The leaf, a photosynthetic organ that plays an indispensable role in plant development and growth, has a certain ability to adapt to the environment and exhibits tremendous diversity among angiosperms. Liriodendron chinense, an ancestral angiosperm species, is very popular in landscaping. The leaf of this species has two lobes and resembles a Qing Dynasty Chinese robe; thus, leaf shape is the most valuable ornamental trait of the tree. In this work, to determine the candidate genes associated with leaf development in L. chinense, scanning electron microscopy (SEM) was employed to distinguish the developmental stages of tender leaves. Four stages were clearly separated, and transcriptome sequencing was performed for two special leaf stages. Altogether, there were 48.23 G clean reads in the libraries of the two leaf developmental stages, and 48,107 assembled unigenes were annotated with five databases. Among four libraries, 3118 differentially expressed genes (DEGs) were enriched in expression profiles. We selected ten DEGs associated with leaf development and validated their expression patterns via quantitative real-time PCR (qRT-PCR) assays. Most validation results were closely correlated with the RNA-sequencing data. Taken together, we examined the dynamic process of leaf development and indicated that several transcription factors and phytohormone metabolism genes may participate in leaf shape development. The transcriptome data analysis presented in this work aims to provide basic insights into the mechanisms mediating leaf development, and the results serve as a reference for the genetic breeding of ornamental traits in L. chinense.

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.

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 a common ornamental tree that has attractive leaves, which is a valuable trait for use in landscape architecture. In this work, we aimed to identify the potential genes that control and regulate the development of L. chinense leaf lobes. Based on the transcriptome data for the leaf developmental stages we previously generated, two candidate genes were identified in this study. KNOTTED-LIKE HOMEOBOX(KNOX), encoding homeobox family proteins, play a large role in leaf lobe and leaf complexity regulation. Here, two full length KNOX genes from L. chinense were amplified and named LcKNOX1 and LcKNOX6 according to their sequence similarities with the respective Arabidopsis thaliana KNOX family genes. Overexpression vectors were constructed and subsequently transformed into wild type (WT) A. thaliana. Additionally, LcKNOX6 was expressed in tobacco leaves to examine its subcellular localization, and the 35S::LcKNOX6 transgenic A. thaliana leaf cells were imaged with the use of SEM. The expression of several genes that participate in KNOX gene regulation were validated by quantitative real-time PCR. The results show that LcKNOX1 produces almost the same phenotype as that found in WT A. thaliana. Notably, the LcKNOX6-1 lines presented deep leaf lobes that were similar to L. chinense leaf lobes. Two 35S::LcKNOX6 lines induced an abnormal growth phenotype whose seeds were abortive. In short, these results indicate that the LcKNOX6 gene might affect leaf development in A. thaliana and provide insights into the regulation of L. chinense leaf shaping.

BackgroundNectar is a major floral attractant and reward for insects that ensures pollination. Liriodendron, a genus of the Magnoliaceae family, includes only two relict species, L. chinense and L. tulipifera, which are considered “basal angiosperms” according to plant evolutionary history. The flowers of Liriodendron plants are insect pollinated and secrete nectar to attract pollinators. To date, the morphology and anatomy of nectaries, the mechanism of nectar secretion and the molecular mechanism of nectary development in Liriodendron remain poorly understood.MethodsIn this study, we examined the nectary surface cells and change in starch in L. tulipifera by using scanning electron microscopy and periodic acid-Schiff techniques to select appropriate samples for subsequent research. Transcriptome sequencing was of the top and middle parts of immature nectaries and the middle part of mature and postsecretory nectaries in L. tulipifera was performed. We evaluated the expression profiles of 21 DEGs that are closely related to nectary development and nectar secretion for real-time quantitative PCR analysis.ResultsL. tulipifera nectaries are starch-storing nectaries and are located in the top and middle parts of L. tulipifera petals. After analyzing the RNA-seq data, we obtained 115.26 Gb of clean data in 12 libraries and mapped the results to the L. chinense reference genome with 71.02–79.77% efficiency. In total, 26,955 DEGs were identified by performing six pairwise comparisons. The flavonoid biosynthesis, phenylpropanoid biosynthesis, anthocyanin biosynthesis and starch and sucrose metabolism pathways were enriched and related to nectar secretion and pigment change. We identified 56 transcription factor families, and members of the TCP, Trihelix, C2H2, ERF, and MADS families changed dynamically during nectary development. Moreover, to further verify the accuracy of the RNA-seq results, we validated the expression profiles of 21 candidate genes.ConclusionsWe evaluated the nectary development and secretion processes comprehensively and identified many related candidate genes in L. tulipifera. These findings suggest that nectaries play important roles in flavonoid synthesis and petal color presentation.