Exogenous Melatonin Enhances Dihydrochalcone Accumulation in Lithocarpus litseifolius Leaves via Regulating Hormonal Crosstalk and Transcriptional Profiling.

Dihydrochalcones (DHCs) constitute a specific class of flavonoids widely known for their various health-related advantages. Melatonin (MLT) has received attention worldwide as a master regulator in plants, but its roles in DHC accumulation remain unclear. Herein, the elicitation impacts of MLT on DHC biosynthesis were examined in Lithocarpus litseifolius, a valuable medicinal plant famous for its sweet flavor and anti-diabetes effect. Compared to the control, the foliar application of MLT significantly increased total flavonoid and DHC (phlorizin, trilobatin, and phloretin) levels in L. litseifolius leaves, especially when 100 µM MLT was utilized for 14 days. Moreover, antioxidant enzyme activities were boosted after MLT treatments, resulting in a decrease in the levels of intracellular reactive oxygen species. Remarkably, MLT triggered the biosynthesis of numerous phytohormones linked to secondary metabolism (salicylic acid, methyl jasmonic acid (MeJA), and ethylene), while reducing free JA contents in L. litseifolius. Additionally, the flavonoid biosynthetic enzyme activities were enhanced by the MLT in leaves. Multiple differentially expressed genes (DEGs) in RNA-seq might play a crucial role in MLT-elicited pathways, particularly those associated with the antioxidant system (SOD, CAT, and POD), transcription factor regulation (MYBs and bHLHs), and DHC metabolism (4CL, C4H, UGT71K1, and UGT88A1). As a result, MLT enhanced DHC accumulation in L. litseifolius leaves, primarily by modulating the antioxidant activity and co-regulating the physiological, hormonal, and transcriptional pathways of DHC metabolism.

Chitosan elicitation of saponin accumulation in Psammosilene tunicoides hairy roots by modulating antioxidant activity, nitric oxide production and differential gene expression.

Elicitation is one of the most effective strategies for enhancing plant bioactive compounds, such as triterpenoid saponins. Chitosan gained worldwide attention for biostimulant activity, but little is known about its roles in the elicitation of triterpenoid saponin in medicinal plants. Here, we explored the regulatory network of chitosan on saponin accumulation in hairy root cultures of Psammosilene tunicoides, a valuable medicinal herb known for its pain-relieving properties endemic to China. Compared with control, the highest total saponin accumulation exhibited a 4.55-fold enhancement in hairy roots elicited by 200 mg L-1 chitosan for nine days. High-performance liquid chromatography (HPLC) revealed the yields of quillaic acid, gypsogenin and gypsogenin-3-O-ß-D-glucuronopyranoside were significantly increased after chitosan treatments. Moreover, exogenous chitosan application dramatically triggered the reactive oxygen species (ROS) scavenging enzyme activities and nitric oxide (NO) content in hairy roots. Comparative transcriptome analysis from chitosan-treated (1 and 9 d) or control groups revealed that differentially expressed genes (DEGs) were greatly enriched in plant-pathogen interaction and metabolic processes. The transcriptions of candidate DEGs involved in chitosan-elicited saponin metabolism were increased, especially genes encoding antioxidant enzymes (SOD, POD and GR), stress-responsive transcription factors (WRKYs and NACs) and terpenoid biosynthetic enzymes (DXS, GPPS and SE). Taken together, these results indicate that chitosan elicitor promotes triterpenoid saponin biosynthesis by enhancing antioxidant activities, NO production and differential gene expression in P. tunicoides hairy roots.

Full-Length Transcriptome Analyses of Genes Involved in Triterpenoid Saponin Biosynthesis of Psammosilene tunicoides Hairy Root Cultures With Exogenous Salicylic Acid.

