tulipifera and characterized its function in leaf morphogenesis through multidimensional approaches. The spatiotemporal expression pattern of LtuBOP2 indicated the high expression of LtuBOP2 in stems and leaf buds. We constructed LtuBOP2 promoter, fused the promoter sequences to the β-glucuronidase (GUS) gene, and then transformed them into Arabidopsis thaliana. Histochemical GUS staining results indicated that GUS activity was higher in petioles and the main vein. LtuBOP2 overexpression in A. thaliana caused moderate serration in the leaf tip, owing to the increased number of abnormal lamina epidermal cells and defective vascular tissue, thus indicating a novel role of BOP2. The ectopic expression of LtuBOP2 in A. thaliana promoted the expression of the lateral organ boundary gene ASYMMETRIC LEAVES2 (AS2) and inhibited JAGGED (JAG) and CUP-SHAPED COTYLEDON2 (CUC2) expression to establish leaf proximal–distal polarity. Moreover, LtuBOP2 participated in leaf serration formation by promoting the antagonistic relationship between KNOX I and hormones during leaf margin development. Our findings revealed the role of LtuBOP2 in the proximal–distal polarity formation and development of leaf margin morphology, providing new insights into the regulatory mechanisms of the leaf formation development of L. tulipifera.

Background The unique ‘mandarin jacket’ leaf shape is the most famous trait of Liriodendron chinense and this characteristic gives L. chinense aesthetic and landscaping value. However, the underlying regulatory mechanism of genes involved in the leaf development of L. chinense has remained unclear. Methods Based on transcriptome data of leaves at different developmental stages from L. chinense, we identified differentially expression genes (DEGs) functioning in leaf development. A candidate gene named LcCUC2-like (LcCUC2L) had high similarity in sequence with Arabidopsis thaliana CUC2, and used for further research. We isolated the full-length LcCUC2L gene and its promoter from L. chinense. Subsequently, we analyzed the function of the LcCUC2L gene and its promoter activity via transformation into A. thaliana. Results In this study, we found that the LcCUC2L and AtCUC2 are homologous in sequence but not homologous in function. Unlike the role of AtCUC2 in leaf serration and SAM formation, the LcCUC2L mainly regulates cotyledon development and rosette leaf number. Histochemical β-glucuronidase (GUS) staining revealed that LcCUC2L was expressed in the cotyledons of A. thaliana seedlings, indicating that the LcCUC2L may play a role in cotyledon development. Ectopic expression of LcCUC2L resulted in long, narrow cotyledons without petioles, abnormal lamina epidermis cells and defective vascular tissue in cotyledons, and these results were consistent with the LcCUC2L expression pattern. Further analysis showed that overexpression of LcCUC2L also induced numerous rosette leaves. Also, LcCUC2L and other related genes showed a severe response in L. chinense by introducing exogenous auxin stimulation, partly revealed that LcCUC2L affects the leaf development by regulating the auxin content. Conclusions These results suggest that LcCUC2L may play a critical role in leaf development and morphogenesis in L. chinense, and our findings provide insight into the molecular mechanisms of leaf development in L. chinense.

Liriodendron chinense is an economically and ecologically important deciduous tree species. Although the reference genome has been revealed, alternative polyadenylation (APA), transcription factors (TFs), long non-coding RNAs (lncRNAs), and co-expression networks of tissue-specific genes remain incompletely annotated. In this study, we used the bracts, petals, sepals, stamens, pistils, leaves, and shoot apex of L. chinense as materials for hybrid sequencing. On the one hand, we improved the annotation of the genome. We detected 13,139 novel genes, 7,527 lncRNAs, 1,791 TFs, and 6,721 genes with APA sites. On the other hand, we found that tissue-specific genes play a significant role in maintaining tissue characteristics. In total, 2,040 tissue-specific genes were identified, among which 9.2% of tissue-specific genes were affected by APA, and 1,809 tissue-specific genes were represented in seven specific co-expression modules. We also found that bract-specific hub genes were associated plant defense, leaf-specific hub genes were involved in energy metabolism. Moreover, we also found that a stamen-specific hub TF Lchi25777 may be involved in the determination of stamen identity, and a shoot-apex-specific hub TF Lchi05072 may participate in maintaining meristem characteristic. Our study provides a landscape of APA, lncRNAs, TFs, and tissue-specific gene co-expression networks in L. chinense that will improve genome annotation, strengthen our understanding of transcriptome complexity, and drive further research into the regulatory mechanisms of tissue-specific genes.

In Arabidopsis thaliana, JAGGED (JAG) is a transcription inhibitor that controls the development of leaf polarity and regulates the expression of genes controlling lateral organ formation. Liriodendron tulipifera is an ornamental tree with extraordinary tulip-shaped flowers and goose web-like leaves, this is one of the suitable plants for morphological development research. To investigate the potential functions of the LtuJAG gene, we isolated the full-length LtuJAG from L. tulipifera, transferred it into A. thaliana via agrobacterium-mediated transformation, and monitored its expression pattern. Subcellular localization showed that LtuJAG was located in the nucleus. RT-qPCR assays indicated that LtuJAG was expressed mainly in leaf buds and flowers, but not in mature leaves and stems. GUS staining results showed that LtuJAG was expressed in the shoot apical meristem (SAM). Overexpressing LtuJAG changed A. thaliana leaf shapes, causing a moderate serration and a slight asymmetric distribution in the medio-lateral and proximal-distal axes. Ectopic expression of LtuJAG induced the expression of lateral organ boundary suppressors JAGGED LATERAL ORGANS (JLO) and ARABIDOPSIS THALIANA HOMEOBOX1 (ATH1). It also repressed the expression of the apical meristem suppressor class-1 KNOX gene (KNOX I) and altered endogenous hormone levels. Our results suggest that LtuJAG plays a role in negatively regulating leaf polarity formation in L. tulipifera.

Alternative splicing (AS) plays pivotal roles in regulating plant growth and development, flowering, biological rhythms, signal transduction, and stress responses. However, no studies on AS have been performed in Liriodendron chinense, a deciduous tree species that has high economic and ecological value. In this study, we used multiple tools and algorithms to analyze transcriptome data derived from seven tissues via hybrid sequencing. Although only 17.56% (8,503/48,408) of genes in L. chinense were alternatively spliced, these AS genes occurred in 37,844 AS events. Among these events, intron retention was the most frequent AS event, producing 1,656 PTC-containing and 3,310 non-PTC-containing transcripts. Moreover, 183 long noncoding RNAs (lncRNAs) also underwent AS events. Furthermore, weighted gene coexpression network analysis (WGCNA) revealed that there were great differences in the activities of transcription and post-transcriptional regulation between pistils and leaves, and AS had an impact on many physiological and biochemical processes in L. chinense, such as photosynthesis, sphingolipid metabolism, fatty acid biosynthesis and metabolism. Moreover, our analysis showed that the features of genes may affect AS, as AS genes and non-AS genes had differences in the exon/intron length, transcript length, and number of exons/introns. In addition, the structure of AS genes may impact the frequencies and types of AS because AS genes with more exons or introns tended to exhibit more AS events, and shorter introns tended to be retained, whereas shorter exons tended to be skipped. Furthermore, eight AS genes were verified, and the results were consistent with our analysis. Overall, this study reveals that AS and gene interaction are mutual—on one hand, AS can affect gene expression and translation, while on the other hand, the structural characteristics of the gene can also affect AS. This work is the first to comprehensively report on AS in L. chinense, and it can provide a reference for further research on AS in L. chinense.