Leaf morphology related genes revealed by integrating Pan-transcriptome, GWAS and eQTL analyses in a Liriodendron population.

Leaf plays an indispensable role in plant development and growth. Although many known genes related to leaf morphology development have been identified, elucidating the complex genetic basis of leaf morphological traits remains a challenge. Liriodendron plants are common ornamental trees due to their unique leaf shapes, while the molecular mechanism underlying Liriodendron leaf morphogenesis has remained unknown. Herein, we firstly constructed a population-level pan-transcriptome of Liriodendron from 81 accessions to explore the expression presence or absence variations (ePAVs), global expression differences at the population level, as well as differentially expressed genes (DEGs) between the Liriodendron chinense and Liriodendron tulipifera accessions. Subsequently, we integrated a genome-wide association study (GWAS), expression quantitative trait loci (eQTL), and transcriptome-wide association study (TWAS) to identify candidate genes related to leaf morphology. Through GWAS analysis, we identified 18 and 17 significant allelic loci in the leaf size and leaf shape modules, respectively. In addition, we discerned 16 candidate genes in relation to leaf morphological traits via TWAS. Further, integrating the co-localization results of GWAS and eQTL, we determined two regulatory hotspot regions, hot88 and hot758, related to leaf size and leaf shape, respectively. Finally, co-expression analysis, eQTL, and linkage mapping together demonstrated that Lchi_4g10795 regulate their own expression levels through cis-eQTL to affect the expression of downstream genes and cooperatively participate in the development of Liriodendron leaf morphology. These findings will improve our understanding of the molecular regulatory mechanism of Liriodendron leaf morphogenesis and will also accelerate molecular breeding of Liriodendron.

Integrated metabolomics and transcriptomics reveal that HhERF9 positively regulates salt tolerance in Hibiscus hamabo Siebold & Zuccarini.

Hibiscus hamabo Siebold & Zuccarini is one of the few semi-mangrove plants in the genus Hibiscus that can survive in saline-alkali soil and flooded land, but the mechanism underlying its adaptation to salt soil remains unknown. Here, to uncover this unsolved mystery, we characterized the changes in the accumulation of specific metabolites under salt stress in H. hamabo by integrating physiological, metabolic, and transcriptomic data, and found that osmotic adjustment and abscisic acid (ABA) is highly associated with the salt stress response. Further, a weighted gene co-expression network analysis was performed on the root transcriptome data, which identified three key candidate transcription factors responsive to salt stress. Among them, the expression HhERF9 was significantly upregulated under salt stress and ABA treatment and was involved in regulating the expression of genes related to the salt stress response. Further research indicated that HhERF9 enhances the accumulation of proline and soluble sugars by regulating the expression of genes such as NHX2 and P5CS. These findings provide a reference for improving H. hamabo through targeted genetic engineering and lay a theoretical foundation for its future promotion and cultivation in saline-alkali areas.

Transcriptome analysis and functional validation reveal the novel role of LhCYCL in axillary bud development in hybrid Liriodendron.

Shoot branching significantly influences yield and timber quality in woody plants, with hybrid Liriodendron being particularly valuable due to its rapid growth. However, understanding of the mechanisms governing shoot branching in hybrid Liriodendron remains limited. In this study, we systematically examined axillary bud development using morphological and anatomical approaches and selected four distinct developmental stages for an extensive transcriptome analysis. A total of 9,449 differentially expressed genes have been identified, many of which are involved in plant hormone signal transduction pathways. Additionally, we identified several transcription factors downregulated during early axillary bud development, including a noteworthy gene annotated as CYC-like from the TCP TF family, which emerged as a strong candidate for modulating axillary bud development. Quantitative real-time polymerase chain reaction results confirmed the highest expression levels of LhCYCL in hybrid Liriodendron axillary buds, while histochemical ß-glucuronidase staining suggested its potential role in Arabidopsis thaliana leaf axil development. Ectopic expression of LhCYCL in A. thaliana led to an increase of branches and a decrease of plant height, accompanied by altered expression of genes involved in the plant hormone signaling pathways. This indicates the involvement of LhCYCL in regulating shoot branching through plant hormone signaling pathways. In summary, our results emphasize the pivotal role played by LhCYCL in shoot branching, offering insights into the function of the CYC-like gene and establishing a robust foundation for further investigations into the molecular mechanisms governing axillary bud development in hybrid Liriodendron.

