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.

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.

Genomic survey and expression analysis of LcARFs reveal multiple functions to somatic embryogenesis in Liriodendron.

BACKGROUND: Auxin response factors (ARFs) are critical transcription factors that mediate the auxin signaling pathway and are essential for regulating plant growth. However, there is a lack of understanding regarding the ARF gene family in Liriodendron chinense, a vital species in landscaping and economics. Thus, further research is needed to explore the roles of ARFs in L. chinense and their potential applications in plant development. RESULT: In this study, we have identified 20 LcARF genes that belong to three subfamilies in the genome of L. chinense. The analysis of their conserved domains, gene structure, and phylogeny suggests that LcARFs may be evolutionarily conserved and functionally similar to other plant ARFs. The expression of LcARFs varies in different tissues. Additionally, they are also involved in different developmental stages of somatic embryogenesis. Overexpression of LcARF1, LcARF2a, and LcARF5 led to increased activity within callus. Additionally, our promoter-GFP fusion study indicated that LcARF1 may play a role in embryogenesis. Overall, this study provides insights into the functions of LcARFs in plant development and embryogenesis, which could facilitate the improvement of somatic embryogenesis in L. chinense. CONCLUSION: The research findings presented in this study shed light on the regulatory roles of LcARFs in somatic embryogenesis in L. chinense and may aid in accelerating the breeding process of this tree species. By identifying the specific LcARFs involved in different stages of somatic embryogenesis, this study provides a basis for developing targeted breeding strategies aimed at optimizing somatic embryogenesis in L. chinense, which holds great potential for improving the growth and productivity of this economically important species.

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.

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.

Identification and characteristics of SnRK genes and cold stress-induced expression profiles in Liriodendron chinense.

BACKGROUND: The sucrose non-fermenting 1 (SNF1)-related protein kinases (SnRKs) play a vivid role in regulating plant metabolism and stress response, providing a pathway for regulation between metabolism and stress signals. Conducting identification and stress response studies on SnRKs in plants contributes to the development of strategies for tree species that are more tolerant to stress conditions. RESULTS: In the present study, a total of 30 LcSnRKs were identified in Liriodendron chinense (L. chinense) genome, which was distributed across 15 chromosomes and 4 scaffolds. It could be divided into three subfamilies: SnRK1, SnRK2, and SnRK3 based on phylogenetic analysis and domain types. The LcSnRK of the three subfamilies shared the same Ser/Thr kinase structure in gene structure and motif composition, while the functional domains, except for the kinase domain, showed significant differences. A total of 13 collinear gene pairs were detected in L. chinense and Arabidopsis thaliana (A. thaliana), and 18 pairs were detected in L. chinense and rice, suggesting that the LcSnRK family genes may be evolutionarily more closely related to rice. Cis-regulation element analysis showed that LcSnRKs were LTR and TC-rich, which could respond to different environmental stresses. Furthermore, the expression patterns of LcSnRKs are different at different times under low-temperature stress. LcSnRK1s expression tended to be down-regulated under low-temperature stress. The expression of LcSnRK2s tended to be up-regulated under low-temperature stress. The expression trend of LcSnRK3s under low-temperature stress was mainly up-or down-regulated. CONCLUSION: The results of this study will provide valuable information for the functional identification of the LcSnRK gene in the future.

Genome-wide identification of the Liriodendron chinense WRKY gene family and its diverse roles in response to multiple abiotic stress.

