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 Analysis of the Liriodendron chinense Hsf Gene Family under Abiotic Stress and Characterization of the LcHsfA2a Gene.

Heat shock factors (Hsfs) play a crucial role in plant defense processes. However, the distribution and functional characteristics of Hsf genes in the relict plant Liriodendron chinense are still unclear. In this study, a total of 19 LcHsfs were identified and divided into three separate subgroups, comprising 10 LcHsfA, 7 LcHsfB, and 2 LcHsfC genes, respectively, based on their phylogenetic tree and the presence/absence of conserved protein domains. Whole-genome duplication and segmental duplication led to an expansion of the LhHsf gene family. The promoters of LcHsf genes are enriched for different types of cis-acting elements, including hormone responsive and abiotic-stress-responsive elements. The expression of LcHsfA3, LcHsfA4b, LcHsfA5, LcHsfB1b, and LcHsfB2b increased significantly as a result of both cold and drought treatments. LcHsfA2a, LcHsfA2b, and LcHsfA7 act as important genes whose expression levels correlate strongly with the expression of the LcHsp70, LcHsp110, and LcAPX genes under heat stress. In addition, we found that transiently transformed 35S:LcHsfA2a seedlings showed significantly lower levels of hydrogen peroxide (H2O2) after heat stress and showed a stronger thermotolerance. This study sheds light on the possible functions of LcHsf genes under abiotic stress and identifies potentially useful genes to target for molecular breeding, in order to develop more stress-resistant varieties.

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.

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.

Nitrate reduces copper toxicity by preventing oxidative stress and inhibiting copper translocation from roots to shoots in Liriodendron Chinense.

Nitrogen forms can affect metal accumulation in plants and tolerance to metals, but a few published studies on the effects on Cu toxicity and Cu accumulation in plants are scarce. Thus, the objective of this study was to evaluate the responses of Liriodendron chinense to different nitrogen forms, by the oxidative stress, antioxidant enzymes system, GSH-AsA cycle, Cu uptake, translocation, and accumulation under Cu stress. We found that Cu-induced growth inhibiting was alleviated by added exclusive NO3--N. Adding N as NH4+-N with or without NO3--N was aggravated as evidenced by significantly elevated malonaldehyde (MDA) and hydrogen peroxide (H2O2) compared to N-Null. Cu exposure and adding NH4+-N inhibited superoxide dismutase activity, but remarkably stimulated the activities of catalase and peroxidase, the efficiency of glutathione-ascorbate (GSH-AsA) cycle, and the activity of glutathione reductase and nitrate reductase, with respect to the control. However, adding exclusive NO3--N progressively restored the alteration of antioxidant to prevent Cu-induced oxidative stress. Additionally, adding exclusive NO3--N significantly promoted the Cu uptake and accumulation in roots, but reduced Cu concentration in leaves, accompanied by the inhibited Cu translocation factor from roots to shoots by 36.7%, when compared with N-Null. Overall, adding NO3--N alleviated its Cu toxicity by preventing Cu-induced oxidative stress and inhibiting Cu translocation from roots to shoots, which provides an effective strategy for phytostabilization in Cu-contaminated lands.

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.

Liriogerphines E-U, further unique sesquiterpene-alkaloid hybrids from the rare Chinese tulip tree.

Seventeen undescribed sesquiterpene-alkaloid hybrids (liriogerphines E-U, 1-17) were isolated and identified during a further phytochemical investigation on the branches and leaves of Chinese tulip tree (Liriodendron chinense), a rare medicinal and ornamental plant endemic to China. These unique heterodimers are conjugates of germacranolide-type sesquiterpenoids with structurally diverse alkaloids [i.e., aporphine- (1-15), proaporphine- (16), and benzyltetrahydroisoquinoline-type (17)] via the formation of a C-N bond. The previously undescribed structures were elucidated by comprehensive spectroscopic data analyses and electronic circular dichroism calculations. Such a class of sesquiterpene-alkaloid hybrids presumably biosynthesized via an aza-Michael addition is quite rare from terrestrial plants. In particular, the sesquiterpene-benzyltetrahydroisoquinoline hybrid skeleton has never been reported until the present study. All the isolates were evaluated for their cytotoxic effects against a small panel of leukemia cell lines (Raji, Jeko-1, Daudi, Jurkat, MV-4-11 and HL-60), and some of them exhibited considerable activities.

Genome-Wide Identification and Characterization of the YTH Domain-Containing RNA-Binding Protein Family in Liriodendron chinense.

