Single-cell RNA sequencing analysis of the embryogenic callus clarifies the spatiotemporal developmental trajectories of the early somatic embryo in Dimocarpus longan.

Plant embryogenic calli (ECs) can undergo somatic embryogenesis to regenerate plants. This process is mediated by regulatory factors, such as transcription factors and specifically expressed genes, but the precise molecular mechanisms underlying somatic embryogenesis at the single-cell level remain unclear. In this study, we performed high-resolution single-cell RNA sequencing analysis to determine the cellular changes in the EC of the woody plant species Dimocarpus longan (longan) and clarify the continuous cell differentiation trajectories at the transcriptome level. The highly heterogeneous cells in the EC were divided into 12 putative clusters (e.g., proliferating, meristematic, vascular, and epidermal cell clusters). We determined cluster-enriched expression marker genes and found that overexpression of the epidermal cell marker gene GDSL ESTERASE/LIPASE-1 inhibited the hydrolysis of triacylglycerol. In addition, the stability of autophagy was critical for the somatic embryogenesis of longan. The pseudo-timeline analysis elucidated the continuous cell differentiation trajectories from early embryonic cell division to vascular and epidermal cell differentiation during the somatic embryogenesis of longan. Moreover, key transcriptional regulators associated with cell fates were revealed. We found that ETHYLENE RESPONSIVE FACTOR 6 was characterized as a heat-sensitive factor that negatively regulates longan somatic embryogenesis under high-temperature stress conditions. The results of this study provide new spatiotemporal insights into cell division and differentiation during longan somatic embryogenesis at single-cell resolution.

Genome-wide high-throughput chromosome conformation capture analysis reveals hierarchical chromatin interactions during early somatic embryogenesis.

Somatic embryogenesis (SE), like zygotic embryo development, is a progressive process. Early SE is the beginning of a switch from a somatic to an embryogenic state and is an important stage for initiating chromatin reprogramming of SE. Previous studies suggest that changes in chromatin accessibility occur during early SE, although information on the 3D structure of chromatin is not yet available. Here, we present a chromosome-level genome assembly of longan (Dimocarpus longan) using PacBio combined with high-through chromosome conformation capture scaffolding, which resulted in a 446 Mb genome assembly anchored onto 15 scaffolds. During early SE, chromatin was concentrated and then decondensed, and a large number of long terminal repeat retrotransposons (LTR-RTs) were enriched in the local chromatin interaction region, suggesting LTR-RTs were involved in chromatin reorganization. Early SE was accompanied by the transformation from A to B compartments, and the interactions between B compartments were enhanced. Results from chromatin accessibility, monomethylation of histone H3 at lysine 4 (H3K4me1) modification, and transcription analyses further revealed a gene regulatory network for cell wall thickening during SE. Particularly, we found that the H3K4me1 differential peak binding motif showed abnormal activation of ethylene response factor transcription factors and participation in SE. The chromosome-level genomic and multiomics analyses revealed the 3D conformation of chromatin during early SE, providing insight into the molecular mechanisms underlying cell wall thickening and the potential regulatory networks of TFs during early SE in D. longan. These results provide additional clues for revealing the molecular mechanisms of plant SE.

Metabolic Composition and Quality Traits of Polygonatum cyrtonema Hua from Different Germplasms and Age Sections Based on Widely Targeted Metabolomics Analysis.

