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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. [ABSTRACT FROM AUTHOR]

The basic leucine zip (bZIP) transcription factors (TFs) are a group of highly conserved gene families that play important roles in plant growth and resistance to adversity stress. However, studies on hormonal regulatory pathways and functional analysis during somatic embryogenesis (SE) in Dimocarpus longan is still unavailable. In this study, a total of 51 bZIP family members were systematically identified in the whole genome of longan, a comprehensive bioinformatics analysis of DlbZIP (bZIP family members of D. longan) was performed, and subcellular localization and profiles patterns after transiently transformed DlbZIP60 were analyzed. The combined analysis of RNA-seq, ATAC-seq and ChIP-seq showed that four members have different H3K4me1 binding peaks in early SE and differentially expressed with increased chromatin accessibility. Comparative transcriptome analysis of bZIPs expression in early SE, different tissues and under 2,4-D treatment revealed that DlbZIP family might involved in growth and development during longan early SE. The qRT-PCR results implied that DlbZIP family were subjected to multiple hormonal responses and showed different degrees of up-regulated expression under indole-3-acetic acid (IAA), abscisic acid (ABA) and methyl jasmonate (MeJA) treatments, which indicated that they played an important role in the hormone synthesis pathways associated with the early SE of longan. Subcellular localization showed that DlbZIP60 was located in the nucleus, and the contents of endogenous IAA, MeJA and ABA were up-regulated in transiently DlbZIP60 overexpressed cell lines. These results suggest that DlbZIP60 may mediate hormones pathways that functions the development during early SE in longan. [ABSTRACT FROM AUTHOR]

Ran GTPases play essential roles in plant growth and development. Our previous studies revealed the nuclear localization of DlRan3A and DlRan3B proteins and proposed their functional redundancy and distinction in Dimocarpus longan somatic embryogenesis, hormone, and abiotic stress responses. To further explore the possible roles of DlRan3A and DlRan3B, gene expression analysis by qPCR showed that their transcripts were both more abundant in the early embryo and pulp in longan. Heterologous expression of DlRan3A driven by its own previously cloned promoter led to stunted growth, increased root hair density, abnormal fruits, bigger seeds, and enhanced abiotic stress tolerance. Conversely, constitutive promoter CaMV 35S (35S)-driven expression of DlRan3A, 35S, or DlRan3B promoter-controlled expression of DlRan3B did not induce the alterations in growth phenotype, while they rendered different hypersensitivities to abiotic stresses. Based on the transcriptome profiling of longan Ran overexpression in tobacco plants, we propose new mechanisms of the Ran-mediated regulation of genes associated with cell wall biosynthesis and expansion. Also, the transgenic plants expressing DlRan3A or DlRan3B genes controlled by 35S or by their own promoter all exhibited altered mRNA levels of stress-related and transcription factor genes. Moreover, DlRan3A overexpressors were more tolerant to salinity, osmotic, and heat stresses, accompanied by upregulation of oxidation-related genes, possibly involving the Ran-RBOH-CIPK network. Analysis of a subset of selected genes from the Ran transcriptome identified possible cold stress-related roles of brassinosteroid (BR)-responsive genes. The marked presence of genes related to cell wall biosynthesis and expansion, hormone, and defense responses highlighted their close regulatory association with Ran. [ABSTRACT FROM AUTHOR]