N6-methyladenosine (m6A) is becoming one of the most important RNA modifications in plant growth and development, including defense, cell differentiation, and secondary metabolism. YT521-B homology (YTH) domain-containing RNA-binding proteins, identified as m6A readers in epitranscriptomics, could affect the fate of m6A-containing RNA by recognizing and binding the m6A site. Therefore, the identification and study of the YTH gene family in Liriodendron chinense (L. chinense) can provide a molecular basis for the study of the role of m6A in L. chinense, but studies on the YTH gene in L. chinense have not been reported. We identified nine putative YTH gene models in the L. chinense genome, which can be divided into DF subgroups and DC subgroups. Domain sequence analysis showed that the LcYTH protein had high sequence conservation. A LcYTH aromatic cage bag is composed of tryptophan and tryptophan (WWW). PrLDs were found in the protein results of YTH, suggesting that these genes may be involved in the process of liquid-liquid phase separation. LcYTH genes have different tissue expression patterns, but the expression of LcYTHDF2 is absolutely dominant in all tissues. In addition, the expression of the LcYTH genes is changed in response to ABA and MeJA. In this study, We identified and analyzed the expression pattern of LcYTH genes. Our results laid a foundation for further study of the function of the LcYTH gene and further genetic and functional analyses of m6A RNA modification in forest trees.

Genome-wide identification of the NAC gene family and its functional analysis in Liriodendron.

As one of the largest plant specific transcription factor families, NAC family members play an important role in plant growth, development and stress resistance. To investigate the function of NAC transcription factors during abiotic stress, as well as during somatic embryogenesis, we identified and characterized the NAC gene family in Liriodendron chinense. We found that most LcNAC members contain more than three exons, with a relatively conserved gene and motif structure, especially at the N-terminus. Interspecies collinearity analysis revealed a closer relationship between the L. chinense NACs and the P. trichocarpa NACs. We analyzed the expression of LcNAC in different tissues and under three abiotic stresses. We found that 12 genes were highly expressed during the ES3 and ES4 stages of somatic embryos, suggesting that they are involved in the development of somatic embryos. 6 LcNAC genes are highly expressed in flower organs. The expression pattern analysis of LcNACs based on transcriptome data and RT-qPCR obtained from L. chinense leaves indicated differential expression responses to drought, cold, and heat stress. Genes in the NAM subfamily expressed differently during abiotic stress, and LcNAC6/18/41/65 might be the key genes in response to abiotic stress. LcNAC6/18/41/65 were cloned and transiently transformed into Liriodendron protoplasts, where LcNAC18/65 was localized in cytoplasm and nucleus, and LcNAC6/41 was localized only in nucleus. Overall, our findings suggest a role of the NAC gene family during environmental stresses in L. chinense. This research provides a basis for further study of NAC genes in Liriodendron 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.

Characterization of the Liriodendron chinense Pentatricopeptide Repeat (PPR) Gene Family and Its Role in Osmotic Stress Response.

The Pentatricopeptide repeat (PPR) superfamily is a large gene family in plants that regulates organelle RNA metabolism, which is important for plant growth and development. However, a genome-wide analysis of the PPR gene family and its response to abiotic stress has not been reported for the relict woody plant Liriodendron chinense. In this paper, we identified 650 PPR genes from the L. chinense genome. A phylogenetic analysis showed that the LcPPR genes could roughly be divided into the P and PLS subfamilies. We found that 598 LcPPR genes were widely distributed across 19 chromosomes. An intraspecies synteny analysis indicated that duplicated genes from segmental duplication contributed to the expansion of the LcPPR gene family in the L. chinense genome. In addition, we verified the relative expression of Lchi03277, Lchi06624, Lchi18566, and Lchi23489 in the roots, stems, and leaves and found that all four genes had the highest expression in the leaves. By simulating a drought treatment and quantitative reverse transcription PCR (qRT-PCR) analysis, we confirmed the drought-responsive transcriptional changes in four LcPPR genes, two of which responded to drought stress independent of endogenous ABA biosynthesis. Thus, our study provides a comprehensive analysis of the L. chinense PPR gene family. It contributes to research into their roles in this valuable tree species' growth, development, and stress resistance.

Genome-Wide Identification and Expression Analysis of TPS Gene Family in Liriodendron chinense.

Terpenoids play a key role in plant growth and development, supporting resistance regulation and terpene synthase (TPS), which is the last link in the synthesis process of terpenoids. Liriodendron chinense, commonly called the Chinese tulip tree, is a rare and endangered tree species of the family Magnoliaceae. However, the genome-wide identification of the TPS gene family and its transcriptional responses to development and abiotic stress are still unclear. In the present study, we identified a total of 58 TPS genes throughout the L. chinense genome. A phylogenetic tree analysis showed that they were clustered into five subfamilies and unevenly distributed across six chromosomes. A cis-acting element analysis indicated that LcTPSs were assumed to be highly responsive to stress hormones, such as methyl jasmonate (MeJA) and abscisic acid (ABA). Consistent with this, transcriptome data showed that most LcTPS genes responded to abiotic stress, such as cold, drought, and hot stress, at the transcriptional level. Further analysis showed that LcTPS genes were expressed in a tissue-dependent manner, especially in buds, leaves, and bark. Quantitative reverse transcription PCR (qRT-PCR) analysis confirmed that LcTPS expression was significantly higher in mature leaves compared to young leaves. These results provide a reference for understanding the function and role of the TPS family, laying a foundation for further study of the regulation of TPS in terpenoid biosynthesis in L. chinense.