BACKGROUND: Liriodendron chinense (Lchi) is a tree species within the Magnoliaceae family and is considered a basal angiosperm. The too low or high temperature or soil drought will restrict its growth as the adverse environmental conditions, thus improving L. chinense abiotic tolerance was the key issues to study. WRKYs are a major family of plant transcription factors known to often be involved in biotic and abiotic stress responses. So far, it is still largely unknown if and how the LchiWRKY gene family is tied to regulating L. chinense stress responses. Therefore, studying the involvement of the WRKY gene family in abiotic stress regulation in L. chinense could be very informative in showing how this tree deals with such stressful conditions. RESULTS: In this research, we performed a genome-wide analysis of the Liriodendron chinense (Lchi) WRKY gene family, studying their classification relationships, gene structure, chromosomal locations, gene duplication, cis-element, and response to abiotic stress. The 44 members of the LchiWRKY gene family contain a significant amount of sequence diversity, with their lengths ranging from 525 bp to 40,981 bp. Using classification analysis, we divided the 44 LchiWRKY genes into three phylogenetic groups (I, II, II), with group II then being further divided into five subgroups (IIa, IIb, IIc, IId, IIe). Comparative phylogenetic analysis including the WRKY families from 17 plant species suggested that LchiWRKYs are closely related to the Magnolia Cinnamomum kanehirae WRKY family, and has fewer family members than higher plants. We found the LchiWRKYs to be evenly distributed across 15 chromosomes, with their duplication events suggesting that tandem duplication may have played a major role in LchiWRKY gene expansion model. A Ka/Ks analysis indicated that they mainly underwent purifying selection and distributed in the group IId. Motif analysis showed that LchiWRKYs contained 20 motifs, and different phylogenetic groups contained conserved motif. Gene ontology (GO) analysis showed that LchiWRKYs were mainly enriched in two categories, i.e., biological process and molecular function. Two group IIc members (LchiWRKY10 and LchiWRKY37) contain unique WRKY element sequence variants (WRKYGKK and WRKYGKS). Gene structure analysis showed that most LchiWRKYs possess 3 exons and two different types of introns: the R- and V-type which are both contained within the WRKY domain (WD). Additional promoter cis-element analysis indicated that 12 cis-elements that play different functions in environmental adaptability occur across all LchiWRKY groups. Heat, cold, and drought stress mainly induced the expression of group II and I LchiWRKYs, some of which had undergone gene duplication during evolution, and more than half of which had three exons. LchiWRKY33 mainly responded to cold stress and LchiWRKY25 mainly responded to heat stress, and LchiWRKY18 mainly responded to drought stress, which was almost 4-fold highly expressed, while 5 LchiWRKYs (LchiWRKY5, LchiWRKY23, LchiWRKY14, LchiWRKY27, and LchiWRKY36) responded equally three stresses with more than 6-fold expression. Subcellular localization analysis showed that all LchiWRKYs were localized in the nucleus, and subcellular localization experiments of LchiWRKY18 and 36 also showed that these two transcription factors were expressed in the nucleus. CONCLUSIONS: This study shows that in Liriodendron chinense, several WRKY genes like LchiWRKY33, LchiWRKY25, and LchiWRKY18, respond to cold or heat or drought stress, suggesting that they may indeed play a role in regulating the tree's response to such conditions. This information will prove a pivotal role in directing further studies on the function of the LchiWRKY gene family in abiotic stress response and provides a theoretical basis for popularizing afforestation in different regions of China.

Identification, Phylogenetic and Expression Analyses of the AAAP Gene Family in Liriodendron chinense Reveal Their Putative Functions in Response to Organ and Multiple Abiotic Stresses.

In this study, 52 AAAP genes were identified in the L. chinense genome and divided into eight subgroups based on phylogenetic relationships, gene structure, and conserved motif. A total of 48 LcAAAP genes were located on the 14 chromosomes, and the remaining four genes were mapped in the contigs. Multispecies phylogenetic tree and codon usage bias analysis show that the LcAAAP gene family is closer to the AAAP of Amborella trichopoda, indicating that the LcAAAP gene family is relatively primitive in angiosperms. Gene duplication events revealed six pairs of segmental duplications and one pair of tandem duplications, in which many paralogous genes diverged in function before monocotyledonous and dicotyledonous plants differentiation and were strongly purification selected. Gene expression pattern analysis showed that the LcAAAP gene plays a certain role in the development of Liriodendron nectary and somatic embryogenesis. Low temperature, drought, and heat stresses may activate some WRKY/MYB transcription factors to positively regulate the expression of LcAAAP genes to achieve long-distance transport of amino acids in plants to resist the unfavorable external environment. In addition, the GAT and PorT subgroups could involve gamma-aminobutyric acid (GABA) transport under aluminum poisoning. These findings could lay a solid foundation for further study of the biological role of LcAAAP and improvement of the stress resistance of Liriodendron.

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.