Triterpenoid saponins constitute a diverse class of bioactive compounds in medicinal plants. Salicylic acid (SA) is an efficient elicitor for secondary metabolite production, but a transcriptome-wide regulatory network of SA-promoted triterpenoid saponin biosynthesis remains little understood. In the current study, we described the establishment of the hairy root culture system for Psammosilene tunicoides, a triterpenoid saponin-producing medicinal herb in China, using genetic transformation by Agrobacterium rhizogenes. Compared to controls, we found that total saponin content was dramatically increased (up to 2.49-fold) by the addition of 5 mg/L SA in hairy roots for 1 day. A combination of single-molecule real-time (SMRT) and next-generation sequencing (Illumina RNA-seq) was generated to analyze the full-length transcriptome data for P. tunicoides, as well as the transcript profiles in treated (8 and 24 h) and non-treated (0 h) groups with 5 mg/L SA in hairy roots. A total of 430,117 circular consensus sequence (CCS) reads, 16,375 unigenes and 4,678 long non-coding RNAs (lncRNAs) were obtained. The average length of unigenes (2,776 bp) was much higher in full-length transcriptome than that derived from single RNA-seq (1,457 bp). The differentially expressed genes (DEGs) were mainly enriched in the metabolic process. SA up-regulated the unigenes encoding SA-binding proteins and antioxidant enzymes in comparison with controls. Additionally, we identified 89 full-length transcripts encoding enzymes putatively involved in saponin biosynthesis. The candidate transcription factors (WRKY, NAC) and structural genes (AACT, DXS, SE, CYP72A) might be the key regulators in SA-elicited saponin accumulation. Their expression was further validated by quantitative real-time PCR (qRT-PCR). These findings preliminarily elucidate the regulatory mechanisms of SA on triterpenoid saponin biosynthesis in the transcriptomic level, laying a foundation for SA-elicited saponin augmentation in P. tunicoides.

The transcription factor VaNAC17 from grapevine (Vitis amurensis) enhances drought tolerance by modulating jasmonic acid biosynthesis in transgenic Arabidopsis.

KEY MESSAGE: Expression of VaNAC17 improved drought tolerance in transgenic Arabidopsis by upregulating stress-responsive genes, modulating JA biosynthesis, and enhancing ROS scavenging. Water deficit severely affects the growth and development of plants such as grapevine (Vitis spp.). Members of the NAC (NAM, ATAF1/2, and CUC2) transcription factor (TF) family participate in drought-stress-induced signal transduction in plants, but little is known about the roles of NAC genes in drought tolerance in grapevine. Here, we explored the role of VaNAC17 in Vitis amurensis, a cold-hardy, drought-tolerant species of grapevine. VaNAC17 was strongly induced in grapevine by drought, exogenous abscisic acid (ABA), and methyl jasmonate (MeJA). A transient expression assay in yeast indicated that VaNAC17 functions as a transcriptional activator. Notably, heterologous expression of VaNAC17 in Arabidopsis thaliana enhanced drought tolerance. VaNAC17-expressing Arabidopsis plants showed decreased reactive oxygen species (ROS) accumulation compared to wild-type plants under drought conditions. RNA-seq analysis indicated that VaNAC17 expression increased the transcription of downstream stress-responsive genes after 5 days of drought treatment, especially genes involved in jasmonic acid (JA) biosynthesis (such as LOX3, AOC1 and OPR3) and signaling (such as MYC2, JAZ1, VSP1 and CORI3) pathways. Endogenous JA levels increased in VaNAC17-OE plants under drought stress. Taken together, these results indicate that VaNAC17 plays a positive role in drought tolerance by modulating endogenous JA biosynthesis and ROS scavenging.

Hepatoprotective Effect of Polysaccharides Isolated from Dendrobium officinale against Acetaminophen-Induced Liver Injury in Mice via Regulation of the Nrf2-Keap1 Signaling Pathway.