GATA transcription factors, which are DNA-binding proteins with type IV zinc finger binding domains, have a role in transcriptional regulation in biological organisms. They have an indispensable role in the growth and development of plants, as well as in improvements in their ability to face various environmental stresses. To date, GATAs have been identified in many gene families, but the GATA gene in longan (Dimocarpus longan Lour) has not been studied in previous explorations. Various aspects of genes in the longan GATA family, including their identification and classification, the distribution of their positions on chromosomes, their exon/intron structures, a synteny analysis, their expression at different temperatures, concentration of PEG, early developmental stages of somatic embryos and their expression levels in different tissues, and concentrations of exogenous hormones, were investigated in this study. This study showed that the 22 DlGATAs could be divided into four subfamilies. There were 10 pairs of homologous GATA genes in the synteny analysis of DlGATA and AtGATA. Four segmental replication motifs and one pair of tandem duplication events were present among the DlGATA family members. The cis-acting elements located in promoter regions were also found to be enriched with light-responsive elements, which contained related hormone-responsive elements. In somatic embryos, DlGATA4 is upregulated for expression at the globular embryo (GE) stage. We also found that DlGATA expression was strongly up-regulated in roots and stems. The study demonstrated the expression of DlGATA under hormone (ABA and IAA) treatments in embryogenic callus of longan. Under ABA treatment, DlGATA4 was up-regulated and the other DlGATA genes did not respond significantly. Moreover, as demonstrated with qRT-PCR, the expression of DlGATA genes showed strong up-regulated expression levels under 100 μ m o l · L − 1 concentration IAA treatment. This experiment further studied these and simulated their possible connections with a drought response mechanism, while correlating them with their expression under PEG treatment. Overall, this experiment explored the GATA genes and dug into their evolution, structure, function, and expression profile, thus providing more information for a more in-depth study of the characteristics of the GATA family of genes. [ABSTRACT FROM AUTHOR]

B3 family transcription factors play an essential regulatory role in plant growth and development processes. This study performed a comprehensive analysis of the B3 family transcription factor in longan (Dimocarpus longan Lour.), and a total of 75 DlB3 genes were identified. DlB3 genes were unevenly distributed on the 15 chromosomes of longan. Based on the protein domain similarities and functional diversities, the DlB3 family was further clustered into four subgroups (ARF, RAV, LAV, and REM). Bioinformatics and comparative analyses of B3 superfamily expression were conducted in different light and with different temperatures and tissues, and early somatic embryogenesis (SE) revealed its specific expression profile and potential biological functions during longan early SE. The qRT-PCR results indicated that DlB3 family members played a crucial role in longan SE and zygotic embryo development. Exogenous treatments of 2,4-D (2,4-dichlorophenoxyacetic acid), NPA (N-1-naphthylphthalamic acid), and PP333 (paclobutrazol) could significantly inhibit the expression of the DlB3 family. Supplementary ABA (abscisic acid), IAA (indole-3-acetic acid), and GA3 (gibberellin) suppressed the expressions of DlLEC2, DlARF16, DlTEM1, DlVAL2, and DlREM40, but DlFUS3, DlARF5, and DlREM9 showed an opposite trend. Furthermore, subcellular localization indicated that DlLEC2 and DlFUS3 were located in the nucleus, suggesting that they played a role in the nucleus. Therefore, DlB3s might be involved in complex plant hormone signal transduction pathways during longan SE and zygotic embryo development. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: Plant embryogenic calli can undergo somatic embryogenesis to regenerate plants. The single‐cell RNA‐seq results described herein provide new spatiotemporal insights into cell division and differentiation in the Dimocarpus longan (longan) embryogenic callus. The generated gene expression map will enable us to investigate how embryonic cells differentiate into vascular and epidermal cells. The results may provide the foundation for future molecular and cellular investigations into the mechanisms mediating longan somatic embryogenesis and zygotic embryogenesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) are widely involved in plant growth and development. However, the function of circRNAs in plant somatic embryogenesis (SE) remains elusive. Here, by using high-throughput sequencing, a total of 5029 circRNAs were identified in the three stages of longan (Dimocarpus longan Lour.) early SE. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that differentially expressed (DE) circRNA host genes were enriched in the 'non-homologous end-joining' (NHEJ) and 'butanoate metabolism' pathways. In addition, the reactive oxygen species (ROS) content during longan early SE was determined. The results indicated that ROS-induced DNA double-strand breaks may not depend on the NHEJ repair pathway. Correlation analyses of the levels of related metabolites (glutamate, γ-aminobutyrate and pyruvate) and the expression levels of circRNAs and their host genes involved in butanoate metabolism were performed. The results suggested that circRNAs may act as regulators of the expression of cognate mRNAs, thereby affecting the accumulation of related compounds. A competing endogenous RNA (ceRNA) network of DE circRNAs, DE mRNAs, DE long noncoding RNAs (lncRNAs) and DE microRNAs (miRNAs) was constructed. The results showed that the putative targets of the noncoding RNA (ncRNAs) were significantly enriched in the KEGG pathways 'mitogen-activated protein kinase signaling' and 'nitrogen metabolism'. Furthermore, the expression patterns of the candidate circRNAs, lncRNAs, miRNAs and mRNAs confirmed the negative correlation between miRNAs and ceRNAs. In addition, two circRNA overexpression vectors were constructed to further verify the ceRNA network correlations in longan early SE. Our study revealed the potential role of circRNAs in longan early SE, providing new insights into the intricate regulatory mechanism underlying plant SE. [ABSTRACT FROM AUTHOR]