Comprehensive deciphering the alternative splicing patterns involved in leaf morphogenesis of Liriodendron chinense.

Alternative splicing (AS), a pivotal post-transcriptional regulatory mechanism, profoundly amplifies diversity and complexity of transcriptome and proteome. Liriodendron chinense (Hemsl.) Sarg., an excellent ornamental tree species renowned for its distinctive leaf shape, which resembles the mandarin jacket. Despite the documented potential genes related to leaf development of L. chinense, the underlying post-transcriptional regulatory mechanisms remain veiled. Here, we conducted a comprehensive analysis of the transcriptome to clarify the genome-wide landscape of the AS pattern and the spectrum of spliced isoforms during leaf developmental stages in L. chinense. Our investigation unveiled 50,259 AS events, involving 10,685 genes (32.9%), with intron retention as the most prevalent events. Notably, the initial stage of leaf development witnessed the detection of 804 differentially AS events affiliated with 548 genes. Although both differentially alternative splicing genes (DASGs) and differentially expressed genes (DEGs) were enriched into morphogenetic related pathways during the transition from fishhook (P2) to lobed (P7) leaves, there was only a modest degree of overlap between DASGs and DEGs. Furthermore, we conducted a comprehensively AS analysis on homologous genes involved in leaf morphogenesis, and most of which are subject to post-transcriptional regulation of AS. Among them, the AINTEGUMENTA-LIKE transcript factor LcAIL5 was characterization in detailed, which experiences skipping exon (SE), and two transcripts displayed disparate expression patterns across multiple stages. Overall, these findings yield a comprehensive understanding of leaf development regulation via AS, offering a novel perspective for further deciphering the mechanism of plant leaf morphogenesis.

Genome-wide investigation of SQUAMOSA promoter binding protein-like genes in Liriodendron and functional characterization of LcSPL2.

The plant-specific SQUAMOSA promoter-binding protein-like (SPL) transcription factors play a pivotal role in various developmental processes, including leaf morphogenesis and vegetative to reproductive phase transition. Liriodendron chinense and Liriodendron tulipifera are widely used in landscaping due to their tulip-like flowers and peculiar leaves. However, the SPL gene family in Liriodendron has not been identified and systematically characterized. We systematically identified and characterized the SPL family members in Liriodendron, including phylogeny, gene structure and syntenic analyses. Subsequently, we quantified the expression patterns of LcSPLs across various tissue sites through transcription-quantitative polymerase chain reaction (RT-qPCR) assays and identified the target gene, LcSPL2. Finally, we characterized the functions of LcSPL2 via ectopic transformation. Altogether, 17 LcSPL and 18 LtSPL genes were genome-widely identified in L. chinense and L. tulipifera, respectively. All the 35 SPLs were grouped into 9 clades. Both species had three SPL gene pairs arising from segmental duplication events, and the LcSPLs displayed high collinearity with the L. tulipifera genome. RT-qPCR assays showed that SPL genes were differentially expressed in different tissues, especially. Because LcSPL2 is highly expressed in pistils and leaves, it was selected to describe the SPL gene family of L. chinense by ectopic expression. We showed that overexpression of LcSPL2 in Arabidopsis thaliana resulted in earlier flowering and fewer rosette leaves. Moreover, we observed that overexpression of LcSPL2 in A. thaliana up-regulated the expression levels of four genes related to flower development. This study identified SPL genes in Liriodendron and characterized the function of LcSPL2 in advancing flower development.

The identification and functional characterization of the LcMCT gene from Liriodendron chinense reveals its potenatial role in carotenoids biosyanthesis.