Polygonatum rhizomes are rich in various compounds with many biological activities and are widely used in functional foods and pharmaceutical products. In order to screen for superior Polygonatum cyrtonema Hua (P. cyrtonema) germplasm and also to elucidate the nutritional and medicinal values of rhizomes, the metabolic composition and quality traits of rhizomes from different germplasms and age sections of P. cyrtonema were analysed by widely targeted metabolomics, and the molecular mechanism of triacylglycerol synthesis was explored. The results showed that the different germplasms and age sections of P. cyrtonema were rich in different nutritional and medicinal components. Of these, the broad-leaved green stem (GK) germplasm is rich in polysaccharides, alkaloids, and lipids; the pointed-leaved green stem (JL) germplasm is rich in flavonoids, steroids, and amino acids, while the pointed-leaved purple stem (JZ) germplasm contains more phenolic acids. The one-year (AT) age section is rich in polysaccharides, steroids, organic acids, and lipids; the three years (CT) age section contains more flavonoids, alkaloids, and amino acid metabolites. Lipids were significantly enriched in the broad-leaved green stem germplasm and the one-year age section. Interestingly, the highest accumulation of triacylglycerols, an important component of lipids, was also found in the GK germplasm and the AT age section. Nineteen, 14, and 13 members of the glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAT), and diacylglycerol acyltransferase (DGAT) gene families, respectively, involved in triacylglycerol synthesis were also identified. The quantitative real-time PCR (qRT-PCR) results further suggested that the differentially expressed PcDGAT1, PcDGAT2.4, PcGPAT9.1, PcLPAT2.9, and PcLPAT4.3 genes may play important roles in triacylglycerol synthesis in P. cyrtonema. Therefore, this study provides a new theoretical reference for product development and the breeding of new varieties of Polygonatum species.

Whole Genome Analysis of SLs Pathway Genes and Functional Characterization of DlSMXL6 in Longan Early Somatic Embryo Development.

Strigolactones (SLs), a new class of plant hormones, are implicated in the regulation of various biological processes. However, the related family members and functions are not identified in longan (Dimocarpus longan Lour.). In this study, 23 genes in the CCD, D27, and SMXL family were identified in the longan genome. The phylogenetic relationships, gene structure, conserved motifs, promoter elements, and transcription factor-binding site predictions were comprehensively analysed. The expression profiles indicated that these genes may play important roles in longan organ development and abiotic stress responses, especially during early somatic embryogenesis (SE). Furthermore, GR24 (synthetic SL analogue) and Tis108 (SL biosynthesis inhibitor) could affect longan early SE by regulating the levels of endogenous IAA (indole-3-acetic acid), JA (jasmonic acid), GA (gibberellin), and ABA (abscisic acid). Overexpression of SMXL6 resulted in inhibition of longan SE by regulating the synthesis of SLs, carotenoids, and IAA levels. This study establishes a foundation for further investigation of SL genes and provides novel insights into their biological functions.

Genome-Wide Identification and Expression Analysis of Bx Involved in Benzoxazinoids Biosynthesis Revealed the Roles of DIMBOA during Early Somatic Embryogenesis in Dimocarpus longan Lour.

Benzoxazinoids (BXs) are tryptophan-derived indole metabolites and play a role in various physiological processes, such as auxin metabolism. Auxin is essential in the process of somatic embryogenesis (SE) in plants. In this study, we used bioinformatics, transcriptome data, exogenous treatment experiments, and qPCR analysis to study the evolutionary pattern of Bx genes in green plants, the regulatory mechanism of DlBx genes during early SE, and the effect of 2,4-dihydroxy-7-methoxy-1,4-benzoxazine-3-one (DIMBOA) on the early SE in Dimocarpus longan Lour. The results showed that 27 putative DlBxs were identified in the longan genome; the Bx genes evolved independently in monocots and dicots, and the main way of gene duplication for the DlBx was tandem duplication (TD) and the DlBx were strongly constrained by purification selection during evolution. The transcriptome data indicated varying expression levels of DlBx during longan early SE, and most DlBxs responded to light, temperature, drought stress, and 2,4-dichlorophenoxyacetic acid (2,4-D) treatment; qRT-PCR results showed DlBx1, DlBx6g and DlBx6h were responsive to auxin, and treatment with 0.1mg/L DIMBOA for 9 days significantly upregulated the expression levels of DlBx1, DlBx3g, DlBx6c, DlBx6f, DlB6h, DlBx7d, DlBx8, and DlBx9b. The correlation analysis showed a significantly negative correlation between the expression level of DlBx1 and the endogenous IAA contents; DIMBOA significantly promoted the early SE and significantly changed the endogenous IAA content, and the IAA content increased significantly at the 9th day and decreased significantly at the 13th day. Therefore, the results suggested that DIMBOA indirectly promote the early SE by changing the endogenous IAA content via affecting the expression level of DlBx1 and hydrogen peroxide (H2O2) content in longan.