DNA methylation plays key roles in regulating genes which control somatic embryogenesis (SE) in plant. 5-Azacytidine (5-AzaC) was considered as a common inhibitor of DNA methylation. Longan (Dimocarpus longan Lour.) SE has been established and used as model system for studying mechanism of SE in woody plants. However, effects of 5-AzaC on longan EC and the underlying mechanism at molecular level are rarely understood. In this study, RNA-seq was performed for transcriptome analysis of longan EC treated with or without 5-AzaC were analysed. A total of 29,232 expressed genes were obtained by aligned to longan reference genome and 1,617 differentially expressed genes (DEGs) were got in the pairwise comparisons of CK_vs_T. Integrating the KEGG enrichment pathway analysis showed that 'butanoate metabolism', 'C5-branched dibasic acid metabolism', 'sulphur metabolism', 'selenocompound metabolism', 'pantothenate and CoA biosynthesis', 'phagosome', and 'plant hormone signal transduction' were significantly enriched pathways, while the analysis of DEGs revealed that 5-AzaC-treatment could alter genes expression which related to the SE to adjust the complicated metabolic pathways. Therefore, these results indicated that 5-AzaC-treatment positively impact the development of early SE from longan and a decrease in the global DNA methylation level during early SE in longan. [ABSTRACT FROM AUTHOR]

DNA methylation plays essential roles in gene regulation, chromatin structure stability, gene imprinting, X chromosome inactivation and embryonic development. However, the dynamics and functions of DNA methylation during the early stage of longan (Dimocarpus longan) somatic embryogenesis (SE) are still unclear. In this study, we carried out whole genome bisulphite sequencing and transcriptome sequencing analyses for embryogenic callus (EC), incomplete compact pro-embryogenic cultures (ICpEC) and globular embryos (GE) in an early SE system. At a global level, the DNA 5-methylcytosine content in EC, ICpEC and GE was 24.59, 19.65 and 19.74%, respectively, suggesting a global decrease in DNA methylation from EC to ICpEC and then a slight increase from ICpEC to GE. Differentially methylated region (DMR) analysis showed that hypomethylation mainly occurred in CHH contexts. Gene ontology and Kyoto encyclopedia of genes and genomes analysis of hypomethylated-CHH-DMR-associated genes revealed that zein biosynthesis, fatty acid biosynthesis, circadian rhythm and mitophagy pathways were involved in longan early SE. Expression patterns of DNA methyltransferase and demethylase genes during longan early SE suggested that the decrease in DNA methylation was probably regulated by DNA methyltransferase genes and the DNA demethylase gene REPRESSOR OF SILENCING 1 (ROS1). The correlation between DNA hypomethylation and gene expression revealed that decreased DNA methylation did not cause extensive changes in gene expression during early longan SE and that gene expression may be affected by methylation changes in gene and downstream regions. Inhibiting DNA methylation with 5-azacytidine treatment in EC promoted the formation of GE and enhanced the capability of longan SE. Our results suggest that DNA demethylation has important roles in longan SE development. [ABSTRACT FROM AUTHOR]