Terpenoids are not only important component of plant floral scent, but also indispensable elements in the formation of floral color. The petals of Liriodendron chinense are rich in tetraterpene carotenoids and release large amounts of volatile monoterpene and sesquiterpene compounds during full blooming stage. However, the mechanism of terpenoid synthesis is not clear in L. chinense. In this study, we identified a LcMCT gene and characterized its potential function in carotenoids biosynthesis. A total of 2947 up-regulated differentially expressed genes (DEGs) were discerned from the transcriptomic data of L. chinense petals, with a significant enrichment of DEGs related to plant hormone signal transduction and terpenoid backbone biosynthesis. After comprehensive analysis on these DEGs, the LcMCT gene was selected for subsequent function characterization. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results showed that LcMCT was expressed at the highest level in the petals during full blooming stage, suggesting a possible role in carotenoids biosynthesis and volatile terpenoid biosynthesis. Subcellular localization showed that the LcMCT protein was localized in the chloroplast. Overexpression of LcMCT in Arabidopsis thaliana affected the expression levels of MEP pathway genes. Moreover, the MCT enzyme activity and carotenoids contents in transgenic A. thaliana were increased by 69.27% and 15.57%, respectively. These results suggest that LcMCT promotes the biosynthesis of terpenoid precursors via the MEP pathway. Our work lays a foundation for exploring the mechanism of terpenoid synthesis in L. chinense.

Genome-wide identification of GROWTH-REGULATING FACTORs in Liriodendron chinense and functional characterization of LcGRF2 in leaf size regulation.

GROWTH-REGULATING FACTORs (GRFs) play a pivotal role in the regulation of leaf size in plants and have been widely reported in plants. However, their specific functions in leaf size regulation in Liriodendron chinense remains unclear. Therefore, in this study, we identified GRF genes on a genome-wide scale in L. chinense to characterize the roles of LcGRFs in regulating leaf size. A total of nine LcGRF genes were identified, and these genes exhibited weak expression in mature leaves but strong expression in shoot apex. Notably, LcGRF2 exhibited the highest expression level in the shoot apex of L. chinense. Further RT-qPCR assay revealed that the expression level of LcGRF2 gradually decreased along with the leaf development process, and also displayed a gradient along the leaf proximo-distal and medio-lateral axes. Furthermore, overexpression of LcGRF2 in Arabidopsis thaliana resulted in increased leaf size, and significantly up-regulated the expression of genes involved in cell division like AtCYCD3;1, AtKNOLLE, and AtCYCB1;1, indicating that LcGRF2 may influence leaf size by promoting cell proliferation. This work contributes to a better understanding of the roles and molecular mechanisms of LcGRFs in the regulation of leaf size in L. chinense.

Genome-wide association study of multiyear dynamic growth traits in hybrid Liriodendron identifies robust genetic loci associated with growth trajectories.

The genetic factors underlying growth traits differ over time points or stages. However, most current studies of phenotypes at single time points do not capture all loci or explain the genetic differences underlying growth trajectories. Hybrid Liriodendron exhibits obvious heterosis and is widely cultivated, although its complex genetic mechanism underlying growth traits remains unknown. A genome-wide association study (GWAS) is an effective method for elucidating the genetic architecture by identifying genetic loci underlying complex quantitative traits. In the present study, using a GWAS, we identified robust loci associated with growth trajectories in hybrid Liriodendron populations. We selected 233 hybrid progenies derived from 25 crosses for resequencing, and measured their tree height (H) and diameter at breast height (DBH) for 11 consecutive years; 192 972 high-quality single nucleotide polymorphisms (SNPs) were obtained. The dynamics of the multiyear single-trait GWAS showed that year-specific SNPs predominated, and only five robust SNPs for DBH were identified in at least three different years. Multitrait GWAS analysis with model parameters as latent variables also revealed 62 SNPs for H and 52 for DBH associated with the growth trajectory, displaying different biomass accumulation patterns, among which four SNPs exerted pleiotropic effects. All identified SNPs also exhibited temporal variations in effect sizes and inheritance patterns potentially related to different growth and developmental stages. The haplotypes resulting from these significant SNPs might pyramid favorable loci, benefitting the selection of superior genotypes. The present study provides insights into the genetic architecture of dynamic growth traits and lays a basis for future molecular-assisted breeding.

Overexpression of the Liriodendron tulipifera BOP2 Gene (LtuBOP2) Affects Leaf Margin Development in Transgenic Arabidopsis thaliana.

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 ß-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.

Overexpression of LtuHB6 from Liriodendron tulipifera causes lobed-leaf formation in Arabidopsis thaliana.