Differences in leaf gas exchange strategies explain Quercus rubra and Liriodendron tulipifera intrinsic water use efficiency responses to air pollution and climate change.

Trees continuously regulate leaf physiology to acquire CO2 while simultaneously avoiding excessive water loss. The balance between these two processes, or water use efficiency (WUE), is fundamentally important to understanding changes in carbon uptake and transpiration from the leaf to the globe under environmental change. While increasing atmospheric CO2 (iCO2 ) is known to increase tree intrinsic water use efficiency (iWUE), less clear are the additional impacts of climate and acidic air pollution and how they vary by tree species. Here, we couple annually resolved long-term records of tree-ring carbon isotope signatures with leaf physiological measurements of Quercus rubra (Quru) and Liriodendron tulipifera (Litu) at four study locations spanning nearly 100 km in the eastern United States to reconstruct historical iWUE, net photosynthesis (Anet ), and stomatal conductance to water (gs ) since 1940. We first show 16%-25% increases in tree iWUE since the mid-20th century, primarily driven by iCO2 , but also document the individual and interactive effects of nitrogen (NOx ) and sulfur (SO2 ) air pollution overwhelming climate. We find evidence for Quru leaf gas exchange being less tightly regulated than Litu through an analysis of isotope-derived leaf internal CO2 (Ci ), particularly in wetter, recent years. Modeled estimates of seasonally integrated Anet and gs revealed a 43%-50% stimulation of Anet was responsible for increasing iWUE in both tree species throughout 79%-86% of the chronologies with reductions in gs attributable to the remaining 14%-21%, building upon a growing body of literature documenting stimulated Anet overwhelming reductions in gs as a primary mechanism of increasing iWUE of trees. Finally, our results underscore the importance of considering air pollution, which remains a major environmental issue in many areas of the world, alongside climate in the interpretation of leaf physiology derived from tree rings.

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.

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.

Effect of the photoperiod on bud dormancy in Liriodendron chinense.

Bud dormancy and its release are complex physiological phenomena in plants. The molecular mechanisms of bud dormancy in Liriodendron chinense are mainly unknown. Here, we studied bud dormancy and the related physiological and molecular phenomena in Liriodendron under long-day (LD) and short-day (SD). Bud burst was released faster under LD than under SD. Abscisic acid (ABA), superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) activities were increased significantly under LD in Liriodendron buds. In contrast, the contents of gibberellic acid (GA3), ascorbic acid (AsA), glutathione (GSH), malondialdehyde (MDA), and ascorbate peroxidase (APX) activity decreased under LD but increased under SD. Differentially expressed genes (DEGs) were up-regulated under LD and down-regulated under SD and these changes correspondingly promoted (LD) or repressed (SD) cell division and the number and/or size of cells in the bud. Transcriptomic analysis of Liriodendron buds under different photoperiods identified 187 DEGs enriched in several pathways such as flavonoid biosynthesis and phenylpropanoid biosynthesis, plant hormone and signal transduction, etc. that are associated with antioxidant enzymes, non-enzymatic antioxidants, and subsequently promote the growth of the buds. Our findings provide novel insights into regulating bud dormancy via flavonoid and phenylpropanoid biosynthesis, plant hormone and signal transduction pathways, and ABA content. These physiological and biochemical traits would help detect bud dormancy in plants.

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.

Characterization of benzylisoquinoline alkaloid methyltransferases in Liriodendron chinense provides insights into the phylogenic basis of angiosperm alkaloid diversity.

Benzylisoquinoline alkaloids (BIAs) are a class of plant secondary metabolites with great pharmacological value. Their biosynthetic pathways have been extensively elucidated in the species from the Ranunculales order, such as poppy and Coptis japonica, in which methylation events play central roles and are directly responsible for BIA chemodiversity. Here, we combined BIA quantitative profiling and transcriptomic analyses to identify novel BIA methyltransferases (MTs) from Liriodendron chinense, a basal angiosperm plant. We identified an N-methyltransferase (LcNMT1) and two O-methyltransferases (LcOMT1 and LcOMT3), and characterized their biochemical functions in vitro. LcNMT1 methylates (S)-coclaurine to produce mono- and dimethylated products. Mutagenesis experiments revealed that a single-residue alteration is sufficient to change its substrate selectivity. LcOMT1 methylates (S)-norcoclaurine at the C6 site and LcOMT3 methylates (S)-coclaurine at the C7 site, respectively. Two key residues of LcOMT3, A115 and T301, are identified as important contributors to its catalytic activity. Compared with Ranunculales-derived NMTs, Magnoliales-derived NMTs were less abundant and had narrower substrate specificity, indicating that NMT expansion has contributed substantially to BIA chemodiversity in angiosperms, particularly in Ranunculales species. In summary, we not only characterized three novel enzymes that could be useful in the biosynthetic production of valuable BIAs but also shed light on the molecular origin of BIAs during angiosperm evolution.

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.