The Sm proteins are a conserved protein family with Sm motifs. The family includes Sm and Sm-like proteins, which play important roles in pre-mRNA splicing. Most research on the Sm proteins have been conducted in herbaceous plants, and less in woody plants such as Dimocarpus longan (longan). And the embryo development status significantly affects the quality and yield of longan. In this study, we conducted a genome-wide analysis of longan Sm genes (DlSm) to clarify their roles during somatic embryogenesis (SE) and identified 29 Sm genes. Phylogenetic analysis deduced longan Sm proteins clustered into 17 phylogenetic groups with the homologous Sm proteins of Arabidopsis thaliana. We also analyzed the gene structures, motif compositions, and conserved domains of the longan Sm proteins. The promoter sequences of the DlSm genes contained many light, endosperm development, hormone, and temperature response elements, which suggested their possible functions. In the non-embryogenic callus(NEC) and during early SE in longan, the alternative splicing(AS) events of DlSm genes indicated that these genes may influence SE development by changing gene structures and sequences. The kinetin(KT) hormone, and blue and white light treatments affected the differentiation and growth of longan embryonic callus(EC) probably by affecting the AS events of DlSm genes. Expression profiles showed the possible functional divergence among Sm genes, and different hormones and light qualities affected their expression levels. The expression trends of the DlSm genes determined by RNA sequencing as fragments per kilobase of exon model per million mapped reads (FPKM) and by real-time quantitative PCR(qRT-PCR) during early SE in longan showed that the expression of the DlSm genes was affected by the growth and differentiation of longan SE, and decreased as the somatic embryo differentiation progressed. The results will contributed to understanding the longan Sm gene family and provide a basis for future functional validation studies. [ABSTRACT FROM AUTHOR]

miRNAs are endogenous regulatory factors that play pivotal roles in post-transcriptional regulation. However, their specific roles in early somatic embryogenesis (SE) remain unclear. Study of the SE system is fundamental for clarifying the molecular mechanisms in Dimocarpus longan. We identified 289 known miRNAs from 106 different miRNA families and 1087 novel miRNAs during early longan SE, including embryogenic callus (EC), incomplete pro-embryogenic culture (ICpEC), globular embryo (GE), and non-embryogenic callus (NEC). The abundances of known miRNAs were concentrated in GE. The differentially expression (DE) miRNAs showed five expression patterns during early SE. Largely miRNAs were expressed highly and specially in EC, ICpEC, and GE, respectively. Some miRNAs and putative target genes were enriched in lignin metabolism. Most potential targets were related to the pathways of plant hormone signal transduction, alternative splicing, tyrosine metabolism and sulfur metabolism in early longan SE. The regulatory relationships between dlo-miR166a-3p and DlHD-zip8, dlo-miR397a and DlLAC7, dlo-miR408-3p and DlLAC12 were confirmed by RNA ligase-mediated rapid amplification of cDNA ends. The expression patterns of eight DE miRNAs detected by qRT-PCR were consistent with RNA-seq. Finally, the miRNA regulatory network in early SE was constructed, which provided new insight into molecular mechanism of early SE in longan. [ABSTRACT FROM AUTHOR]

Background: The APETALA2/ethylene responsive factor (AP2/ERF) superfamily members are transcription factors that regulate diverse developmental processes and stress responses in plants. They have been identified in many plants. However, little is known about the AP2/ERF superfamily in longan (Dimocarpus longan Lour.), which is an important tropical/subtropical evergreen fruit tree that produces a variety of bioactive compounds with rich nutritional and medicinal value. We conducted a genome-wide analysis of the AP2/ERF superfamily and its roles in somatic embryogenesis (SE) and developmental processes in longan. Results: A genome-wide survey of the AP2/ERF superfamily was carried out to discover its evolution and function in longan. We identified 125 longan AP2/ERF genes and classified them into the ERF (101 members), AP2 (19 members), RAV (four members) families, and one Soloist. The AP2 and Soloist genes contained one to ten introns, whereas 87 genes in the ERF and RAV families had no introns. Hormone signaling molecules such as methyl jasmonate (MeJA), abscisic acid (ABA), gibberellin, auxin, and salicylic acid (SA), and stress response cis-acting element low-temperature (55) and defense (49) boxes also were identified. We detected diverse single nucleotide polymorphisms (SNPs) between the 'Hong He Zi' (HHZ) and 'SI JI MI' (SJM) cultivars. The number of insertions and deletions (InDels) was far fewer than SNPs. The AP2 family members exhibited more alternative splicing (AS) events in different developmental processes of longan than members of the other families. Expression pattern analysis revealed that some AP2/ERF members regulated early SE and developmental processes in longan seed, root, and flower, and responded to exogenous hormones such as MeJA, SA, and ABA, and 2,4-D, a synthetic auxin. Protein interaction predictions indicated that the Baby Boom (BBM) transcription factor, which was up-regulated at the transcriptional level in early SE, may interact with the LALF/AGL15 network. Conclusions: The comprehensive analysis of molecular evolution and expression patterns suggested that the AP2/ERF superfamily may plays an important role in longan, especially in early SE, and in seed, root, flower, and young fruit. This systematic analysis provides a foundation for further functional characterization of the AP2/ERF superfamily with the aim of longan improvement. [ABSTRACT FROM AUTHOR]