Liriodendron tulipifera L. is an ornamental tree species with extraordinarily lobed leaves. However, the mechanisms underlying lobed leaf formation in plants remain unclear. The transcription factor, ARABIDOPSIS THALIANA HOMEBOX 6 (HB6), plays a role in regulating leaf margin development. HB6 is involved in cell division and differentiation of developmental organs and negatively regulates abscisic acid (ABA) signal transmission under external abiotic stress; it is unclear whether HB6 performs a pivotal role in leaf morphogenesis in L. tulipifera. In this study, full-length LtuHB6 from L. tulipifera was heterologously expressed in tobacco and Arabidopsis thaliana; its expression pattern was analyzed to determine its potential role in leaf development. In addition, LtuHB6 is localized in the nucleus and cell membrane of tobacco leaves. The expression of LtuHB6 was highest in mature leaves compared to the other stages of leaf development (bud growth, young leaves, and leaf senescence). Transgenic A. thaliana plants overexpressing LtuHB6 exhibited an abnormal phenotype with lobed leaves. Moreover, LtuHB6 overexpression significantly affected the expression of seven genes related to leaf serration in the initial stage of leaf primordia and altered the expression levels of hormonal genes. Our findings indicate that LtuHB6 is an essential regulatory factor in L. tulipifera lobed-leaf formation and is involved in regulating and responding to hormones. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01254-9.

Genome-wide identification of XTH genes in Liriodendron chinense and functional characterization of LcXTH21.

Liriodendron chinense is a relic tree species of the family Magnoliaceae with multiple uses in timber production, landscape decoration, and afforestation. L. chinense often experiences drought stress in arid areas. However, the molecular basis underlying the drought response of L. chinense remains unclear. Many studies have reported that the xyloglucan endotransglucosylase/hydrolase (XTH) family plays an important role in drought stress resistance. Hereby, to explore the drought resistance mechanism of L. chinense, we identify XTH genes on a genome-wide scale in L. chinense. A total of 27 XTH genes were identified in L. chinense, and these genes were classified into three subfamilies. Drought treatment and RT-qPCR analysis revealed that six LcXTH genes significantly responded to drought stress, especially LcXTH21. Hence, we cloned the LcXTH21 gene and overexpressed it in tobacco via gene transfer to analyze its function. The roots of transgenic plants were more developed than those of wild-type plants under different polyethylene glycol (PEG) concentration, and further RT-qPCR analysis showed that LcXTH21 highly expressed in root compared to aboveground organs, indicating that LcXTH21 may play a role in drought resistance through promoting root development. The results of this study provide new insights into the roles of LcXTH genes in the drought stress response. Our findings will also aid future studies of the molecular mechanisms by which LcXTH genes contribute to the drought response.

Liriodendron chinense LcMAX1 regulates primary root growth and shoot branching in Arabidopsis thaliana.

Strigolactones (SLs) play prominent roles in regulating shoot branching and root architecture in model plants. However, their roles in non-model (particularly woody) plants remain unclear. Liriodendron chinense is a timber tree species widely planted in southern China. The outturn percentage and wood quality of L. chinense are greatly affected by the branching characteristics of its shoot, and the rooting ability of the cuttings is key for its vegetative propagation. Here, we isolated and analyzed the function of the MORE AXILLARY GROWTH 1 (LcMAX1) gene, which is involved in L. chinense SL biosynthesis. RT-qPCR showed that LcMAX1 was highly expressed in the roots and axillary buds. LcMAX1 was located in the endoplasmic reticulum (ER) and nucleus. LcMAX1 ectopic expression promoted primary root growth, whereas there were no phenotypic differences in shoot branching between transgenic and wild-type (WT) A. thaliana plants. LcMAX1 overexpression in the max1 mutant restored them to the WT A. thaliana phenotypes. Additionally, AtPIN1, AtPIN2, and AtBRC1 expressions were significantly upregulated in transgenic A. thaliana and the max1 mutant. It was therefore speculated that LcMAX1 promotes primary root growth by regulating expression of auxin transport-related genes in A. thaliana, and LcMAX1 inhibits shoot branching by upregulating expression of AtBRC1 in the max1 mutant. Altogether, these results demonstrated that the root development and shoot branching functions of LcMAX1 were similar to those of AtMAX1. Our findings provide a foundation for obtaining further insights into root and branch development in L. chinense.

Predicting the impact of climate change on the distribution of two relict Liriodendron species by coupling the MaxEnt model and actual physiological indicators in relation to stress tolerance.