Genomic Survey of Heat Shock Proteins in Liriodendron chinense Provides Insight into Evolution, Characterization, and Functional Diversities.

Heat shock proteins (HSPs) are conserved molecular chaperones whose main role is to facilitate the regulation of plant growth and stress responses. The HSP gene family has been characterized in most plants and elucidated as generally stress-induced, essential for their cytoprotective roles in cells. However, the HSP gene family has not yet been analyzed in the Liriodendron chinense genome. In current study, 60 HSP genes were identified in the L. chinense genome, including 7 LchiHSP90s, 23 LchiHSP70s, and 30 LchiHSP20s. We investigated the phylogenetic relationships, gene structure and arrangement, gene duplication events, cis-acting elements, 3D-protein structures, protein-protein interaction networks, and temperature stress responses in the identified L. chinense HSP genes. The results of the comparative phylogenetic analysis of HSP families in 32 plant species showed that LchiHSPs are closely related to the Cinnamomum kanehirae HSP gene family. Duplication events analysis showed seven segmental and six tandem duplication events that occurred in the LchiHSP gene family, which we speculated to have played an important role in the LchiHSP gene expansion and evolution. Furthermore, the Ka/Ks analysis indicated that these genes underwent a purifying selection. Analysis in the promoter region evidenced that the promoter region LchiHSPs carry many stress-responsive and hormone-related cis-elements. Investigations in the gene expression patterns of the LchiHSPs using transcriptome data and the qRT-PCR technique indicated that most LchiHSPs were responsive to cold and heat stress. In total, our results provide new insights into understanding the LchiHSP gene family function and their regulatory mechanisms in response to abiotic stresses.

Gibberellin Oxidase Gene Family in L. chinense: Genome-Wide Identification and Gene Expression Analysis.

GAox is a key enzyme for the transformation of gibberellins, and belongs to the 2-ketoglutarate dependent dioxygenase gene family (2ODD). However, a systematic analysis of GAox in the angiosperm L. chinense has not yet been reported. Here, we identified all LcGAox gene family members in L. chinense, which were classified into the three subgroups of GA20ox, C19GA2ox, and C20GA2ox. Comparison of the gene structure, conserve motifs, phylogenetic relationships, and syntenic relationships of gibberellin oxidase gene families in different species indicated that the gene functional differences may be due to the partial deletion of their domains during evolution. Furthermore, evidence for purifying selection was detected between orthologous GAox genes in rice, grape, Arabidopsis, and L. chinense. Analysis of the codon usage patterns showed that mutation pressure and natural selection might have induced codon usage bias in angiosperms; however, the LcGAox genes in mosses, lycophytes, and ambarella plants exhibited no obvious codon usage preference. These results suggested that the gibberellin oxidase genes were more primitive. The gene expression pattern was analyzed in different organs subjected to multiple abiotic stresses, including GA, abscisic acid (ABA), and chlormequat (CCC) treatment, by RNA-seq and qRT-PCR, and the stress- and phytohormone-responsive cis-elements were counted. The results showed that the synthesis and decomposition of GA were regulated by different LcGAox genes in the vegetative and reproductive organs of L. chinense, and only LcGA2ox1,4, and 7 responded to the NaCl, polyethylene glycol, 4 °C, GA, ABA, and CCC treatment in the roots, stems, and leaves of seedlings at different time periods, revealing the potential role of LcGAox in stress resistance.

Genome-Wide Identification and Expression Analysis of R2R3-MYB Family Genes Associated with Petal Pigment Synthesis in Liriodendron.

