Dendrobium officinale Kimura et Migo (D. officinale) is one of the most important traditional Chinese medicinal herbs, celebrated for its abundant bioactive ingredients. This study demonstrated that the diurnal temperature difference (DIF) (T1: 13/13 °C, T2: 25/13 °C, and T3: 25/25 °C) was more favorable for high chlorophyll, increased polysaccharide, and total flavonoid contents compared to constant temperature treatments in D. officinale PLBs. The transcriptome analysis revealed 4251, 4404, and 4536 differentially expressed genes (DEGs) in three different comparisons (A: 25/13 °C vs. 13/13 °C, B: 13/13 °C vs. 25/25 °C, and C: 25/13 °C vs. 25/25 °C, respectively). The corresponding up-/down-regulated DEGs were 1562/2689, 2825/1579, and 2310/2226, respectively. GO and KEGG enrichment analyses of DEGs showed that the pathways of biosynthesis of secondary metabolites, carotenoid biosynthesis, and flavonoid biosynthesis were enriched in the top 20; further analysis of the sugar- and flavonol-metabolism pathways in D. officinale PLBs revealed that the DIF led to a differential gene expression in the enzymes linked to sugar metabolism, as well as to flavonol metabolism. Certain key metabolic genes related to ingredient accumulation were identified, including those involved in polysaccharide metabolism (SUS, SUT, HKL1, HGL, AMY1, and SS3) and flavonol (UGT73C and UGT73D) metabolism. Therefore, these findings indicated that these genes may play an important role in the regulatory network of the DIF in the functional metabolites of D. officinale PLBs. In a MapMan annotation of abiotic stress pathways, the DEGs with significant changes in their expression levels were mainly concentrated in the heat-stress pathways, including heat-shock proteins (HSPs) and heat-shock transcription factors (HSFs). In particular, the expression levels of HSP18.2, HSP70, and HSF1 were significantly increased under DIF treatment, which suggested that HSF1, HSP70 and HSP18.2 may respond to the DIF. In addition, they can be used as candidate genes to study the effect of the DIF on the PLBs of D. officinale. The results of our qPCR analysis are consistent with those of the transcriptome-expression analysis, indicating the reliability of the sequencing. The results of this study revealed the transcriptome mechanism of the DIF on the accumulation of the functional metabolic components of D. officinale. Furthermore, they also provide an important theoretical basis for improving the quality of D. officinale via the DIF in production.

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.

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.

Methyl jasmonic acid (MeJA) is a vital phytohormone that plays a key role in plant growth and adaptation to various environmental stresses. In the present study, on the basis of the longan genome, we identified a total of seven versatile putative abscisic acid-insensitive genes, which are the key players in plant growth and stress response. On the basis of bioinformatics analysis, transcriptome data, exogenous treatment experiments, and RT-qPCR findings, a comprehensive evolutionary pattern of ABI genes in different plant species and the effect of different MeJA treatments during early somatic embryogenesis in D. longan was carried out. The phylogeny results revealed that the seven DlABI genes evolved independently in monocots and dicots, having high protein sequence similarity, especially with Arabidopsis ABI genes. The comparative findings of gene structure, motif prediction, and synteny analysis suggest that DlABI genes disperse mainly through duplication events rather than localized tandem repeats. Furthermore, the correlations among the expressions of DlABI genes propose that the organization of the cis-regulatory elements in the promoter regions may regulate the temporal and spatial transcription activation of these genes. The qRT-PCR results revealed that the 50 µM MeJA treatment significantly upregulated the expression of DlABI3, followed by DlABI1, DlABI2, DlABI5, DlABI4, and DlABI8, respectively. The ROS findings clearly revealed that MeJA distinctly elevated the SOD, POD, and H₂O₂ activities while reducing catalase and MDA contents. The subcellular localization of DlABI3 further confirmed its presence in the nucleus, suggesting its predicated transcriptional regulatory role in MeJA-mediated early SE in longan. Our findings reveal that the ABI genes are integral to the mechanism of MeJA-induced early somatic embryogenesis in longan by maintaining the ROS activity.

Dimocarpus longan Lour. is an evergreen tree of the genus Longan in the Sapindaceae family, native to tropical and subtropical regions. Longan embryonic development is closely related to fruit set and fruit quality. An in-depth study of the mechanism of longan embryonic development could therefore contribute to the development of the longan industry. DIMBOA is the principal compound representing benzoxazinoids (BXs), and is closely linked to auxin biosynthesis and signal transduction. Auxin is one of the crucial hormones for inducing somatic embryogenesis (SE) in plants. Previous research has shown that DIMBOA promotes morphogenesis in the early somatic embryogenesis of longan, but the specific regulatory mechanism has not yet been clarified. To elucidate the molecular mechanism by which DIMBOA affects early somatic embryogenesis in longan, we chose longan embryogenic cultures grown under 0 mg/L DIMBOA as the control group (the check, CK), and longan embryogenic cultures grown under 0.1 mg/L DIMBOA as the treatment group (D) to be analyzed by transcriptomic sequencing. A total of 478 differentially expressed genes (DEGs) are detected in check vs. D, of which 193 are upregulated and 285 are downregulated. These DEGs are significantly enriched in the biosynthetic and metabolic functions of various substances such as vitamin B₆ (VB₆) biosynthesis, phenylpropanoid pathways, and carbohydrate metabolism. DIMBOA affects SE processes in longan via TFs, including MYB, ZF, bHLH, LBD, NAC, WRKY, etc. After DIMBOA treatment, the expression of most of the key genes for IAA synthesis was significantly downregulated, VB₆ content was significantly reduced, and H₂O₂ content was significantly increased. Therefore, it is suggested that DIMBOA directly or indirectly affects the H₂O₂ content through the VB₆ metabolic pathway, thereby regulating the endogenous IAA level to modulate the early SE morphogenesis of longan.

The PP2C (protein phosphates 2C) are key regulators of abscisic acid (ABA) signaling that play a crucial role in plant stress responses. In this study, we performed a comprehensive genome-wide analysis and identified 71 DlPP2C genes in Dimocarpus longan which is an economically important fruit crop. The evolutionary analysis revealed that DlPP2C genes were classified into distinct subgroups based on phylogenetic relationships with Arabidopsis thaliana and Oryza sativa. Structural analysis demonstrated conserved motif composition and gene organization within subgroups, whereas chromosomal distribution and synteny analysis revealed that segmental duplication events contributed to gene family expansion. Promoter analysis findings identified numerous cis-acting elements related to hormone and stress responsiveness especially abscisic acid-responsive elements (ABREs), suggesting their potential involvement in ABA signaling pathways. Under exogenous ABA treatments, expression profiling of the DlPP2C genes exhibited dynamic, dose and time dependent response with several genes showing peak expression at 10 μM ABA after 16 h, especially the DlPP2C1 displayed a strong transcriptional response, indicating its potential role as a key regulator. Overexpression and GUS staining assays revealed enhanced activity under ABA treatment, further supporting its involvement in ABA-responsive regulation. Moreover, RNA sequencing analysis revealed a total of 1799 differentially expressed genes, with prevalence of downregulated genes, showing extensive transcriptional reprograming. Functional enrichment analysis demonstrated that these genes were largely associated with plant hormone signaling, stress response and metabolic pathways. Together, these findings propose that DlPP2C genes, especially DlPP2C1, play a key role in ABA-mediated regulatory networks and provide valuable insights intro stress adaption mechanisms during early somatic embryogenesis in longan.