Climate change has a crucial impact on the distributions of plants, especially relict species. Hence, predicting the potential impact of climate change on the distributions of relict plants is critical for their future conservation. Liriodendron plants are relict trees, and only two natural species have survived: L. chinense and L. tulipifera. However, the extent of the impact of future climate change on the distributions of these two Liriodendron species remains unclear. Therefore, we predicted the suitable habitat distributions of two Liriodendron species under present and future climate scenarios using MaxEnt modeling. The results showed that the area of suitable habitats for two Liriodendron species would significantly decrease. However, the two relict species presented different habitat shift patterns, with a local contraction of suitable habitat for L. chinense and a northward shift in suitable habitat for L. tulipifera, indicating that changes in environmental factors will affect the distributions of these species. Among the environmental factors assessed, May precipitation induced the largest impact on the L. chinense distribution, while L. tulipifera was significantly affected by precipitation in the driest quarter. Furthermore, to explore the relationship between habitat suitability and Liriodendron stress tolerance, we analyzed six physiological indicators of stress tolerance by sampling twelve provenances of L. chinense and five provenances of L. tulipifera. The composite index of six physiological indicators was significantly negatively correlated with the habitat suitability of the species. The stress tolerance of Liriodendron plants in highly suitable areas was lower than that in areas with moderate or low suitability. Overall, these findings improve our understanding of the ecological impacts of climate change, informing future conservation efforts for Liriodendron species.

Genome-wide study of the GRAS gene family in Hibiscus hamabo Sieb. et Zucc and analysis of HhGRAS14-induced drought and salt stress tolerance in Arabidopsis.

GRAS proteins are widely distributed plant-specific transcription factors. In this study, we identified 59 GRAS proteins (HhGRASs) from the genomic and transcriptomic datasets of Hibiscus hamabo Sieb. et Zucc. These proteins were phylogenetically divided into nine subfamilies. RNA-seq analysis revealed that most HhGRASs were expressed in response to abiotic stresses. Results from quantitative real-time PCR analysis of nine selected HhGRASs suggested that HhGRAS14 was significantly upregulated under multiple abiotic stresses; therefore, this gene was selected for further study. Silencing HhGRAS14 in H. hamabo reduced the tolerance to drought and salt stress, while overexpression in Arabidopsis thaliana significantly increased the tolerance to drought and salt and reduced the sensitivity to abscisic acid (ABA). In summary, we analyzed the GRAS family of proteins in semi-mangrove plants for the first time and identified a gene that responds to drought and salt stress, which provided the basis for a comprehensive analysis of GRAS genes and insight into the abiotic stress response mechanism in H. hamabo.

Genome-Wide Identification and Characterization of NAC Family in Hibiscus hamabo Sieb. et Zucc. under Various Abiotic Stresses.

NAC transcription factor is one of the largest plant gene families, participating in the regulation of plant biological and abiotic stresses. In this study, 182 NAC proteins (HhNACs) were identified based on genomic datasets of Hibiscus hamabo Sieb. et Zucc (H. hamabo). These proteins were divided into 19 subfamilies based on their phylogenetic relationship, motif pattern, and gene structure analysis. Expression analysis with RNA-seq revealed that most HhNACs were expressed in response to drought and salt stress. Research of quantitative real-time PCR analysis of nine selected HhNACs supported the transcriptome data's dependability and suggested that HhNAC54 was significantly upregulated under multiple abiotic stresses. Overexpression of HhNAC54 in Arabidopsis thaliana (A. thaliana) significantly increased its tolerance to salt. This study provides a basis for a comprehensive analysis of NAC transcription factor and insight into the abiotic stress response mechanism in H. hamabo.

Overexpression of the LcCUC2-like gene in Arabidopsis thaliana alters the cotyledon morphology and increases rosette leaf number.

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 ß-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.

Overexpression of Liriodendron tulipifera JAG Gene (LtuJAG) Changes Leaf Shapes in Transgenic Arabidopsis thaliana.

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.

The Roles of microRNA-Long Non-coding RNA-mRNA Networks in the Regulation of Leaf and Flower Development in Liriodendron chinense.