Background: Long non-coding RNAs (lncRNAs) are involved in variable cleavage, transcriptional interference, regulation of DNA methylation and protein modification. However, the regulation of lncRNAs in plant somatic embryos remains unclear. The longan (Dimocarpus longan) somatic embryogenesis (SE) system is a good system for research on longan embryo development. Results: In this study, 7643 lncRNAs obtained during early SE in D. longan were identified by high-throughput sequencing, among which 6005 lncRNAs were expressed. Of the expressed lncRNAs, 4790 were found in all samples and 160 were specifically expressed in embryogenic callus (EC), 154 in incomplete embryogenic compact structures (ICpECs), and 376 in globular embryos (GEs). We annotated the 6005 expressed lncRNAs, and 1404 lncRNAs belonged to 506 noncoding RNA (ncRNA) families and 4682 lncRNAs were predicted to target protein-coding genes. The target genes included 5051 cis-regulated target genes (5712 pairs) and 1605 trans-regulated target genes (3618 pairs). KEGG analysis revealed that most of the differentially expressed target genes (mRNAs) of the lncRNAs were enriched in the "plant-pathogen interaction" and "plant hormone signaling" pathways during early longan SE. Real-time quantitative PCR confirmed that 20 selected lncRNAs showed significant differences in expression and that five lncRNAs were related to auxin response factors. Compared with the FPKM expression trends, 16 lncRNA expression trends were the same in qPCR. In lncRNA-miRNA-mRNA relationship prediction, 40 lncRNAs were predicted to function as eTMs for 15 miRNAs and 7 lncRNAs were identified as potential miRNA precursors. In addition, we verified the lncRNA-miRNA-mRNA regulatory relationships by transient expression of miRNAs (miR172a, miR159a.1 and miR398a). Conclusion: Analyses of lncRNAs during early longan SE showed that differentially expressed lncRNAs were involved in expression regulation at each SE stage, and may form a regulatory network with miRNAs and mRNAs. These findings provide new insights into lncRNAs and lay a foundation for future functional analysis of lncRNAs during early longan SE. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Somatic embryogenesis (SE), which resembles zygotic embryogenesis, is an essential component of the process of plant cell differentiation and embryo development. Although microRNAs (miRNAs) are important regulators of many plant develop- mental processes, their roles in SE have not been thoroughly investigated. In this study, we used deep-sequencing, computational, and qPCR methods to identify, profile, and describe conserved and novel miRNAs involved in longan (Dimocarpus longan) SE. A total of 643 conserved and 29 novel miRNAs (including star strands) from more than 169 miRNA families were identified in longan embryogenic tissue using Solexa sequencing. By combining computational and degradome sequencing approaches, we were able to predict 2063 targets of 272 miRNAs and verify 862 targets of 181 miRNAs. Target annotation revealed that the putative targets were involved in a broad variety of biological processes, including plant metabolism, signal transduction, and stimulus response. Analysis of stage- and tissue-specific expressions of 20 conserved and 4 novel miRNAs indicated their possible roles in longan SE. These miRNAs were dlo-miR156 family members and dlo-miR166c* associated with early embryonic culture developmental stages; dlo-miR26, dlo-miR160a, and families dlo-miR159, dlo-miR390, and dlo-miR398b related to heart-shaped and torpedo- shaped embryo formation; dlo-miR4a, dlo-miR24, dlo-miR167a, dlo-miR168a*, dlo-miR397a, dlo-miR398b.1, dlo-miR398b.2, dlo-miR808 and dlo-miR5077 involved in cotyledonary embryonic development; and dlo-miR17 and dlo-miR2089*-1 that have regulatory roles during longan SE. In addition, dlo-miR167a, dlo-miR808, and dlo-miR5077 may be required for mature embryo formation. This study is the first reported investigation of longan SE involving large-scale cloning, characterization, and expression profiling of miRNAs and their targets. The reported results contribute to our knowledge of somatic embryo miRNAs and provide insights into miRNA biogenesis and expression in plant somatic embryo development. [ABSTRACT FROM AUTHOR]