The MYB transcription factor family is one of the largest families in plants, and its members have various biological functions. R2R3-MYB genes are involved in the synthesis of pigments that yield petal colors. Liriodendron plants are widely cultivated as ornamental trees owing to their peculiar leaves, tulip-like flowers, and colorful petals. However, the mechanism underlying petal coloring in this species is unknown, and minimal information about MYB genes in Liriodendron is available. Herein, this study aimed to discern gene(s) involved in petal coloration in Liriodendron via genome-wide identification, HPLC, and RT-qPCR assays. In total, 204 LcMYB superfamily genes were identified in the Liriodendron chinense genome, and 85 R2R3-MYB genes were mapped onto 19 chromosomes. Chromosome 4 contained the most (10) R2R3-MYB genes, and chromosomes 14 and 16 contained the fewest (only one). MEME analysis showed that R2R3-MYB proteins in L. chinense were highly conserved and that their exon-intron structures varied. The HPLC results showed that three major carotenoids were uniformly distributed in the petals of L. chinense, while lycopene and ß-carotene were concentrated in the orange band region in the petals of Liriodendron tulipifera. Furthermore, the expression profiles via RT-qPCR assays revealed that four R2R3-MYB genes were expressed at the highest levels at the S3P/S4P stage in L. tulipifera. This result combined with the HPLC results showed that these four R2R3-MYB genes might participate in carotenoid synthesis in the petals of L. tulipifera. This work laid a cornerstone for further functional characterization of R2R3-MYB genes in Liriodendron plants.

Development of nuclear SSR and chloroplast genome markers in diverse Liriodendron chinense germplasm based on low-coverage whole genome sequencing.

BACKGROUND: Liriodendron chinense ranges widely in subtropical China and northern Vietnam; however, it inhabits several small, isolated populations and is now an endangered species due to its limited seed production. The objective of this study was to develop a set of nuclear SSR (simple sequence repeats) and multiple chloroplast genome markers for genetic studies in L. chinense and their characterization in diverse germplasm. RESULTS: We performed low-coverage whole genome sequencing of the L. chinense from four genotypes, assembled the chloroplast genome and identified nuclear SSR loci by searching in contigs for SSR motifs. Comparative analysis of the four chloroplast genomes of L. chinense revealed 45 SNPs, 17 indels, 49 polymorphic SSR loci, and five small inversions. Most chloroplast intraspecific polymorphisms were located in the interspaces of single-copy regions. In total, 6147 SSR markers were isolated from low-coverage whole genome sequences. The most common SSR motifs were dinucleotide (70.09%), followed by trinucleotide motifs (23.10%). The motif AG/TC (33.51%) was the most abundant, followed by TC/AG (25.53%). A set of 13 SSR primer combinations were tested for amplification and their ability to detect polymorphisms in a set of 109 L. chinense individuals, representing distinct varieties or germplasm. The number of alleles per locus ranged from 8 to 28 with an average of 21 alleles. The expected heterozygosity (He) varied from 0.19 to 0.93 and the observed heterozygosity (Ho) ranged from 0.11 to 0.79. CONCLUSIONS: The genetic resources characterized and tested in this study provide a valuable tool to detect polymorphisms in L. chinense for future genetic studies and breeding programs.

Transcriptomic and microstructural analyses in Liriodendron tulipifera Linn. reveal candidate genes involved in nectary development and nectar secretion.

BACKGROUND: Nectar 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. METHODS: In 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. RESULTS: L. 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. CONCLUSIONS: We 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.

Interspecies evolutionary divergence in Liriodendron, evidence from the nucleotide variations of LcDHN-like gene.

BACKGROUND: Liriodendron is a genus of Magnoliaceae, which consists of two relict species, Liriodendron chinense and L. tulipifera. Although the morphologies are highly similar, the two species exhibit different adaptive capacity. Dehydrins (DHNs) are abiotic stresses resistant proteins in planta, which are associated with adaptive evolution. To better understand the evolution divergence between L. chinense and L. tulipifera and how DHN genes are associated with adaptation evolution, we firstly investigated the DNA polymorphisms of the LcDHN-like gene in 21 L. chinense and 6 L. tulipifera populations. RESULTS: A 707 bp LcDHN-like gene was cloned, which included a 477 bp open reading frame (ORF) and coding 158 amino acids. 311 LcDHN-like gDNA sequences were obtained from 70 L. chinense and 35 L. tulipifera individuals. The AMOVA and phylogenetic relationship analysis showed significant differences between the two species. A higher genetic diversity was observed in L. tulipifera compared to L. chinense, in consistent with the higher adaptive capacity of L. tulipifera. Our data also suggested that the LcDHN-like genes' polymorphisms were under neutral mutation and purifying selection model in the L. chinense and L. tulipifera populations, respectively. The distinct expanding range and rate between the two species, haplotypes shared only in L.chinense's nearby populations, and wide dispersals in L. tulipifera could contribute to the obscure east-west separation in L. chinense and entirely unordered phylogeny in L. tulipifera. The completely separated nonsynonymous substitution at position 875 and the higher range scope of aliphatic index in L. tulipifera populations may be related with its higher adaptive capacity. Taken together, our study suggests LcDHN-like gene is a potential mark gene responsible for adaptive evolution divergence in Liriodendron. CONCLUSIONS: Significant differences and completely distinct haplogroups between L. chinense and L. tulipifera showed that the two species have evolved into different directions. The more widely distribution, earlier haplogroups divergence events, and richer SNPs variations in L. tulipifera could imply its stronger adaptation in this species. And potential effect of the allelic variations in LcDHN-like gene may reflect the difference of water stress and chill tolerance between L. chinense and L. tulipifera, which could provide some information for further adaption evolution studies of Liriodendron.