The effect of polysaccharides isolated from Dendrobium officinale (DOP) on acetaminophen- (APAP-) induced hepatotoxicity and the underlying mechanisms involved are investigated. Male Institute of Cancer Research (ICR) mice were randomly assigned to six groups: (1) control, (2) vehicle (APAP, 230 mg/kg), (3) N-acetylcysteine (100 mg/kg), (4) 50 mg/kg DOP, (5) 100 mg/kg DOP, and (6) 200 mg/kg DOP. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the serum and glutathione (GSH), malondialdehyde (MDA), catalase (CAT), total antioxidant capacity (T-AOC), myeloperoxidase (MPO), and reactive oxygen species (ROS) levels in the liver were determined after the death of the mice. The histological examination of the liver was also performed. The effect of DOP on the Kelch-like ECH-associated protein 1- (Keap1-) nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway was evaluated using Western blot analysis and real-time polymerase chain reaction (PCR). The results showed that DOP treatment significantly alleviated the hepatic injury. The decrease in ALT and AST levels in the serum and ROS, MDA, and MPO contents in the liver, as well as the increases in GSH, CAT, and T-AOC in the liver, were observed after DOP treatment. DOP treatment significantly induced the dissociation of Nrf2 from the Nrf2-Keap1 complex and promoted the Nrf2 nuclear translocation. Subsequently, DOP-mediated Nrf2 activation triggered the transcription and expressions of the glutamate-cysteine ligase catalytic (GCLC) subunit, glutamate-cysteine ligase regulatory subunit (GCLM), heme oxygenase-1 (HO-1), and NAD(P)H dehydrogenase quinone 1 (NQO1) in APAP-treated mice. The present study revealed that DOP treatment exerted potentially hepatoprotective effects against APAP-induced liver injury. Further investigation about mechanisms indicated that DOP exerted the hepatoprotective effect by suppressing the oxidative stress and activating the Nrf2-Keap1 signaling pathway.

An efficient method for transgenic callus induction from Vitis amurensis petiole.

Transformation is the main platform for genetic improvement and gene function studies in plants. However, the established somatic embryo transformation system for grapevines is time-consuming and has low efficiency, which limits its utilization in functional genomics research. Vitis amurensis is a wild Vitis species with remarkable cold tolerance. The lack of an efficient genetic transformation system for it has significantly hindered the functional identification of cold stress related genes in the species. Herein, an efficient method was established to produce transformed calli of V. amurensis. Segments of petioles from micropropagated plantlets of V. amurensis exhibited better capacity to differentiate calli than leaf-discs and stem segments, and thus was chosen as target tissue for Agrobacterium-mediated transformation. Both neomycin phosphotransferase II (NPTII) and enhanced green fluorescent protein (eGFP) genes were used for simultaneous selection of transgenic calli based on kanamycin resistance and eGFP fluorescence. Several parameters affecting the transformation efficiency were optimized including the concentration of kanamycin, Agrobacterium stains, bacterial densities, infection treatments and co-cultivation time. The transgenic callus lines were verified by checking the integration of NPTII gene into calli genomes, the expression of eGFP gene and the fluorescence of eGFP. Up to 20% of the petiole segments produced transformed calli after 2 months of cultivation. This efficient transformation system will facilitate the functional analysis of agronomic characteristics and related genes not only in V. amurensis but also in other grapevine species.

Expression of Vitis amurensis NAC26 in Arabidopsis enhances drought tolerance by modulating jasmonic acid synthesis.

The growth and fruit quality of grapevines are widely affected by abnormal climatic conditions such as water deficits, but many of the precise mechanisms by which grapevines respond to drought stress are still largely unknown. Here, we report that VaNAC26, a member of the NAC transcription factor family, was upregulated dramatically during cold, drought and salinity treatments in Vitis amurensis, a cold and drought-hardy wild Vitis species. Heterologous overexpression of VaNAC26 enhanced drought and salt tolerance in transgenic Arabidopsis. Higher activities of antioxidant enzymes and lower concentrations of H2O2 and O2 (-) were found in VaNAC26-OE lines than in wild type plants under drought stress. These results indicated that scavenging by reactive oxygen species (ROS) was enhanced by VaNAC26 in transgenic lines. Microarray-based transcriptome analysis revealed that genes related to jasmonic acid (JA) synthesis and signaling were upregulated in VaNAC26-OE lines under both normal and drought conditions. VaNAC26 showed a specific binding ability on the NAC recognition sequence (NACRS) motif, which broadly exists in the promoter regions of upregulated genes in transgenic lines. Endogenous JA content significantly increased in the VaNAC26-OE lines 2 and 3. Our data suggest that VaNAC26 responds to abiotic stresses and may enhance drought tolerance by transcriptional regulation of JA synthesis in Arabidopsis.

Identification of cold-inducible microRNAs in grapevine.