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. • Kac is involved in the early regulation of longan embryogenesis with various regulatory pathways. • Sodium butyrate led to reduced the proliferation and delayed the differentiation of ECs by regulating the homeostasis of ROS and IAA. • Carbon metabolism, fatty acid degradation, and oxidative phosphorylation pathways were mainly influenced by Kac. [ABSTRACT FROM AUTHOR]

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. • DlSAUR genes were sensitive to IAA and NPA treatments. • The regulatory network of transcription factors and DlSAUR genes was predicted. • DlWRKY75-1 and DlWRKY75-2 can significantly reduce the expression of DlSAUR32. • Overexpression of DlWRKY75-1 and DlWRKY75-2 decreased IAA content. [ABSTRACT FROM AUTHOR]

• 327 CYP were found from the longan genome, and they exhibited a strong expansion with mainly tandem duplication (TD) events. • DlCYPs expressed higher in globular embryos (GE) compared with those in incomplete compact pro-embryogenic (ICpEC) stage and the embryogenic callus (EC) stage in longan. • DlCYPs had more AS events in ICpEC compared to those in EC and GE. Their transcript isoforms showed expression patterns during early SE in longan. Cytochrome P450 (CYP), a multi-gene superfamily, is involved in a broad range of physiological processes, including hormone responses and secondary metabolism throughout the plant life cycle. Longan (Dimocarpus longan), a subtropical and tropical evergreen fruit tree, its embryonic development is closely related to the yield and quality of fruits. And a large number of secondary metabolites, such as flavonoids and carotenoids, are also produced during the longan somatic embryogenesis (SE). It is important, therefore, to study potential functions of CYPs in longan. However, the knowledge of longan CYP s is still very limited. Here, a total of 327 DlCYPs were identified using the genome-search method, which could be classified into nine clans. The expansion of the DlCYP family was mainly caused by tandem duplication (TD) events. Promoter cis -acting elements analysis elucidated that DlCYPs played important roles in hormonal responses. A total of 246 DlCYPs exhibited six different expression patterns during the early SE based on longan transcriptomic data. Eight DlCYPs underwent alternative splicing (AS) events, and they might produce one to six isoforms. And the AS transcript of DlCYP97C1 might act as an alternative to the full-length transcript in ICpEC and GE stages. Finally, protein–protein interaction (PPI) networks and miRNA target prediction elucidated that DlCYPs might be involved in the phenylpropanoid metabolic pathway and primarily regulated and targeted by miR413. In summary, our results provided valuable inventory for understanding the classification and biological functions of DlCYPs and provided insight into further functional verification of DlCYPs during the longan early SE. [ABSTRACT FROM AUTHOR]

• A total of 69 core cell cycle genes (CCCs) family members were identified in the longan genome. • Either silencing of CKS or CYCD3;2 increased the expression of RB-E2F pathway-related genes. • Silencing of CYCD3;2 might lead to apoptosis in longan embryogenic callus. • ABA, 2,4-D and PEG4000 could increase the expression of all selected longan CCCs , while NaCl did not. Core cell cycle genes (CCCs) are essential regulators of cell cycle operation. In this study, a total of 69 CCCs family members, including 37 CYCs , 20 CDKs , five E2F/DPs , three KRPs , two RBs , one CKS and one Wee1 , were identified from the longan genome. Phylogenetic and motifs analysis showed the evolutionary conservation of CCCs. Transcriptome dataset showed that CCCs had various expression patterns during longan early somatic embryogenesis (SE). Either CKS or CYCD3;2 silencing increased the expression of RB-E2F pathway genes, and the silencing of CYCD3;2 might induce the process of apoptosis in longan embryogenic callus (EC) cells. In addition, The qRT-PCR results showed that the expression levels of CDKG2 , CYCD3;2 , CYCT1;2 , CKS and KRP1 were elevated by ABA, 2,4-D and PEG4000 treatments, while CDKG2 and CYCT1;2 were inhibited by NaCl treatment. In conclusion, our study provided valuable information for understanding the characterization and biological functions of longan CCCs. [ABSTRACT FROM AUTHOR]

