NtMYB12 requires for competition between flavonol and (pro)anthocyanin biosynthesis in Narcissus tazetta tepals.

The color of flowers is one of the main characteristics adopted for plants to attract pollinators to ensure the reproductive success of the plant, they are also important in their ornamental appeal in Narcissus plant. In this study, we identified a NtMYB12 locus encoding an R2R3-MYB transcription factor. Comparative transcriptome analysis of loss- and gain- of NtMYB12 tissue relative to wild-type narcissus showed NtMYB12 was mainly involved in flavonol and phenylpropanoid metabolic pathways. Biochemical evidences of dual-luciferase activity and chromatin immunoprecipitation assay supported that MYB12 directly bound to promoters of NtFLS, NtLAR, and NtDFR that were cloned by genome walking assay, and activated NtFLS and NtLAR expression but repressed NtDFR expression. More interestingly, NtMYB12 can interact with NtbHLH1 and NtWD40-1 proteins via R3 domain that were selected by transcriptome-based WGCNA and confirmed by yeast two hybrid, bimolecular fluorescence complementation and coimmunoprecipitation assay. Interaction of NtMYB12 with NtbHLH1 and NtWD40-1 forming MYB-bHLH-WD40 triplex specially activated NtDFR and NtANS expression and promoted (pro)anthocyanin accumulation, while NtMYB12 alone activated NtFLS and NtLAR expression and accumulated flavonols, but repressed NtDFR expression. These results indicated that NtMYB12 alone or NtMYB12-bHLH1-WD40-1 triplex requires for competition of metabolism fluxes between flavonol and (pro)anthocyanin biosynthesis. NtMYB12 dually functions on flavonol and proanthocyanin biogenesis via physically binding to NtFLS and NtLAR promoter activating their expression and on (pro)anthocyanin biosynthesis via NtMYB12-NtWD40-NtbHLH (MBW) triplex activating NtDFR and NtANS expression. Requirement of NtMYB12 alone or MBW complex for the competition between flavonol and anthocyanin biosynthesis results in narcissus colorized petal traits.

Screening and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR under PEG, NaCl and ZnSO4 Treatments in Broussonetia papyrifera.

Real-time quantitative PCR (RT-qPCR) has a high sensitivity and strong specificity, and is widely used in the analysis of gene expression. Selecting appropriate internal reference genes is the key to accurately analyzing the expression changes of target genes by RT-qPCR. To find out the most suitable internal reference genes for studying the gene expression in Broussonetia papyrifera under abiotic stresses (including drought, salt, and ZnSO4 treatments), seven different tissues of B. papyrifera, as well as the roots, stems, and leaves of B. papyrifera under the abiotic stresses were used as test materials, and 15 candidate internal reference genes were screened based on the transcriptome data via RT-qPCR. Then, the expression stability of the candidate genes was comprehensively evaluated through the software geNorm (v3.5), NormFinder (v0.953), BestKeeper (v1.0), and RefFinder. The best internal reference genes and their combinations were screened out according to the analysis results. rRNA and Actin were the best reference genes under drought stress. Under salt stress, DOUB, HSP, NADH, and rRNA were the most stable reference genes. Under heavy metal stress, HSP and NADH were the most suitable reference genes. EIF3 and Actin were the most suitable internal reference genes in the different tissues of B. papyrifera. In addition, HSP, rRNA, NADH, and UBC were the most suitable internal reference genes for the abiotic stresses and the different tissues of B. papyrifera. The expression patterns of DREB and POD were analyzed by using the selected stable and unstable reference genes. This further verified the reliability of the screened internal reference genes. This study lays the foundation for the functional analysis and regulatory mechanism research of genes in B. papyrifera.

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.

Characterization of the Complete Chloroplast Genome and Phylogenetic Implications of Euonymus microcarpus (Oliv.) Sprague.

Euonymus microcarpus (Oliv.) Sprague, is a species of evergreen shrub of the genus Euonymus, family Celastraceae. Here, we extracted the genomic DNA from the leaves of E. microcarpus and constructed a paired-end library. The chloroplast genome of E. microcarpus was generated with the high-throughput sequencing by the illumina Hiseq X Ten platform and de novo assembly. The chloroplast genome had a quadripartite structure, containing a long single copy region with a size of 85,386 bp and a short single copy region with a size of 18,456 bp, separated by two inverted repeat regions of 26,850 bp. The chloroplast genome contained 133 genes identified in total, including 87 potential protein-coding genes, 38 transfer RNA genes, and eight ribosomal RNA genes. A total of 282 simple sequence repeats and 63 long repeats were found. Furthermore, the phylogenetic relationships inferred that E. microcarpus is sister to E. japonicus and E. schensianus. A comparison of the structure of the chloroplast genomes of eight Euonymus species suggests a nucleotide variability of the junction sites and a higher divergence of non-coding regions, compared to the coding regions. The original findings of the study serves as a good reference for chloroplast genome assembly and a valuable foundation for the genetic diversity and evolution of E. microcarpus.

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.

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.