The "fossilized" mitochondrial genome of Liriodendron tulipifera: ancestral gene content and order, ancestral editing sites, and extraordinarily low mutation rate.

BACKGROUND: The mitochondrial genomes of flowering plants vary greatly in size, gene content, gene order, mutation rate and level of RNA editing. However, the narrow phylogenetic breadth of available genomic data has limited our ability to reconstruct these traits in the ancestral flowering plant and, therefore, to infer subsequent patterns of evolution across angiosperms. RESULTS: We sequenced the mitochondrial genome of Liriodendron tulipifera, the first from outside the monocots or eudicots. This 553,721 bp mitochondrial genome has evolved remarkably slowly in virtually all respects, with an extraordinarily low genome-wide silent substitution rate, retention of genes frequently lost in other angiosperm lineages, and conservation of ancestral gene clusters. The mitochondrial protein genes in Liriodendron are the most heavily edited of any angiosperm characterized to date. Most of these sites are also edited in various other lineages, which allowed us to polarize losses of editing sites in other parts of the angiosperm phylogeny. Finally, we added comprehensive gene sequence data for two other magnoliids, Magnolia stellata and the more distantly related Calycanthus floridus, to measure rates of sequence evolution in Liriodendron with greater accuracy. The Magnolia genome has evolved at an even lower rate, revealing a roughly 5,000-fold range of synonymous-site divergence among angiosperms whose mitochondrial gene space has been comprehensively sequenced. CONCLUSIONS: Using Liriodendron as a guide, we estimate that the ancestral flowering plant mitochondrial genome contained 41 protein genes, 14 tRNA genes of mitochondrial origin, as many as 7 tRNA genes of chloroplast origin, >700 sites of RNA editing, and some 14 colinear gene clusters. Many of these gene clusters, genes and RNA editing sites have been variously lost in different lineages over the course of the ensuing ∽200 million years of angiosperm evolution.

Mitochondrial genomes as living 'fossils'.

The huge variation between mitochondrial genomes makes untangling their evolutionary histories difficult. Richardson et al. report on the remarkably unaltered 'fossil' genome of the tulip tree, giving us many clues as to how the mitochondrial genomes of flowering plants have evolved over the last 150 million years, and raising questions about how such extraordinary sequence conservation can be maintained.

Physiological and transcriptomic analysis revealed that the accumulation of reactive oxygen species caused the low temperature sensitivity of Liriodendron × sinoamericanum.

Liriodendron × sinoamericanum is widely cultivated in southern China as an excellent wood and garden ornamental trees. However, its intolerance to low temperature limits its application to high latitudes. Understanding the molecular mechanism of low temperature sensitivity of Liriodendron × sinoamericanum is very important for its further application. In this study, combined with physiological and transcriptomic analysis, it was revealed that low temperature stress can lead to water loss and decreased photosynthetic capacity of Liriodendron × sinoamericanum leaves. The accelerated accumulation of reactive oxygen species (ROS) caused by the imbalance of cell REDOX homeostasis is one of the important reasons for the low temperature sensitivity. Further analysis showed that several transcription factors could be involved in regulating the synthesis and degradation of ROS, among which LsNAC72 and LsNAC73a could regulate the accumulation of O2- and H2O2 in leaves by affecting the expression level of LsAPX, LsSOD, LsPAO, and LsPOD.

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.