Low temperature is one of the most important environmental factors that limits the geographical distribution and productivity of grapevine. However, the molecular mechanisms on how grapevine responds to cold stress remains to be elucidated. MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that play an essential role during plant development and stress responses. Although miRNAs and their targets have been identified in several Vitis species, their participation during cold accumulation in grapevine remains unknown. In this study, two small RNA libraries were generated from micropropagated 'Muscat Hamburg' (V. vinifera) plantlets under normal and low temperatures (4°C). A total of 163 known miRNAs and 67 putative novel miRNAs were detected from two small RNA libraries by Solexa sequencing. Forty-four cold-inducible miRNAs were identified through differentially expressed miRNAs (DEMs) analysis; among which, 13 belonged to upregulated DEMs while 31 belonged downregulated DEMs. The expression patterns of the 13 DEMs were verified by real-time RT-PCR analysis. The prediction of the target genes for DEMs indicated that miRNA may regulate transcription factors, including AP2, SBP, MYB, bHLH, GRAS, and bZIP under cold stress. The 5'-RLM RACE were conducted to verify the cleavage site of predicted targets. Seven predicted target genes for four known and three novel vvi-miRNAs showed specific cleavage sites corresponding to their miRNA complementary sequences. The expression pattern of these seven target genes revealed negative correlation with the expression level of the corresponding vvi-miRNAs. Our results indicated that a diverse set of miRNAs in V. vinifera are cold-inducible and may play an important role in cold stress response.

A novel system for evaluating drought-cold tolerance of grapevines using chlorophyll fluorescence.

BACKGROUND: Grape production in continental climatic regions suffers from the combination of drought and cold stresses during winter. Developing a reliable system to simulate combined drought-cold stress and to determine physiological responses and regulatory mechanisms is important. Evaluating tolerance to combined stress at germplasm level is crucial to select parents for breeding grapevines. RESULTS: In the present study, two species, namely, Vitis amurensis and V. vinifera cv. 'Muscat Hamburg', were used to develop a reliable system for evaluating their tolerance to drought-cold stress. This system used tissue -cultured grapevine plants, 6% PEG solution, and gradient cooling mode to simulate drought-cold stress. V. amurensis had a significantly lower LT50 value (the temperature of 50% electrolyte leakage) than 'Muscat Hamburg' during simulated drought-cold stress. Thus, the former had higher tolerance than the latter to drought-cold stress based on electrolyte leakage (EL) measurements. Moreover, the chlorophyll fluorescence responses of V. amurensis and 'Muscat Hamburg' were also analyzed under drought-cold stress. The maximum photochemical quantum yield of PS II (Fv/Fm) exhibited a significant linear correlationship with EL. The relationship of EL with Fv/Fm in the other four genotypes of grapevines under drought-cold stress was also detected. CONCLUSIONS: A novel LT50 estimation model was established, and the LT50 values can be well calculated based on Fv/Fm in replacement of EL measurement. The Fv/Fm-based model exhibits good reliability for evaluating the tolerance of different grapevine genotypes to drought-cold stress.

Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera.

BACKGROUND: WRKY transcription factors are one of the largest families of transcriptional regulators in plants. WRKY genes are not only found to play significant roles in biotic and abiotic stress response, but also regulate growth and development. Grapevine (Vitis vinifera) production is largely limited by stressful climate conditions such as cold stress and the role of WRKY genes in the survival of grapevine under these conditions remains unknown. RESULTS: We identified a total of 59 VvWRKYs from the V. vinifera genome, belonging to four subgroups according to conserved WRKY domains and zinc-finger structure. The majority of VvWRKYs were expressed in more than one tissue among the 7 tissues examined which included young leaves, mature leaves, tendril, stem apex, root, young fruits and ripe fruits. Publicly available microarray data suggested that a subset of VvWRKYs was activated in response to diverse stresses. Quantitative real-time PCR (qRT-PCR) results demonstrated that the expression levels of 36 VvWRKYs are changed following cold exposure. Comparative analysis was performed on data from publicly available microarray experiments, previous global transcriptome analysis studies, and qRT-PCR. We identified 15 VvWRKYs in at least two of these databases which may relate to cold stress. Among them, the transcription of three genes can be induced by exogenous ABA application, suggesting that they can be involved in an ABA-dependent signaling pathway in response to cold stress. CONCLUSIONS: We identified 59 VvWRKYs from the V. vinifera genome and 15 of them showed cold stress-induced expression patterns. These genes represented candidate genes for future functional analysis of VvWRKYs involved in the low temperature-related signal pathways in grape.