Genome-wide identification and expression analysis of the GAD family reveal their involved in embryogenesis and hormones responses in Dimocarpus longan Lour.

Glutamate decarboxylase (GAD) is involved in GABA metabolism and plays an essential regulatory role in plant growth, abiotic stresses, and hormone response. This study investigated the expression mechanism of the GAD family during longan early somatic embryogenesis (SE) and identified 6 GAD genes based on the longan genome. Homology analysis indicated that DlGAD genes had a closer relationship with dicotyledonous plants. The analysis of cis-acting elements in the promoter region suggests that the GAD genes were associated with various stress responses and hormones. RNA sequencing (RNA-Seq) and the qRT-PCR data indicated that most DlGAD genes were highly expressed in the incomplete compact pro-embryogenic cultures (ICpEC) and upregulated in longan embryogenic callus (EC) after treatments with 2,4-D, high temperature (35 °C), IAA, and ABA. Moreover, the RNA-Seq analysis also revealed that DlGADs exhibit different expression patterns in various tissues and organs. The subcellular localization results showed that DlGAD5 was localized in the cytoplasm, suggesting that it played a role in the cytoplasm. Transient overexpression of DlGAD5 enhanced the expression levels of DlGADs and increased the activity of glutamate decarboxylase in longan embryogenic callus (EC), while the content of glutamic acid decreased. Thus, the DlGAD gene can play an important role in the early somatic embryogenesis of longan by responding to hormones such as IAA and ABA. DlGAD5 can affect the growth and development of longan by stimulating the expression of the DlGAD gene family, thereby increasing the GAD activity in the early SE of longan, participating in hormone synthesis and signaling pathways.

Genome-wide analysis of the SAUR gene family and function exploration of DlSAUR32 during early longan somatic embryogenesis.

The early auxin responsive small auxin up-regulated RNA (SAUR) family is an important gene family in the auxin signal transduction pathway. This study focused on the regulatory mechanism of DlSAUR genes during early somatic embryogenesis (SE) and its response to hormone treatment and abiotic stress. Mining of the available Dimocarpus longan Lour. (D. longan) genome sequence yielded 68 putative SAUR genes. Transcript profiles based on RNA-seq data showed that most of the 24 detected DlSAUR genes were highly expressed in the globular embryos (GE) (10) and most of them responded to heat stress and 2,4-D treatment. The results of qRT-PCR showed that most of DlSAUR genes were up-regulated under auxin inhibitor N-1-naphthylphthalamic acid (NPA) and auxin indole-3-acetic acid (IAA) treatments. Moreover, NPA could promote longan SE. The assay for ATAC-seq data analysis showed that chromatin accessibility of 19 of the 24 DlSAUR genes were open during early SE, and most DlSAUR genes differentially expressed during early SE were not associated with H3K4me1 signal enrichment. The DlSAUR32 was selected for subcellular localization and RNA-seq analysis, which encode a cell nuclear-localized protein. Dual-luciferase assays and transient transformation showed that the transcription factors (TFs) DlWRKY75-1 and DlWRKY75-2 might bind to the DlSAUR32 promoters to inhibition gene transcription. Transient overexpression of DlWRKY75-1 and DlWRKY75-2 decreased IAA content in N. benthamiana leaves. Thus, the regulatory network composed of DlSAUR32 and its related TFs may regulate the early longan SE and be involved in the auxin response regulatory pathway of longan.

Integrated proteome and acetylome analyses provide novel insights into early somatic embryogenesis of Dimocarpus longan.