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.

Genome-wide identification and characterization of LcCCR13 reveals its potential role in lignin biosynthesis in Liriodendron chinense.

Introduction: Wood formation is closely related to lignin biosynthesis. Cinnamoyl-CoA reductase (CCR) catalyzes the conversion of cinnamoyl-CoA to cinnamaldehydes, which is the initiation of the lignin biosynthesis pathway and a crucial point in the manipulation of associated traits. Liriodendron chinense is an economically significant timber tree. Nevertheless, the underlying mechanism of wood formation in it remains unknown; even the number of LcCCR family members in this species is unclear. Materials and Results: This study aimed to perform a genome-wide identification of genes(s) involved in lignin biosynthesis in L. chinense via RT-qPCR assays and functional verification. Altogether, 13 LcCCR genes were identified that were divided into four major groups based on structural and phylogenetic features. The gene structures and motif compositions were strongly conserved between members of the same groups. Subsequently, the expression patterns analysis based on RNA-seq data indicated that LcCCR5/7/10/12/13 had high expression in the developing xylem at the stem (DXS). Furthermore, the RT-qPCR assays showed that LcCCR13 had the highest expression in the stem as compared to other tissues. Moreover, the overexpression of the LcCCR13 in transgenic tobacco plants caused an improvement in the CCR activity and lignin content, indicating that it plays a key role in lignin biosynthesis in the stems. Discussion: Our research lays a foundation for deeper investigation of the lignin synthesis and uncovers the genetic basis of wood formation in L. chinense.

Genome-wide survey and identification of AP2/ERF genes involved in shoot and leaf development in Liriodendron 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.

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.

Cloning, Characterization and Functional Analysis of the LtuPTOX Gene, a Homologue of Arabidopsis thalianaIMMUTANS Derived from Liriodendron tulipifera.

Flower colour and colour patterns are crucial traits for ornamental species; thus, a comprehensive understanding of their genetic basis is extremely significant for plant breeders. The tulip tree (Liriodendron tulipifera Linn.) is well known for its flowers, odd leave shape and tree form. However, the genetic basis of its colour inheritance remains unknown. In this study, a putative plastid terminal oxidase gene (LtuPTOX) was identified from L. tulipifera based on multiple databases of differentially expressed genes at various developmental stages. Then, the full-length cDNA of LtuPTOX was derived from tepals and leaves using RACE (rapid amplification of cDNA ends) approaches. Furthermore, gene structure and phylogenetic analyses of PTOX as well as AOXs (alternative oxidases), another highly similar homologue in the AOX family, were used to distinguish between the two subfamilies of genes. In addition, transient transformation and qPCR methods were used to determine the subcellular localization and tissue expression pattern of the LtuPTOX gene. Moreover, the expression of LtuPTOX as well as pigment contents was investigated to illustrate the function of this gene during the formation of orange bands on petals. The results showed that the LtuPTOX gene encodes a 358-aa protein that contains a complete AOX domain (PF01786). Accordingly, the LiriodendronPTOX and AOX genes were identified as only paralogs since they were rather similar in sequence. LtuPTOX showed chloroplast localization and was expressed in coloured organs such as petals and leaves. Additionally, an increasing pattern of LtuPTOX transcripts leads to carotenoid accumulation on the orange-band during flower bud development. Taken together, our results suggest that LtuPTOX is involved in petal carotenoid metabolism and orange band formation in L. tulipifera. The identification of this potentially involved gene will lay a foundation for further uncovering the genetic basis of flower colour in L. tulipifera.

Genetic linkage map construction and QTL mapping of seedling height, basal diameter and crown width of Taxodium 'Zhongshanshan 302' × T. mucronatum.

Taxodium is a genus renowned for its fast growth, good form and tolerance of flooding, salt, alkalinity, disease and strong winds. In this study, a genetic linkage map was constructed using sequence-related amplified polymorphism (SRAP) and simple sequence repeat (SSR) markers based on an F1 population containing 148 individuals generated from a cross between T. 'Zhongshanshan 302' and T. mucronatum. The map has a total length of 976.5 cM, with a mean distance of 7.0 cM between markers, and contains 34 linkage groups with 179 markers (171 SRAPs and 8 SSRs). Quantitative trait loci (QTLs) affecting growth traits, such as seedling height, basal diameter and crown width, were detected based on the constructed linkage map. Four significant QTLs were identified, three of which, namely qtSH-1 for seedling height, qtBD-1 for basal diameter and qtCW-1 for crown width, were located at 2.659 cM of LG7 with logarithm odds values of 3.72, 3.49 and 3.93, respectively, and explained 24.9, 27.0 and 21.7 % of the total variation of the three grown traits, respectively. Another QTL for crown width (qtCW-2) was detected at 1.0 cM on LG13, with a logarithm of odds value of 3.15, and explained 31.7 % of the total variation of crown width. This is the first report on the construction of a genetic linkage map and QTL analysis in Taxodium, laying the groundwork for the construction of a high-density genetic map and QTL mapping in the genus Taxodium.