The leaf and the flower are vital plant organs owing to their roles in photosynthesis and reproduction. Long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and transcription factors (TFs) are very important to the development of these organs. Liriodendron chinense is a common ornamental tree species in southern China with an unusual leaf shape and tulip-like flowers. The genetic mechanisms underlying leaf and flower development in L. chinense and the miRNA-lncRNA-TF regulatory networks are poorly studied. Through the integration and analysis of different types of sequencing data, we identified the miRNA-lncRNA-TF regulatory networks that were related to leaf and flower development. These networks contained 105 miRNAs, 258 lncRNAs, 393 TFs, and 22 endogenous target mimics. Notably, lch-lnc7374-miR156h-SPL3 and lch-lnc7374-miR156j-SPL9 were potential regulators of stamen and pistil development in L. chinense, respectively. miRNA-lncRNA-mRNA regulatory networks were shown to impact anther development, male and female fertility, and petal color by regulating the biosynthesis of phenylpropanoid metabolites. Phenylpropanoid metabolite biosynthesis genes and TFs that were targeted by miRNAs and lncRNAs were differentially expressed in the leaf and flower. Moreover, RT-qPCR analysis confirmed 22 differentially expressed miRNAs, among which most of them showed obvious leaf or flower specificity; miR157a-SPL and miR160a-ARF module were verified by using RLM-RACE, and these two modules were related to leaf and flower development. These findings provide insight into the roles of miRNA-lncRNA-mRNA regulatory networks in organ development and function in L. chinense, and will facilitate further investigation into the regulatory mechanisms of leaf and flower development in L. chinense.

Genetic divergence and local adaptation of Liriodendron driven by heterogeneous environments.

Ecological adaptive differentiation alters both the species diversity and intraspecific genetic diversity in forests, thus affecting the stability of forest ecosystems. Therefore, knowledge of the genetic underpinnings of the ecological adaptive differentiation of forest species is critical for effective species conservation. In this study, single-nucleotide polymorphisms (SNPs) from population transcriptomes were used to investigate the spatial distribution of genetic variation in Liriodendron to assess whether environmental variables can explain genetic divergence. We examined the contributions of environmental variables to population divergence and explored the genetic underpinnings of local adaptation using a landscape genomic approach. Niche models and statistical analyses showed significant niche divergence between L. chinense and L. tulipifera, suggesting that ecological adaptation may play a crucial role in driving interspecific divergence. We detected a new fine-scale genetic structure in L. chinense, and divergence of the six groups occurred during the late Pliocene to early Pleistocene. Redundancy analysis (RDA) revealed significant associations between genetic variation and multiple environmental variables. Environmental association analyses identified 67 environmental association loci (EALs; nonsynonymous SNPs) that underwent interspecific or intraspecific differentiation, 28 of which were associated with adaptive genes. These 28 candidate adaptive loci provide substantial evidence for local adaptation in Liriodendron. Our findings reveal ecological adaptive divergence pattern between Liriodendron species and provide novel insight into the role of heterogeneous environments in shaping genetic structure and driving local adaptation among populations, informing future L. chinense conservation efforts.

Transcriptomic profiling of the floral fragrance biosynthesis pathway of Liriodendron and functional characterization of the LtuDXR gene.

Floral fragrance, which has the function of attracting pollinators, is a class of volatile secondary metabolites mainly released by the secretory tissue of petals. Terpenoids are key components of floral volatile substances. Previous studies have shown that there are significant differences in the concentration and composition of volatile floral fragrances, especially terpenoids, between Liriodendron chinense and L. tulipifera. At present, the mechanism by which the synthesis of floral fragrance is regulated in Liriodendron remains unexplored. In this study, we analyzed the differentially expressed genes (DEGs) of L. chinense and L. tulipifera, and identified 130 DEGs related to terpenoid synthesis. A KEGG enrichment analysis of DEGs related to terpenoid biosynthesis revealed that the monoterpenoid biosynthesis pathway was the most significant. We cloned the LtuDXR gene from L. tulipifera using RACE technology. RT-qPCR results showed that the expression of the LtuDXR gene was the highest in the early florescence petals, indicating that the LtuDXR gene may play a role in the synthesis of volatile terpenoids. Subcellular localization showed that the LtuDXR protein is mainly localized in the chloroplast. Overexpression of LtuDXR in Arabidopsis thaliana significantly increased the plant height, DXR enzyme activity, and carotenoid content. In this study, we identified and functionally characterized LtuDXR, which is involved in terpenoid synthesis in Liriodendron. Our work lays the foundation for further exploration of the molecular mechanism by which terpenoid biosynthesis is regulated in Liriodendron.