Longan (Dimocarpus longan) is a precious subtropical fruit with high nutritional value. The somatic embryogenesis (SE) affects the quality and yield of fruit. Apart from clonal propagation, SE has extensive applications in genetic improvement and mutation. Thus, understanding the molecular basis of embryogenesis in longan will help to develop strategies for mass production of quality planting material. Lysine acetylation (Kac) plays an important role in diverse cellular processes, but limited knowledge is available regarding acetylation modifications in plant early SE. In this study, the proteome and acetylome of longan embryogenic callus (ECs) and globular embryos (GEs) were investigated. In total, 7232 proteins and 14,597 Kac sites were identified, and this resulted in the discovery of 1178 differentially expressed proteins and 669 differentially expressed acetylated proteins. KEGG and GO analysis showed that glucose metabolism, carbon metabolism, fatty acid degradation, and oxidative phosphorylation pathways were influenced by Kac modification. Furthermore, sodium butyrate (Sb, a deacetylase inhibitor) led to reduced the proliferation and delayed the differentiation of ECs by regulating the homeostasis of reactive oxygen species (ROS) andindole-3-acetic acid (IAA). Our study provides a comprehensive proteomic and acetylomic analysis to aid in understanding the molecular mechanisms involved in early SE, representing a potential tool for genetic improvement of longan.

PAs Regulate Early Somatic Embryo Development by Changing the Gene Expression Level and the Hormonal Balance in Dimocarpus longan Lour.

Polyamines (PAs) play an important regulatory role in many basic cellular processes and physiological and biochemical processes. However, there are few studies on the identification of PA biosynthesis and metabolism family members and the role of PAs in the transition of plant embryogenic calli (EC) into globular embryos (GE), especially in perennial woody plants. We identified 20 genes involved in PA biosynthesis and metabolism from the third-generation genome of longan (Dimocarpus longan Lour.). There were no significant differences between longan and other species regarding the number of members, and they had high similarity with Citrus sinensis. Light, plant hormones and a variety of stress cis-acting elements were found in these family members. The biosynthesis and metabolism of PAs in longan were mainly completed by DlADC2, DlSAMDC2, DlSAMDC3, DlSPDS1A, DlSPMS, DlCuAOB, DlCuAO3A, DlPAO2 and DlPAO4B. In addition, 0.01 mmol∙L-1 1-aminocyclopropane-1-carboxylic acid (ACC), putrescine (Put) and spermine (Spm), could promote the transformation of EC into GE, and Spm treatment had the best effect, while 0.01 mmol∙L-1 D-arginine (D-arg) treatment inhibited the process. The period between the 9th and 11th days was key for the transformation of EC into GE in longan. There were higher levels of gibberellin (GA), salicylic acid (SA) and abscisic acid (ABA) and lower levels of indole-3-acetic acid (IAA), ethylene and hydrogen peroxide (H2O2) in this key period. The expression levels in this period of DlADC2, DlODC, DlSPDS1A, DlCuAOB and DlPAO4B were upregulated, while those of DlSAMDC2 and DlSPMS were downregulated. These results showed that the exogenous ACC, D-arg and PAs could regulate the transformation of EC into GE in longan by changing the content of endogenous hormones and the expression levels of PA biosynthesis and metabolism genes. This study provided a foundation for further determining the physicochemical properties and molecular evolution characteristics of the PA biosynthesis and metabolism gene families, and explored the mechanism of PAs and ethylene for regulating the transformation of plant EC into GE.

A new iridoid glycoside and a new cinnamoyl glycoside from Scrophularia ningpoensis Hemsl.

A new iridoid glycoside, namely 8-O-(threo-2, 3-dihydroxyl-3-phenyl-propionoyl)-harpagide (1), along with a new cinnamoyl glycoside named as cis-sibirioside A (2), were isolated from Scrophularia ningpoensis Hemsl. Their chemical structures were completely established by spectroscopic methods and comparison with related literatures. Compound 1 exhibited moderate antifouling effect against the settlement of Balanus amphitrite larvae with IC50 being 13.5 µg/mL and LC50 > 25 µg/mL.