ADP-ribosylation modification considered as a model to study histone post-translational modification in chromatin modification. Despite it was reported in many plants, the study of ARFs gene family in longan was still unclear. In this study, 14 longan ARFs genes were identified using the longan genome (the third-generation genome) and further divided into two major groups, including the DlARF in the I-II group and the ARF-like (DlARL) in the III-V group, according to their structure and evolutionary characteristics. Whole-genome duplication (WGD) and segmental duplication events played a major role in the expansion of the DlARFs gene family, the synteny and phylogenetic analyses provided a deeper insight into the evolutionary characteristics of the DlARFs. Protein-protein interactions suggested that some DlARFs proteins may interact to participate in biological processes. Promoter analysis showed more stress response elements in DlARF5 , DlGB1 , DlARL1 , DlARL2, and DlARL8a , suggesting that they may participate in abiotic stress. Expression profiles of DlARFs by quantitative real-time PCR (qRT-PCR) showed that they were abundant accumulation during early somatic embryogenesis (SE). Expression pattern analysis of RNA-seq and qRT-PCR revealed that some ARFs members regulated early SE, and respond to exogenous hormones and abiotic stress such as abscisic acid (ABA), gibberellin A3 (GA3), salicylic acid (SA), methyl jasmonate (MeJA), cold, and heat. Our study provides new insights for further research on the potential function of DlARFs , which may be useful for the improvement of longan. [ABSTRACT FROM AUTHOR]

• 58 DlDDX family genes were identified during the early SE in longan genome. • DlDDX proteins appeared neofunctionalization in longan, and associated with abiotic stress during longan early SE. • Upregulation of 8 DlDDXs genes might promote abortion of in vitro and in vivo embryogenesis. • DlDDX3, 15, 28, 36 might be contribute to miRNA biosynthesis and DNA demethylation in longan early SE. DEAD-box (DDX) proteins belong to the largest subfamily of RNA helicase SF2, which contributes to all biological processes of RNA metabolism in the plant kingdom. Till now, no significant data are available regarding studies on DDX in Somatic Embryogenesis (SE) of woody plants. It is important to investigate the biological function of the DlDDX family in longan SE. Thus, a comprehensive analysis of 58 longan DEAD-box (DlDDX) genes characterization was performed by genome-wide identification and transcript abundance validation analysis. Homologous evolution has revealed that some DlDDXs in longan had high sequence similarity with Mus musculus , Citrus and Saccharomyces cerevisiae , indicating that DlDDXs were highly conservative in the animal, plant, and microorganism. Remarkably, gene duplication, purifying selection, and alternative splicing events, and new auxiliary domains have likely contributed to the functional evolution of DlDDX, indicating that DlDDX appeared neofunctionalization in longan. Besides, DlDDX3, 15, 28, 36 might interact with protein complex (MAC3A, MAC3B, CDC5, CBP20) of miRNA biosynthesis. Notably, DlDDX28 contained a novel auxiliary domain (CAF-1 p150), which might contribute to DNA demethylation in longan early SE. 4 DlDDX genes significantly expressed not only in early SE and zygotic embryogenesis (ZE) but also up-regulated at high levels in 'Honghezi' and 'Quanlongbaihe' with abortive seeds, which are of great significance. Moreover, some DlDDXs presented abiotic stress-response dynamic expression patterns by ABA, SA, JA, and NaCl treatments during early SE. Hence, DEAD-box is essential to SE development and seed abortive in longan. [ABSTRACT FROM AUTHOR]